Max Costa, Ph.D.

Global Levels of Histone Modifications in Peripheral Blood Mononuclear Cells of Subjects with Exposure to Nickel
Arita A; Niu J; Qu Q; Zhao N; Ruan Y; Nadas A; Chervona Y; Wu F; Sun H; Hayes RB; Costa M
2011 Oct 24;:198-203 #, Environmental health perspectives
Background: Occupational exposure to nickel is associated with an increased risk for lung and nasal cancers. Nickel compounds exhibit weak mutagenic activity, cause gene amplification, and disrupt cellular epigenetic homeostasis. However, the nickel-induced changes in global histone modification levels have only been tested in vitro. Objective: This study was conducted in a Chinese population to determine whether occupational exposure to nickel is associated with alterations of global histone modification levels and to evaluate the inter-and intra-individual variance of global histone modification levels. Method: 45 subjects with occupational exposure to nickel and 75 referents were recruited. Urinary nickel and global H3K4 trimethylation (H3K4me3), H3K9 acetylation (H3K9ac), and H3K9 dimethylation (H3K9me2) levels were measured in peripheral blood mononuclear cells (PBMCs) of subjects. Results: H3K4me3 was elevated (0.25%+/-0.11%, 0.15%+/-0.04%, p=0.0004) and H3K9me2 was decreased (0.11%+/-0.05%, 0.15%+/-0.04%, p=0.003) in Ni-exposed subjects. H3K4me3 was positively (r=0.4, p=0.0008) and H3K9ac was negatively (r=0.1, p=0.01) associated with urinary nickel. Inter-individual variances of H3K4me3, H3K9ac, and H3K9me2 were larger relative to intra-individual variance in both groups, resulting in reliability coefficients, estimate of consistency of a set of measurements, of 0.75, 0.74, and 0.97 for H3K4me3, H3K9ac, and H3K9me2, respectively, for referent subjects. Reliability coefficients of 0.60, 0.67, and 0.79 were found for H3K4me3, H3K9ac, and H3K9me2, respectively, for Ni-exposed subjects. Conclusion: The results of this study indicate that occupational exposure to nickel is associated with alterations of global histone modification levels and that measurements of global levels of histone modifications are relatively stable over time in human PBMCs
— id: 141421, year: 2011, vol: , page: 198, stat: Journal Article,

The control of histone methylation and gene expression by oxidative stress, hypoxia and metals
Costa M.
2011 ;51:S3-S3, Free radical biology & medicine
The oxidative histone demethylases remove a number of transcriptional activating and inhibitory marks on the histone tails. For example, the JARID1 family (KMD5 A-D) removes the transcriptional activating mark H3K4 trimethylation, while JMJD2A removes H3K9 mono and dimethylation transcriptional silencing marks. These enzymes are members of the dioxygenase superfamily of enzymes which have Fe in their active site and decarboxylate oxogluterate releasing CO₂ and formaldehyde as a result of the oxidative demethylation of their substrates. They also use ascorbate to reduce the oxidized Fe. The dependence of these enzymes on oxygen results in their inhibition by hypoxia, and there is an accumulation of transcriptional activating marks such as H3K4 trimethylation and transcriptional silencing marks such as H3K9 dimethylation. In addition, hypoxia results in the increased protein and activity of G9a which also increases H3K9 dimethylation. Thus, there is extensive activation and repression of genes beyond what is attributable to the transcription factor HIF-1 alpha. In addition, exposure to metals such as Ni inhibits the dioxygenase enzymes by displacing the Fe from the active site. We have shown this using the dioxygenase DNA demethylase ABH2. Unlike the histone demethylases, it can be expressed in bacteria and the nickel displacement of ABH2 was studied by XAS and ⁶³Ni binding. We have mapped the changes in H3K4 trimethylation in the whole genome using ChIP-Seq and correlated this with changes in gene expression using RNA-Seq following hypoxia and nickel exposure in BEAS2B cells. With nickel exposure for example, H3K4 trimethylation increases in the promoter and coding region of the most induced genes. Since the dioxygenase superfamily of enzymes also require reduced ascrobate, the depletion of ascorbate by oxidative stress leads to inhibition of the oxidative histone demethylases. Chromate, for example, increases H3K4 trimethylation and H3K9 dimethylation by reacting with and decreasing cellular reduced ascrobate levels and this results in the inhibition of the respective oxidative histone demethylases. Thus, metals, hypoxia and oxidative stress all impact the epigenetic program and result in changes in gene expression, which is likely responsible for their toxic and carcinogenic effects
— id: 141793, year: 2011, vol: 51, page: S3, stat: Journal Article,

Structural comparisons of non-heme Fe enzymes
Giri N.C.; Maroney M.J.; Costa M.; Chen H.; Munck E.; Stoian S.A.
2011 ;16:S218-S218, Journal of biological inorganic chemistry. JBIC
Non-heme Fe enzymes catalyze a wide range of oxidations involving 02. We have used a combination ofspectroscopic techniques to investigate the structure and function of three different kinds of mononuclear non-heme Fe enzymes -ABH2 (a DNA demethylase), histone demethylases (JMJD2A, JMJD2C and JMJD2D), and rat liver cysteine dioxygenase (CDO). The demethylases have the 2-His-1-carboxylate Fe-binding motif, while CDO has a triad of histidines. It has been shown that Ni substitution causes inactivation ofABH2 that leads to transcriptional silencing. X-ray absorption spectroscopy (XAS) has been used to investigate the active site ofABH2 in presence of Fe and Ni. XAS shows that in the presence of both cofactor and substrate Fe is 5-coordinate (leaving an empty coordination site for 02 activation) but Ni is 6-coordinate (no empty coordination site for 02 binding). CDO catalyzes the conversion of cysteine to cysteine sulfinic acid (CSA). XAS of rat liver CDO reconstituted with Fe(ll) shows the presence of a 5- coordinate Fe site both in presence and absence of cysteine. Ligation of cysteine is confirmed by the presence of a S- donor. Mossbauer spectroscopy of rat liver CDO reconstituted with 57Fe shows that without substrate CDO contains mostly Fe(lll), while in the presence ofsubstrate CDO contains mostly Fe(ll). Posttranslational modifications of histone tails regulate chromatin structure and gene expression. It has already been shown that Ni substitution causes inactivation of JMJD2A. It has also been shown that JMJD2A is a H3-K9me3- and H3K36me3-specific demethylase. However, little is known about the basis ofsubstrate selectivity by the JMJD2 family of histone demethylases. We will report on XAS experiments involving the JMJD2 family of histone demethylases. The results will be compared with results obtained from ABH2 and from CDO. These XAS experiments, along with Mossbauer and MCD studies will allow us to compare metal site structures, substrate selectivity and reaction mechanism among different types of nonheme Fe enzymes. (Figure presented)
— id: 149832, year: 2011, vol: 16, page: S218, stat: Journal Article,

X-ray Absorption Spectroscopy Structural Investigation of Early Intermediates in the Mechanism of DNA Repair by Human ABH2
Giri, Nitai Charan; Sun, Hong; Chen, Haobin; Costa, Max; Maroney, Michael J
2011 Jun 7;50(22):5067-5076, Biochemistry
Human ABH2 repairs DNA lesions by using an Fe(II)- and alphaKG-dependent oxidative demethylation mechanism. The structure of the active site features the facial triad of protein ligands consisting of the side chains of two histidine residues and one aspartate residue that is common to many non-heme Fe(II) oxygenases. X-ray absorption spectroscopy (XAS) of metallated (Fe and Ni) samples of ABH2 was used to investigate the mechanism of ABH2 and its inhibition by Ni(II) ions. The data are consistent with a sequential mechanism that features a five-coordinate metal center in the presence and absence of the alpha-ketoglutarate cofactor. This aspect is not altered in the Ni(II)-substituted enzyme, and both metals are shown to bind the cofactor. When the substrate is bound to the native Fe(II) complex with alpha-ketoglutarate bound, a five-coordinate Fe(II) center is retained that features an open coordination position for O(2) binding. However, in the case of the Ni(II)-substituted enzyme, the complex that forms in the presence of the cofactor and substrate is six-coordinate and, therefore, features no open coordination site for oxygen activation at the metal
— id: 134095, year: 2011, vol: 50, page: 5067, stat: Journal Article,

Liprin-alpha4 Is Required for Nickel Induced Receptor Protein Tyrosine Phosphatase-Leukocyte Antigen Related Receptor F (RPTP-LAR) Activity
Kiok, Kathrin; Sun, Hong; Clancy, Hailey; Bose, Sutapa; Kluz, Thomas; Wu, Fen; Costa, Max
2011 ;6(8):e22764-e22764, PLoS ONE
Liprin-alpha4 was strongly induced following nickel (II) chloride exposure in a variety of cell types including BEAS-2B, A549, BEP2D and BL41 cells. Liprin-alpha4, a member of the Liprin alpha family, has seven isoforms but only three of these variants were detected in BEAS-2B cells (004, 201 and 202). The level of Liprin-alpha4 variants 201 and 004 were highly increased in BEAS-2B cells in response to nickel. We showed that Liprin-alpha4 bound directly to the cytoplasmic region of RPTP-LAR (receptor protein tyrosine phosphatase-leukocyte antigen-related receptor F). The cytoplasmic region of RPTP-LAR contains two phosphatase domains but only the first domain shows activity. The second domain interacts with other proteins. The phosphatase activity was increased both following nickel treatment and also in the presence of nickel ions in cell extracts. Liprin-alpha4 knock-down lines with decreased expression of Liprin-alpha4 variants 004 and 201 exhibited greater nickel toxicity compared to controls. The RPTP-LAR phosphatase activity was only slightly increased in a Liprin-alpha4 knock-down line. Liprin-alpha4 appeared necessary for the nickel induced tyrosine phosphatase activity. The presence of Liprin-alpha4 and nickel increased tyrosine phosphatase activity that reduced the global levels of tyrosine phosphorylation in the cell
— id: 136524, year: 2011, vol: 6, page: e22764, stat: Journal Article,

Comparison of Gene Expression Profiles in Chromate Transformed BEAS-2B Cells
Sun, Hong; Clancy, Harriet A; Kluz, Thomas; Zavadil, Jiri; Costa, Max
2011 ;6(3):e17982-e17982, PLoS ONE
BACKGROUND: Hexavalent chromium [Cr(VI)] is a potent human carcinogen. Occupational exposure has been associated with increased risk of respiratory cancer. Multiple mechanisms have been shown to contribute to Cr(VI) induced carcinogenesis, including DNA damage, genomic instability, and epigenetic modulation, however, the molecular mechanism and downstream genes mediating chromium's carcinogenicity remain to be elucidated. METHODS/RESULTS: We established chromate transformed cell lines by chronic exposure of normal human bronchial epithelial BEAS-2B cells to low doses of Cr(VI) followed by anchorage-independent growth. These transformed cell lines not only exhibited consistent morphological changes but also acquired altered and distinct gene expression patterns compared with normal BEAS-2B cells and control cell lines (untreated) that arose spontaneously in soft agar. Interestingly, the gene expression profiles of six Cr(VI) transformed cell lines were remarkably similar to each other yet differed significantly from that of either control cell lines or normal BEAS-2B cells. A total of 409 differentially expressed genes were identified in Cr(VI) transformed cells compared to control cells. Genes related to cell-to-cell junction were upregulated in all Cr(VI) transformed cells, while genes associated with the interaction between cells and their extracellular matrices were down-regulated. Additionally, expression of genes involved in cell proliferation and apoptosis were also changed. CONCLUSION: This study is the first to report gene expression profiling of Cr(VI) transformed cells. The gene expression changes across individual chromate exposed clones were remarkably similar to each other but differed significantly from the gene expression found in anchorage-independent clones that arose spontaneously. Our analysis identified many novel gene expression changes that may contribute to chromate induced cell transformation, and collectively this type of information will provide a better understanding of the mechanism underlying chromate carcinogenicity
— id: 129325, year: 2011, vol: 6, page: e17982, stat: Journal Article,

Effects of Nickel Treatment on H3K4 Trimethylation and Gene Expression
Tchou-Wong, Kam-Meng; Kiok, Kathrin; Tang, Zuojian; Kluz, Thomas; Arita, Adriana; Smith, Phillip R; Brown, Stuart; Costa, Max
2011 ;6(3):e17728-e17728, PLoS ONE
Occupational exposure to nickel compounds has been associated with lung and nasal cancers. We have previously shown that exposure of the human lung adenocarcinoma A549 cells to NiCl(2) for 24 hr significantly increased global levels of trimethylated H3K4 (H3K4me3), a transcriptional activating mark that maps to the promoters of transcribed genes. To further understand the potential epigenetic mechanism(s) underlying nickel carcinogenesis, we performed genome-wide mapping of H3K4me3 by chromatin immunoprecipitation and direct genome sequencing (ChIP-seq) and correlated with transcriptome genome-wide mapping of RNA transcripts by massive parallel sequencing of cDNA (RNA-seq). The effect of NiCl(2) treatment on H3K4me3 peaks within 5,000 bp of transcription start sites (TSSs) on a set of genes highly induced by nickel in both A549 cells and human peripheral blood mononuclear cells were analyzed. Nickel exposure increased the level of H3K4 trimethylation in both the promoters and coding regions of several genes including CA9 and NDRG1 that were increased in expression in A549 cells. We have also compared the extent of the H3K4 trimethylation in the absence and presence of formaldehyde crosslinking and observed that crosslinking of chromatin was required to observe H3K4 trimethylation in the coding regions immediately downstream of TSSs of some nickel-induced genes including ADM and IGFBP3. This is the first genome-wide mapping of trimethylated H3K4 in the promoter and coding regions of genes induced after exposure to NiCl(2). This study may provide insights into the epigenetic mechanism(s) underlying the carcinogenicity of nickel compounds
— id: 130306, year: 2011, vol: 6, page: e17728, stat: Journal Article,

Epigallocatechin-3-gallate (EGCG) protects against chromate-induced toxicity in vitro
Wu F; Sun H; Kluz T; Clancy HA; Kiok K; Costa M
2011 Jan 15;258(2):166-175, Toxicology & applied pharmacology
Hexavalent chromium [Cr(VI)] is a human carcinogen that results in the generation of reactive oxygen species (ROS) and a variety of DNA lesions leading to cell death. Epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea, possesses potent antioxidative activity capable of protecting normal cells from various stimuli-induced oxidative stress and cell death. Here we demonstrated that co-treatment with EGCG protected human normal bronchial epithelial BEAS-2B cells from Cr(VI)-induced cell death in a dose-dependent manner. Cr(VI) induces apoptosis as the primary mode of cell death. Co-treatment of BEAS-2B cells with EGCG dose-dependently suppressed Cr(VI)-induced apoptosis. Fluorescence microscopic analyses and quantitative measurement revealed that EGCG significantly decreased intracellular levels of ROS induced by Cr(VI) exposure. Using a well-established K(+)/SDS precipitation assay, we further showed that EGCG was able to dose-dependently reduce DNA-protein cross-links (DPC), lesions that could be partially attributed to Cr(VI)-induced oxidative stress. Finally, analyses of Affymetrix microarray containing 28,869 well-annotated genes revealed that, among the 3412 genes changed more than 1.5-fold by Cr(VI) treatment, changes of 2404 genes (70%) were inhibited by pretreatment of EGCG. Real-time PCR confirmed the induction of 3 genes involved in cell death and apoptosis by Cr(VI), which was eliminated by EGCG. In contrast, Cr(VI) reduced the expression of 3 genes related to cellular defense, and this reduction was inhibited by EGCG. Our results indicate that EGCG protects BEAS-2B cells from Cr(VI)-induced cytotoxicity presumably by scavenging ROS and modulating a subset of genes. EGCG, therefore, might serve as a potential chemopreventive agent against Cr(VI) carcinogenesis
— id: 141420, year: 2011, vol: 258, page: 166, stat: Journal Article,

Nickel Ions Inhibit Histone Demethylase JMJD1A and DNA Repair Enzyme ABH2 by Replacing the Ferrous Iron in the Catalytic Centers
Chen, Haobin; Giri, Nitai Charan; Zhang, Ronghe; Yamane, Kenichi; Zhang, Yi; Maroney, Michael; Costa, Max
2010 Mar 5;285(10):7374-7383, Journal of biological chemistry
Iron- and 2-oxoglutarate-dependent dioxygenases are a diverse family of non-heme iron enzymes that catalyze various important oxidations in cells. A key structural motif of these dioxygenases is a facial triad of 2-histidines-1-carboxylate that coordinates the Fe(II) at the catalytic site. Using histone demethylase JMJD1A and DNA repair enzyme ABH2 as examples, we show that this family of dioxygenases is highly sensitive to inhibition by carcinogenic nickel ions. We find that, with iron, the 50% inhibitory concentrations of nickel (IC(50) [Ni(II)]) are 25 mum for JMJD1A and 7.5 mum for ABH2. Without iron, JMJD1A is 10 times more sensitive to nickel inhibition with an IC(50) [Ni(II)] of 2.5 mum, and approximately one molecule of Ni(II) inhibits one molecule of JMJD1A, suggesting that nickel causes inhibition by replacing the iron. Furthermore, nickel-bound JMJD1A is not reactivated by excessive iron even up to a 2 mm concentration. Using x-ray absorption spectroscopy, we demonstrate that nickel binds to the same site in ABH2 as iron, and replacement of the iron by nickel does not prevent the binding of the cofactor 2-oxoglutarate. Finally, we show that nickel ions target and inhibit JMJD1A in intact cells, and disruption of the iron-binding site decreases binding of nickel ions to ABH2 in intact cells. Together, our results reveal that the members of this dioxygenase family are specific targets for nickel ions in cells. Inhibition of these dioxygenases by nickel is likely to have widespread impacts on cells (e.g. impaired epigenetic programs and DNA repair) and may eventually lead to cancer development
— id: 107769, year: 2010, vol: 285, page: 7374, stat: Journal Article,

Hypoxia and nickel inhibit histone demethylase JMJD1A and repress Spry2 expression in human bronchial epithelial BEAS-2B cells
Chen, Haobin; Kluz, Thomas; Zhang, Ronghe; Costa, Max
2010 Dec;31(12):2136-2144, Carcinogenesis
Epigenetic silencing of tumor suppressor genes commonly occurs in human cancers via increasing DNA methylation and repressive histone modifications at gene promoters. However, little is known about how pathogenic environmental factors contribute to cancer development by affecting epigenetic regulatory mechanisms. Previously, we reported that both hypoxia and nickel (an environmental carcinogen) increased global histone H3 lysine 9 methylation in cells through inhibiting a novel class of iron- and alpha-ketoglutarate-dependent histone demethylases. Here, we investigated whether inhibition of histone demethylase JMJD1A by hypoxia and nickel could lead to repression/silencing of JMJD1A-targeted gene(s). By using Affymetrix GeneChip and ChIP-on-chip technologies, we identified Spry2 gene, a key regulator of receptor tyrosine kinase/extracellular signal-regulated kinase (ERK) signaling, as one of the JMJD1A-targeted genes in human bronchial epithelial BEAS-2B cells. Both hypoxia and nickel exposure increased the level of H3K9me2 at the Spry2 promoter by inhibiting JMJD1A, which probably led to a decreased expression of Spry2 in BEAS-2B cells. Repression of Spry2 potentiated the nickel-induced ERK phosphorylation, and forced expression of Spry2 in BEAS-2B cells decreased the nickel-induced ERK phosphorylation and significantly suppressed nickel-induced anchorage-independent growth. Taken together, our results suggest that histone demethylases could be targets of environmental carcinogens and their inhibition may lead to altered gene expression and eventually carcinogenesis
— id: 114823, year: 2010, vol: 31, page: 2136, stat: Journal Article,

Effects of cellular iron deficiency on the formation of vascular endothelial growth factor and angiogenesis. Iron deficiency and angiogenesis
Eckard, Jonathan; Dai, Jisen; Wu, Jing; Jian, Jinlong; Yang, Qing; Chen, Haobin; Costa, Max; Frenkel, Krystyna; Huang, Xi
2010 ;10:28-28, Cancer cell international
ABSTRACT: BACKGROUND: Young women diagnosed with breast cancer are known to have a higher mortality rate from the disease than older patients. Specific risk factors leading to this poorer outcome have not been identified. In the present study, we hypothesized that iron deficiency, a common ailment in young women, contributes to the poor outcome by promoting the hypoxia inducible factor-1alpha (HIF-1alpha and vascular endothelial growth factor (VEGF) formation. This hypothesis was tested in an in vitro cell culture model system. RESULTS: Human breast cancer MDA-MB-231 cells were transfected with transferrin receptor-1 (TfR1) shRNA to constitutively impair iron uptake. Cellular iron status was determined by a set of iron proteins and angiogenesis was evaluated by levels of VEGF in cells as well as by a mouse xenograft model. Significant decreases in ferritin with concomitant increases in VEGF were observed in TfR1 knockdown MDA-MB-231 cells when compared to the parental cells. TfR1 shRNA transfectants also evoked a stronger angiogenic response after the cells were injected subcutaneously into nude mice. The molecular mechanism appears that cellular iron deficiency elevates VEGF formation by stabilizing HIF-1alpha. This mechanism is also true in human breast cancer MCF-7 and liver cancer HepG2 cells. CONCLUSIONS: Cellular iron deficiency increased HIF-1alpha, VEGF, and angiogenesis, suggesting that systemic iron deficiency might play an important part in the tumor angiogenesis and recurrence in this young age group of breast cancer patients
— id: 114147, year: 2010, vol: 10, page: 28, stat: Journal Article,

Roles of MAPK pathway activation during cytokine induction in BEAS-2B cells exposed to fine World Trade Center (WTC) dust
Wang, Shang; Prophete, Colette; Soukup, Joleen M; Chen, Lung-Chi; Costa, Max; Ghio, Andrew; Qu, Qingshan; Cohen, Mitchell D; Chen, Haobin
2010 Oct-Dec;7(4):298-307, Journal of immunotoxicology
The World Trade Center (WTC) collapse on September 11, 2001 released copious amounts of particulate matter (PM) into the atmosphere of New York City. Follow-up studies on persons exposed to the dusts have revealed a severely increased rate for asthma and other respiratory illnesses. There have only been a few studies that have sought to discern the possible mechanisms underlying these untoward pathologies. In one study, an increased cytokine release was detected in cells exposed to WTC fine dusts (PM. fraction or WTC.). However, the mechanism(s) for these increases has yet to be fully defined. Because activation of the mitogen-activated protein kinase (MAPK) signaling pathways is known to cause cytokine induction, the current study was undertaken to analyze the possible involvement of these pathways in any increased cytokine formation by lung epithelial cells (as BEAS-2B cells) exposed to WTC.. Our results showed that exposure to WTC. for 5 hr increased interleukin-6 (IL-6) mRNA expression in BEAS-2B cells, as well as its protein levels in the culture media, in a dose-dependent manner. Besides IL-6, cytokine multiplex analyses revealed that formation of IL-8 and -10 was also elevated by the exposure. Both extracellular signal-regulated kinase (ERK) and p38, but not c-Jun N-terminal protein kinase, signaling pathways were found to be activated in cells exposed to WTC.. Inactivation of ERK signaling pathways by PD98059 effectively blocked IL-6, -8, and -10 induction by WTC.; the p38 kinase inhibitor SB203580 significantly decreased induction of IL-8 and -10. Together, our data demonstrated activation of MAPK signaling pathway(s) likely played an important role in the WTC.-induced formation of several inflammatory (and, subsequently, anti-inflammatory) cytokines. The results are important in that they help to define one mechanism via which the WTC dusts may have acted to cause the documented increases in asthma and other inflammation-associated respiratory dysfunctions in the individuals exposed to the dusts released from the WTC collapse
— id: 138258, year: 2010, vol: 7, page: 298, stat: Journal Article,

Temporal reproducibility of taurine measurements in frozen serum of healthy postmenopausal women
Wojcik, Oktawia P; Koenig, Karen L; Zeleniuch-Jacquotte, Anne; Costa, Max; Chen, Yu
2010 Sep;104(5):629-632, British journal of nutrition
Animal studies and small clinical trials have shown that taurine (2-aminoethanesulphonic acid), a sulphur-containing molecule mainly obtained from the diet in human subjects, has a variety of biological actions that are related to atherosclerosis and cardiovascular functions. However, epidemiological studies of taurine and CHD risk are lacking. We evaluated whether a single measurement of serum taurine could serve as an estimate for long-term serum levels. Serum taurine was measured using HPLC in three annual samples from thirty postmenopausal women selected from the New York University Women's Health Study. Overall, serum taurine values ranged from 62.8 to 245.3 nmol/ml, with a mean of 140 nmol/ml. The intraclass correlation coefficient of a single measurement of serum taurine was 0.48 (95 % CI 0.26, 0.68), which can be improved to 0.65 by using the mean of two annual measurements. The CV was 7 %. These results indicate that the mean of two or more annual measurements of serum taurine is a sufficiently reliable measure of long-term serum levels that can be used in epidemiological studies
— id: 132231, year: 2010, vol: 104, page: 629, stat: Journal Article,

The potential protective effects of taurine on coronary heart disease
Wojcik, Oktawia P; Koenig, Karen L; Zeleniuch-Jacquotte, Anne; Costa, Max; Chen, Yu
2010 Jan;208(1):19-25, Atherosclerosis
In humans, taurine (2-aminoethanesulfonic acid) is mainly obtained from diet. Despite the fact that the health effects of taurine are largely unknown, taurine has become a popular supplement and ingredient in energy drinks in recent years. Evidence from mechanistic and animal studies has shown that the main biological actions of taurine include its ability to conjugate bile acids, regulate blood pressure (BP), and act as a potent antioxidant and anti-inflammatory agent. These actions suggest that high levels of taurine may be protective against coronary heart disease (CHD). However, data from epidemiologic and intervention studies in humans are limited. We review what is known about taurine\'s metabolism, its transportation in the body, its food sources, and evidence of its effect on cardiovascular health from in vitro, animal, and epidemiologic studies. We also discuss shortcomings of the human studies that need to be addressed in the future. The identification of taurine as a preventive factor for CHD may be of great public health importance
— id: 101575, year: 2010, vol: 208, page: 19, stat: Journal Article,

Phase II study of darinaparsin in patients with advanced hepatocellular carcinoma
Wu, Jennifer; Henderson, Charles; Feun, Lynn; Van Veldhuizen, Peter; Gold, Philip; Zheng, Hui; Ryan, Theresa; Blaszkowsky, Lawrence S; Chen, Haobin; Costa, Max; Rosenzweig, Barry; Nierodzik, MaryLynn; Hochster, Howard; Muggia, Franco; Abbadessa, Giovanni; Lewis, Jonathan; Zhu, Andrew X
2010 Oct;28(5):670-676, Investigational new drugs
BACKGROUND: Darinaparsin is a novel organic arsenic that reaches higher intracellular concentration with decreased toxicity compared to inorganic arsenic. We conducted a multi-center phase II study with darinaparsin in patients with advanced HCC. METHODS: Eligibility criteria included unresectable or metastatic measurable HCC, up to two prior systemic treatments, ECOG performance status < or = 2, Child Pugh Class A or B and adequate organ functions. Darinaparsin was administered at 420 mg/m(2) intravenously, twice weekly at least 72 h apart for 3 weeks in a 4-week cycle. The primary end point was response rate. A Simon two-stage design was used. RESULTS: Among 15 patients in the first stage, no objective responses were observed. Two patients had stable disease. The median number of cycles on study per patient was 2 (1-6). The median progression free survival and overall survival were 55 days (95% confidence interval: 50-59) and 190 days (95% confidence interval: 93-227), respectively. No treatment related hospitalizations or deaths occurred. Treatment related grade 1-2 toxicities included nausea, vomiting (26.7% each), fatigue (20%), anorexia and diarrhea (13.3% each). Grade 3 anorexia, wheezing, agitation, abdominal pain and SGPT were observed in 1 patient each (6.7%). One patient experienced grade 4 hypoglycemia (6.7%). CONCLUSIONS: Darinaparsin could be safely administered with tolerable toxicity profiles, and no QTc prolongation in patients with advanced HCC. However, at this dose and schedule, it has shown no objective responses in HCC and this trial was terminated as planned after the first stage of efficacy analysis
— id: 138148, year: 2010, vol: 28, page: 670, stat: Journal Article,

Plk3 Functions as an Essential Component of the Hypoxia Regulatory Pathway by Direct Phosphorylation of HIF-1{alpha}
Xu, Dazhong; Yao, Yixin; Lu, Luo; Costa, Max; Dai, Wei
2010 Dec 10;285(50):38944-38950, Journal of biological chemistry
Polo-like kinase 3 (Plk3) plays an important role in the regulation of cell cycle progression and stress responses. Plk3 also has a tumor-suppressing activity as aging PLK3-null mice develop tumors in multiple organs. The growth of highly vascularized tumors in PLK3-null mice suggests a role for Plk3 in angiogenesis and cellular responses to hypoxia. By studying primary isogenic murine embryonic fibroblasts, we tested the hypothesis that Plk3 functions as a component in the hypoxia signaling pathway. PLK3(-/-) murine embryonic fibroblasts contained an enhanced level of HIF-1alpha under hypoxic conditions. Immunoprecipitation and pulldown analyses revealed that Plk3 physically interacted with HIF-1alpha under hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, phosphorylated HIF-1alpha in vitro, resulting in a major mobility shift. Mass spectrometry identified two unique serine residues that were phosphorylated by Plk3. Moreover, ectopic expression followed by cycloheximide or pulse-chase treatment demonstrated that phospho-mutants exhibited a much longer half-life than the wild-type counterpart, strongly suggesting that Plk3 directly regulates HIF-1alpha stability in vivo. Combined, our study identifies Plk3 as a new and essential player in the regulation of the hypoxia signaling pathway
— id: 115272, year: 2010, vol: 285, page: 38944, stat: Journal Article,

JNK1 mediates degradation HIF-1alpha by a VHL-independent mechanism that involves the chaperones Hsp90/Hsp70
Zhang, Dongyun; Li, Jingxia; Costa, Max; Gao, Jimin; Huang, Chuanshu
2010 Jan 15;70(2):813-823, Cancer research
Hypoxia-inducible factor-1alpha (HIF-1alpha) is a master transcription factor that is critical for the regulation of a variety of cellular functions. HIF-1alpha is rapidly degraded under normoxic conditions by ubiquitin-mediated proteasome pathway controlled by the tumor suppressor von Hippel Lindau (VHL). Several recent studies reveal that heat-shock proteins (Hsp) can regulate HIF-1alpha protein degradation by a VHL-independent pathway. Here, we demonstrate that the stress kinase c-Jun NH(2)-terminal kinase 1 (JNK1) is required for Hsp-dependent regulation of HIF-1alpha. Stabilization of HIF-1alpha was impaired in JNK1-/- cells but could be rescued by JNK1 reconstitution under hypoxic conditions. These effects could be phenocopied in other cell settings by JNK1 silencing. Accordingly, HIF-1 transcriptional activity and target gene expression were dramatically reduced in JNK1-/- cells. Further, decreased levels of endogenous Hsp90/Hsp70 proteins in JNK1-/- cells affected the protective roles of these chaperones in stabilizing newly synthesized HIF-1alpha, whereas enforced expression of Hsp90/Hsp70 in JNK1-/- cells increased HIF-1alpha stability relative to parental control cells. Furthering this connection, we also found that defective expression of the Hsp90 acetyltransferase HDAC6 in JNK1-/- cells was associated with reduced Hsp90 chaperone activity. Taken together, our studies define a novel function for JNK1 in regulating HIF-1alpha turnover by a VHL-independent mechanism
— id: 106216, year: 2010, vol: 70, page: 813, stat: Journal Article,

Hypoxia induces trimethylated H3 lysine 4 by inhibition of JARID1A demethylase
Zhou, Xue; Sun, Hong; Chen, Haobin; Zavadil, Jiri; Kluz, Thomas; Arita, Adriana; Costa, Max
2010 May 15;70(10):4214-4221, Cancer research
Histone H3 lysine 4 (H3K4) trimethylation (H3K4me3) at the promoter region of genes has been linked to transcriptional activation. In the present study, we found that hypoxia (1% oxygen) increased H3K4me3 in both normal human bronchial epithelial Beas-2B cells and human lung carcinoma A549 cells. The increase of H3K4me3 from hypoxia was likely caused by the inhibition of H3K4 demethylating activity, as hypoxia still increased H3K4me3 in methionine-deficient medium. Furthermore, an in vitro histone demethylation assay showed that 1% oxygen decreased the activity of H3K4 demethylases in Beas-2B nuclear extracts because ambient oxygen tensions were required for the demethylation reaction to proceed. Hypoxia only minimally increased H3K4me3 in the BEAS-2B cells with knockdown of JARID1A, which is the major histone H3K4 demethylase in this cell line. However, the mRNA and protein levels of JARID1A were not affected by hypoxia. GeneChip and pathway analysis in JARID1A knockdown Beas-2B cells revealed that JARID1A regulates the expression of hundreds of genes involved in different cellular functions, including tumorigenesis. Knocking down of JARID1A increased H3K4me3 at the promoters of HMOX1 and DAF genes. Thus, these results indicate that hypoxia might target JARID1A activity, which in turn increases H3K4me3 at both the global and gene-specific levels, leading to the altered programs of gene expression and tumor progression
— id: 110413, year: 2010, vol: 70, page: 4214, stat: Journal Article,

Nickel binding to histone H4
Zoroddu, Maria Antonietta; Peana, Massimiliano; Medici, Serenella; Casella, Luigi; Monzani, Enrico; Costa, Max
2010 Jan 21;39(3):787-793, Dalton transactions (Cambridge, England : 2003)
Nickel compounds influence carcinogenesis by interfering with a variety of cellular targets. It has been found that nickel is a potent inhibitor in vivo of histone H4 acetylation, in both yeast and mammalian cells. It has preference to specific lysine residues in the H4 N-terminal -S(1)GRGK(5)GGK(8)GLGK(12)GGAK(16)RH(18)RKVL(22) tail, in which the sites of acetylation are clustered. About the nature of the structural changes induced by histone acetylation on H4, it has been recently demonstrated that acetylation induces an increase in alpha-helical conformation of the acetylated N-terminal tail of H4. It causes a shortening of the tail and, such an effect, may have an important structural and functional implication as a mechanism of transcriptional regulation. Here we report a study on the conformational changes induced by carcinogenic nickel compounds on the histone H4 protein. From a circular dichroism study we found that nickel is able to induce a secondary structure in the protein. In particular, nickel has the same effect as acetylation: it induces an increase in alpha-helical conformation of the non-acetylated histone H4. The alpha-helical increase that occurs upon nickel interaction with histone H4 should decrease the ability of histone acetyl transferase to recognize and bind to the histone tail and thus affect the ability of the enzyme to further modify the lysine residues. The shortening of the distance between adjacent amino acids, caused by the translation from an extended to a helical conformation, could disrupt the histone recognition motif; this may eventually compromise the entire 'histone code'
— id: 134096, year: 2010, vol: 39, page: 787, stat: Journal Article,

Epigenetics in metal carcinogenesis: Nickel, Arsenic, Chromium and Cadmium
Arita, Adriana; Costa, Max
2009 ;1(3):222-228, Metallomics : integrated biometal science
Although carcinogenic metals have been known to disrupt a wide range of cellular processes the precise mechanism by which these exert their carcinogenic effects is not known. Over the last decade or two, studies in the field of metal carcinogenesis suggest that epigenetic mechanisms may play a role in metal-induced carcinogenesis. In this review we summarize the evidence demonstrating that exposure to carcinogenic metals such as nickel, arsenic, chromium, and cadmium can perturb DNA methylation levels as well as global and gene specific histone tail posttranslational modification marks. We also wish to emphasize the importance in understanding that gene expression can be regulated by both genetic and epigenetic mechanisms and both these must be considered when studying the mechanism underlying the toxicity and cell-transforming ability of carcinogenic metals and other toxicants, and aberrant changes in gene expression that occur during disease states such as cancer
— id: 122687, year: 2009, vol: 1, page: 222, stat: Journal Article,

A genome-wide deletion mutant screen identifies pathways affected by nickel sulfate in Saccharomyces cerevisiae
Arita, Adriana; Zhou, Xue; Ellen, Thomas P; Liu, Xin; Bai, Jingxiang; Rooney, John P; Kurtz, Adrienne; Klein, Catherine B; Dai, Wei; Begley, Thomas J; Costa, Max
2009 ;10:524-524, BMC genomics
BACKGROUND: The understanding of the biological function, regulation, and cellular interactions of the yeast genome and proteome, along with the high conservation in gene function found between yeast genes and their human homologues, has allowed for Saccharomyces cerevisiae to be used as a model organism to deduce biological processes in human cells. Here, we have completed a systematic screen of the entire set of 4,733 haploid S. cerevisiae gene deletion strains (the entire set of nonessential genes for this organism) to identify gene products that modulate cellular toxicity to nickel sulfate (NiSO(4)). RESULTS: We have identified 149 genes whose gene deletion causes sensitivity to NiSO(4) and 119 genes whose gene deletion confers resistance. Pathways analysis with proteins whose absence renders cells sensitive and resistant to nickel identified a wide range of cellular processes engaged in the toxicity of S. cerevisiae to NiSO(4). Functional categories overrepresented with proteins whose absence renders cells sensitive to NiSO(4) include homeostasis of protons, cation transport, transport ATPases, endocytosis, siderophore-iron transport, homeostasis of metal ions, and the diphthamide biosynthesis pathway. Functional categories overrepresented with proteins whose absence renders cells resistant to nickel include functioning and transport of the vacuole and lysosome, protein targeting, sorting, and translocation, intra-Golgi transport, regulation of C-compound and carbohydrate metabolism, transcriptional repression, and chromosome segregation/division. Interactome analysis mapped seven nickel toxicity modulating and ten nickel-resistance networks. Additionally, we studied the degree of sensitivity or resistance of the 111 nickel-sensitive and 72 -resistant strains whose gene deletion product has a similar protein in human cells. CONCLUSION: We have undertaken a whole genome approach in order to further understand the mechanism(s) regulating the cell's toxicity to nickel compounds. We have used computational methods to integrate the data and generate global models of the yeast's cellular response to NiSO(4). The results of our study shed light on molecular pathways associated with the cellular response of eukaryotic cells to nickel compounds and provide potential implications for further understanding the toxic effects of nickel compounds to human cells
— id: 105506, year: 2009, vol: 10, page: 524, stat: Journal Article,

Iron- and 2-oxoglutarate-dependent Dioxygenases: an emerging group of molecular targets for nickel toxicity and carcinogenicity
Chen, Haobin; Costa, Max
2009 Feb;22(1):191-196, Biometals
Nickel compounds are important occupational and environmental pollutants. Chronic exposure to these pollutants has been connected with increased risks of respiratory cancers and cardiovascular diseases. However, it is still not clear what are the specific molecular targets for nickel toxicity and carcinogenicity. Here, we propose that the iron- and 2-oxoglutarate-dependent dioxygenase family enzymes are important intracellular targets that mediate the toxicity and carcinogenicity of nickel. In support of this hypothesis, our data show that three different classes of enzymes in this iron- and 2-oxoglutarate-dependent dioxygenase family, including HIF-prolyl hydroxylase PHD2, histone demethylase JHDM2A/JMJD1A, and DNA repair enzyme ABH3, are all highly sensitive to nickel inhibition. Inactivation of these enzymes accounts for a number of deleterious effects caused by nickel in cells, namely hypoxia-mimic stress and aberrant epigenetic changes. Future studies on nickel's effects on these iron- and 2-oxoglutarate-dependent dioxygenases would deepen our understanding on nickel toxicity and carcinogenicity
— id: 96242, year: 2009, vol: 22, page: 191, stat: Journal Article,

Heterochromatinization as a potential mechanism of nickel-induced carcinogenesis
Ellen, Thomas P; Kluz, Thomas; Harder, Mark E; Xiong, Judy; Costa, Max
2009 Jun 2;48(21):4626-4632, Biochemistry
Epigenetics refers to heritable patterns of gene expression that do not depend on alterations of the genomic DNA sequence. Nickel compounds have demonstrated carcinogenicity without any associated mutagenesis, suggesting that its mechanism of carcinogenesis is epigenetic in nature. One such potential mechanism is the heterochromatinization of chromatin within a region of the genome containing a gene sequence, inhibiting any further molecular interactions with that underlying gene sequence and effectively inactivating that gene. We report here the observation, by atomic force microscopy and circular dichroism spectropolarimetry, that nickel ion (Ni(2+)) condenses chromatin to a greater extent than the natural divalent cation of the cell, magnesium ion (Mg(2+)). In addition, we use a model experimental system that incorporates a transgene, the bacterial xanthine guanine phosphoribosyl transferase gene (gpt), differentially near, and far from, a heterochromatic region of the genome, in two cell lines, the Chinese hamster V79-derived G12 and G10 cells, respectively, to demonstrate by a DNase I protection assay that nickel treatement protects the gpt gene sequence from DNase I exonuclease digestion in the G12 cells, but not in the G10 cells. We conclude that condensation of chromatin by nickel is a potential mechanism of nickel-mediated gene regulation
— id: 101113, year: 2009, vol: 48, page: 4626, stat: Journal Article,

c-Myc mediates a hypoxia-induced decrease in acetylated histone H4
Li, Qin; Costa, Max
2009 Oct;91(10):1307-1310, Biochimie
Global acetylation of histone H4 is a mark of gene transcriptional activation. The c-Myc transcription factor binds to specific DNA sites in cellular chromatin and induces the acetylation of histone H4. In this study, hypoxia (1% Oxygen) induced a decrease in both global acetylated histone H4 (AcH4) and c-Myc in human lung carcinoma A549 cells and in human bronchial epithelial Beas-2B cells, The decline was more striking in A549 cells compared to Beas2-B cells, when cells were exposed to hypoxic stress for 24 h. Further studies showed that these alterations of global AcH4 can be attributed to the decrease in c-Myc protein levels. While hypoxia-induced gene activation is known to be mediated by Hypoxia Response Elements (HRE), the mechanism for down-regulation of genes by hypoxia is not known. The decrease in c-Myc protein levels induced by hypoxia may contribute to hypoxia-induced gene repression
— id: 102399, year: 2009, vol: 91, page: 1307, stat: Journal Article,

Alterations of histone modifications by cobalt compounds
Li, Qin; Ke, Qingdong; Costa, Max
2009 Jul;30(7):1243-1251, Carcinogenesis
In the present study, we examined the effects of CoCl(2) on multiple histone modifications at the global level. We found that in both human lung carcinoma A549 cells and human bronchial epithelial Beas-2B cells, exposure to CoCl(2) (>/=200 muM) for 24 h increased H3K4me3, H3K9me2, H3K9me3, H3K27me3, H3K36me3, uH2A and uH2B but decreased acetylation at histone H4 (AcH4). Further investigation demonstrated that in A549 cells, the increase in H3K4me3 and H3K27me3 by cobalt ions exposure was probably through enhancing histone methylation processes, as methionine-deficient medium blocked the induction of H3K4me3 and H3K27me3 by cobalt ions, whereas cobalt ions increased H3K9me3 and H3K36me3 by directly inhibiting JMJD2A demethylase activity in vitro, which was probably due to the competition of cobalt ions with iron for binding to the active site of JMJD2A. Furthermore, in vitro ubiquitination and deubiquitination assays revealed that the cobalt-induced histone H2A and H2B ubiquitination is the result of inhibition of deubiquitinating enzyme activity. Microarray data showed that exposed to 200 microM of CoCl(2) for 24 h, A549 cells not only increased but also decreased expression of hundreds of genes involved in different cellular functions, including tumorigenesis. This study is the first to demonstrate that cobalt ions altered epigenetic homeostasis in cells. It also sheds light on the possible mechanisms involved in cobalt-induced alteration of histone modifications, which may lead to altered programs of gene expression and carcinogenesis since cobalt at higher concentrations is a known carcinogen
— id: 100598, year: 2009, vol: 30, page: 1243, stat: Journal Article,

Mechanisms of c-myc degradation by nickel compounds and hypoxia
Li, Qin; Kluz, Thomas; Sun, Hong; Costa, Max
2009 ;4(12):e8531-e8531, PLoS ONE
Nickel (Ni) compounds have been found to cause cancer in humans and animal models and to transform cells in culture. At least part of this effect is mediated by stabilization of hypoxia inducible factor (HIF1a) and activating its downstream signaling. Recent studies reported that hypoxia signaling might either antagonize or enhance c-myc activity depending on cell context. We investigated the effect of nickel on c-myc levels, and demonstrated that nickel, hypoxia, and other hypoxia mimetics degraded c-myc protein in a number of cancer cells (A549, MCF-7, MDA-453, and BT-474). The degradation of the c-Myc protein was mediated by the 26S proteosome. Interestingly, knockdown of both HIF-1alpha and HIF-2alpha attenuated c-Myc degradation induced by Nickel and hypoxia, suggesting the functional HIF-1alpha and HIF-2alpha was required for c-myc degradation. Further studies revealed two potential pathways mediated nickel and hypoxia induced c-myc degradation. Phosphorylation of c-myc at T58 was significantly increased in cells exposed to nickel or hypoxia, leading to increased ubiquitination through Fbw7 ubiquitin ligase. In addition, nickel and hypoxia exposure decreased USP28, a c-myc de-ubiquitinating enzyme, contributing to a higher steady state level of c-myc ubiquitination and promoting c-myc degradation. Furthermore, the reduction of USP28 protein by hypoxia signaling is due to both protein degradation and transcriptional repression. Nickel and hypoxia exposure significantly increased the levels of dimethylated H3 lysine 9 at the USP28 promoter and repressed its expression. Our study demonstrated that Nickel and hypoxia exposure increased c-myc T58 phosphorylation and decreased USP28 protein levels in cancer cells, which both lead to enhanced c-myc ubiquitination and proteasomal degradation
— id: 106102, year: 2009, vol: 4, page: e8531, stat: Journal Article,

Nickel compounds induce apoptosis in human bronchial epithelial Beas-2B cells by activation of c-Myc through ERK pathway
Li, Qin; Suen, Ting-Chung; Sun, Hong; Arita, Adriana; Costa, Max
2009 Mar 1;235(2):191-198, Toxicology & applied pharmacology
Nickel compounds are carcinogenic to humans and have been shown to alter epigenetic homeostasis. The c-Myc protein controls 15% of human genes and it has been shown that fluctuations of c-Myc protein alter global epigenetic marks. Therefore, the regulation of c-Myc by nickel ions in immortalized but not tumorigenic human bronchial epithelial Beas-2B cells was examined in this study. It was found that c-Myc protein expression was increased by nickel ions in non-tumorigenic Beas-2B and human keratinocyte HaCaT cells. The results also indicated that nickel ions induced apoptosis in Beas-2B cells. Knockout of c-Myc and its restoration in a rat cell system confirmed the essential role of c-Myc in nickel ion-induced apoptosis. Further studies in Beas-2B cells showed that nickel ion increased the c-Myc mRNA level and c-Myc promoter activity, but did not increase c-Myc mRNA and protein stability. Moreover, nickel ion upregulated c-Myc in Beas-2B cells through the MEK/ERK pathway. Collectively, the results demonstrate that c-Myc induction by nickel ions occurs via an ERK-dependent pathway and plays a crucial role in nickel-induced apoptosis in Beas-2B cells
— id: 96241, year: 2009, vol: 235, page: 191, stat: Journal Article,

Soluble and insoluble nickel compounds exert a differential inhibitory effect on cell growth through IKKalpha-dependent cyclin D1 down-regulation
Ouyang, Weiming; Zhang, Dongyun; Li, Jingxia; Verma, Udit N; Costa, Max; Huang, Chuanshu
2009 Jan;218(1):205-214, Journal of cellular physiology
It is well-known that insoluble nickel compounds possess much more potent carcinogenic activities as compared with soluble nickel compounds. Although it is assumed that the different entry and clearance rate are responsible for the difference, the mechanisms underlying the different carcinogenic activities are still not well understood yet. In the present study, we found that exposure to soluble, but not insoluble nickel compounds, caused a significant inhibition of cell growth and G1/G0 cell cycle arrest, which was concomitant with a marked down-regulation of cylin D1, an essential nuclear protein for controlling G1/S transition, while both soluble and insoluble nickel compounds showed similar effects on NFkappaB activation, HIF-1alpha protein accumulation and TNF-alpha transcription and CAP43 protein expression at same doses range. The down-regulation of cyclin D1 is due to protein degradation rather than inhibition of transcription, because the nickel compounds treatment did not change cyclin D1 mRNA level, while MG132, the proteasome inhibitor, can rescue the degradation of cyclin D1 caused by soluble nickel compound. Moreover, the soluble nickel-induced cyclin D1 degradation is dependent on its Thr286 residue and requires IKKalpha, but not HIF-1alpha, which are both reported to be involved in cyclin D1 down-regulation. Taken together, we demonstrate that soluble, but not insoluble nickel compound, is able to cause cyclin D1 degradation and a cell growth arrest in an IKKalpha-dependent manner. Given the role of cyclin D1 and cell proliferation in carcinogenesis, we anticipate that the different effects of soluble and insoluble nickel compounds on cyclin D1 degradation and cell growth arrest may at least partially account for their different carcinogenic activities
— id: 93369, year: 2009, vol: 218, page: 205, stat: Journal Article,

Modulation of histone methylation and MLH1 gene silencing by hexavalent chromium
Sun, Hong; Zhou, Xue; Chen, Haobin; Li, Qin; Costa, Max
2009 Jun 15;237(3):258-266, Toxicology & applied pharmacology
Hexavalent chromium [Cr(VI)] is a mutagen and carcinogen, and occupational exposure can lead to lung cancers and other adverse health effects. Genetic changes resulting from DNA damage have been proposed as an important mechanism that mediates chromate's carcinogenicity. Here we show that chromate exposure of human lung A549 cells increased global levels of di- and tri-methylated histone H3 lysine 9 (H3K9) and lysine 4 (H3K4) but decreased the levels of tri-methylated histone H3 lysine 27 (H3K27) and di-methylated histone H3 arginine 2 (H3R2). Most interestingly, H3K9 dimethylation was enriched in the human MLH1 gene promoter following chromate exposure and this was correlated with decreased MLH1 mRNA expression. Chromate exposure increased the protein as well as mRNA levels of G9a a histone methyltransferase that specifically methylates H3K9. This Cr(VI)-induced increase in G9a may account for the global elevation of H3K9 dimethylation. Furthermore, supplementation with ascorbate, the primary reductant of Cr(VI) and also an essential cofactor for the histone demethylase activity, partially reversed the H3K9 dimethylation induced by chromate. Thus our studies suggest that Cr(VI) may target histone methyltransferases and demethylases, which in turn affect both global and gene promoter specific histone methylation, leading to the silencing of specific tumor suppressor genes such as MLH1
— id: 99233, year: 2009, vol: 237, page: 258, stat: Journal Article,

Covalent modifications of histones during mitosis and meiosis
Xu, Dazhong; Bai, Jingxiang; Duan, Qing; Costa, Max; Dai, Wei
2009 Nov 15;8(22):3688-3694, Cell cycle
Higher order chromosome structures are the hallmark of mitotic and meiotic cells. Chromatin condensation and compaction are essential for rapid chromosome congression and accurate chromosome segregation during cell division. The core histones possess tails at their amino-termini. These tails, which extend from the surface of the nucleosomes, are highly dynamic and subject to an extensive array of covalent modifications. Modified histone tails play an important role, not only in the folding of nucleosomal arrays into higher order chromatin structures but also in gene regulation. The combination of these distinct covalent modifications of histones constitutes 'the histone code' that regulates various cellular processes, including mitotic and meiotic progression
— id: 105250, year: 2009, vol: 8, page: 3688, stat: Journal Article,

A genome-wide screen in Saccharomyces cerevisiae reveals pathways affected by arsenic toxicity
Zhou, Xue; Arita, Adriana; Ellen, Thomas P; Liu, Xin; Bai, Jingxiang; Rooney, John P; Kurtz, Adrienne D; Klein, Catherine B; Dai, Wei; Begley, Thomas J; Costa, Max
2009 Nov;94(5):294-307, Genomics
We have used Saccharomyces cerevisiae to identify toxicologically important proteins and pathways involved in arsenic-induced toxicity and carcinogenicity in humans. We performed a systemic screen of the complete set of 4733 haploid S. cerevisiae single-gene-deletion mutants to identify those that have decreased or increased growth, relative to wild type, after exposure to sodium arsenite (NaAsO(2)). IC(50) values for all mutants were determined to further validate our results. Ultimately we identified 248 mutants sensitive to arsenite and 5 mutants resistant to arsenite exposure. We analyzed the proteins corresponding to arsenite-sensitive mutants and determined that they belonged to functional categories that include protein binding, phosphate metabolism, vacuolar/lysosomal transport, protein targeting, sorting, and translocation, cell growth/morphogenesis, cell polarity and filament formation. Furthermore, these data were mapped onto a protein interactome to identify arsenite-toxicity-modulating networks. These networks are associated with the cytoskeleton, ubiquitination, histone acetylation and the MAPK signaling pathway. Our studies have potential implications for understanding toxicity and carcinogenesis in arsenic-induced human conditions, such as cancer and aging
— id: 104719, year: 2009, vol: 94, page: 294, stat: Journal Article,

Effects of nickel, chromate, and arsenite on histone 3 lysine methylation
Zhou, Xue; Li, Qin; Arita, Adriana; Sun, Hong; Costa, Max
2009 Apr 1;236(1):78-84, Toxicology & applied pharmacology
Occupational exposure to nickel (Ni), chromium (Cr), and arsenic (As) containing compounds has been associated with lung cancer and other adverse health effects. Their carcinogenic properties may be attributable in part, to activation and/or repression of gene expression induced by changes in the DNA methylation status and histone tail post-translational modifications. Here we show that individual treatment with nickel, chromate, and arsenite all affect the gene activating mark H3K4 methylation. We found that nickel (1 mM), chromate (10 microM), and arsenite (1 microM) significantly increase tri-methyl H3K4 after 24 h exposure in human lung carcinoma A549 cells. Seven days of exposure to lower levels of nickel (50 and 100 microM), chromate (0.5 and 1 microM) or arsenite (0.1, 0.5 and 1 microM) also increased tri-methylated H3K4 in A549 cells. This mark still remained elevated and inherited through cell division 7 days following removal of 1 microM arsenite. We also demonstrate by dual staining immunofluorescence microscopy that both H3K4 tri-methyl and H3K9 di-methyl marks increase globally after 24 h exposure to each metal treatment in A549 cells. However, the tri-methyl H3K4 and di-methyl H3K9 marks localize in different regions in the nucleus of the cell. Thus, our study provides further evidence that a mechanism(s) of carcinogenicity of nickel, chromate, and arsenite metal compounds may involve alterations of various histone tail modifications that may in turn affect the expression of genes that may cause transformation
— id: 97963, year: 2009, vol: 236, page: 78, stat: Journal Article,

Phosphorylation of H3S10 blocks the access of H3K9 by specific antibodies and histone methyltransferase. Implication in regulating chromatin dynamics and epigenetic inheritance during mitosis
Duan, Qing; Chen, Haobin; Costa, Max; Dai, Wei
2008 Nov 28;283(48):33585-33590, Journal of biological chemistry
Post-translational modifications of histones play a critical role in regulating genome structures and integrity. We have focused on the regulatory relationship between covalent modifications of histone H3 lysine 9 (H3K9) and H3S10 during the cell cycle. Immunofluorescence microscopy revealed that H3S10 phosphorylation in HeLa, A549, and HCT116 cells was high during prophase, prometaphase, and metaphase, whereas H3K9 monomethylation (H3K9me1) and dimethylation (H3K9me2), but not H3K9 trimethylation (H3K9me3), were significantly suppressed. When H3S10 phosphorylation started to diminish during anaphase, H3K9me1 and H3K9me2 signals reemerged. Western blot analyses confirmed that mitotic histones, extracted in an SDS-containing buffer, had little H3K9me1 and H3K9me2 signals but abundant H3K9me3 signals. However, when mitotic histones were extracted in the same buffer without SDS, the difference in H3K9me1 and H3K9me2 signals between interphase and mitotic cells disappeared. Removal of H3S10 phosphorylation by pretreatment with lambda-phosphatase unmasked mitotic H3K9me1 and H3K9me2 signals detected by both fluorescence microscopy and Western blotting. Further, H3S10 phosphorylation completely blocked methylation of H3K9 but not demethylation of the same residue in vitro. Given that several conserved motifs consisting of a Lys residue immediately followed by a Ser residue are present in histone tails, our studies reveal a potential new mechanism by which phosphorylation not only regulates selective access of methylated lysines by cellular factors but also serves to preserve methylation patterns and epigenetic programs during cell division
— id: 91974, year: 2008, vol: 283, page: 33585, stat: Journal Article,

NDRG1, a growth and cancer related gene: regulation of gene expression and function in normal and disease states
Ellen, Thomas P; Ke, Qingdong; Zhang, Ping; Costa, Max
2008 Jan;29(1):2-8, Carcinogenesis
N-myc downstream-regulated gene 1 (NDRG1) is an intracellular protein that is induced under a wide variety of stress and cell growth-regulatory conditions. NDRG1 is up-regulated by cell differentiation signals in various cancer cell lines and suppresses tumor metastasis. Despite its specific role in the molecular cause of Charcot-Marie-Tooth type 4D disease, there has been more interest in the gene as a marker of tumor progression and enhancer of cellular differentiation. Because it is strongly up-regulated under hypoxic conditions, and this condition is prevalent in solid tumors, its regulation is somewhat complex, governed by hypoxia-inducible factor 1 alpha (HIF-1alpha)- and p53-dependent pathways, as well as its namesake, neuroblastoma-derived myelocytomatosis, and probably many other factors, at the transcriptional and translational levels, and through mRNA stability. We survey the data for clues to the NDRG1 gene's mechanism and for indications that the NDRG1 gene may be an efficient diagnostic tool and therapy in many types of cancers
— id: 76760, year: 2008, vol: 29, page: 2, stat: Journal Article,

Nickel compounds induce histone ubiquitination by inhibiting histone deubiquitinating enzyme activity
Ke, Qingdong; Ellen, Thomas P; Costa, Max
2008 Apr 15;228(2):190-199, Toxicology & applied pharmacology
Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of cell lines. Investigations of the mechanism by which nickel increases histone ubiquitination in cells reveal that nickel does not affect cellular levels of the substrates of this modification, i.e., ubiquitin, histones, and other non-histone ubiquitinated proteins. In vitro ubiquitination and deubiquitination assays have been developed to further investigate possible effects of nickel on enzymes responsible for histone ubiquitination. Results from the in vitro assays demonstrate that the presence of nickel did not affect the levels of ubiquitinated histones in the ubiquitinating assay. Instead, the addition of nickel significantly prevents loss of uH2A and uH2B in the deubiquitinating assay, suggesting that nickel-induced histone ubiquitination is the result of inhibition of (a) putative deubiquitinating enzyme(s). Additional supporting evidence comes from the comparison of the response to nickel ions with a known deubiquitinating enzyme inhibitor, iodoacetamide (IAA). This study is the first to demonstrate such effects of nickel ions on histone ubiquitination. It also sheds light on the possible mechanisms involved in altering the steady state of this modification. The study provides further evidence that supports the notion that nickel ions alter epigenetic homeostasis in cells, which may lead to altered programs of gene expression and carcinogenesis
— id: 79243, year: 2008, vol: 228, page: 190, stat: Journal Article,

Nickel compounds induce phosphorylation of histone H3 at serine 10 by activating JNK-MAPK pathway
Ke, Qingdong; Li, Qin; Ellen, Thomas P; Sun, Hong; Costa, Max
2008 Jun;29(6):1276-1281, Carcinogenesis
Nickel (Ni) is a known carcinogen, although the mechanism of its carcinogenicity is not clear. Here, we provide evidence that Ni can induce phosphorylation of histone H3 at its serine 10 residue in a c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)-dependent manner. Ni induces the phosphorylation of JNK, with no effect on the phosphorylation states of the extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinases. An inhibitor of JNK eliminated the Ni-initiated JNK-mediated induction of histone H3 phosphorylation at serine 10, whereas inhibitors specific for ERK or p38 kinases had no effect on the phosphorylation levels of histone H3 at serine 10 (P-H3S10) in Ni-treated cells. A complete loss of Ni ion-induced phosphorylation of H3S10 was observed when JNK was specifically knocked down with RNAi. These results are the first to show the specific JNK-mediated phosphorylation of histone H3 at its serine 10 residue. We show that addition of Ni to an in vitro P-H3S10 dephosphorylation reaction does not change the loss of phosphorylation in the reaction, supporting the notion that Ni causes H3S10 phosphorylation via the JNK/SAPK pathway. It is likely that modification of H3S10 is one of a growing number of epigenetic changes believed to be involved in the carcinogenesis caused by Ni
— id: 80302, year: 2008, vol: 29, page: 1276, stat: Journal Article,

CrVI exposure and biomarkers: Cr in erythrocytes in relation to exposure and polymorphisms of genes encoding anion transport proteins
Qu, Qingshan; Li, Xiaomei; An, Feiyun; Jia, Guang; Liu, Lanzeng; Watanabe-Meserve, Hiroko; Koenig, Karen; Cohen, Beverly; Costa, Max; Roy, Nirmal; Zhong, Mianhua; Chen, Lung Chi; Liu, Suhua; Yan, Lei
2008 Aug;13(5):467-477, Biomarkers
A total of 195 subjects, including 141 exposed workers and 54 farmers, were recruited in China to evaluate the usefulness of chromium (Cr) in erythrocytes as a biomarker of exposure to CrVI. The levels of Cr in red blood cells (RBC) were remarkably elevated even in a group of workers routinely exposed to CrVI as low as 5-15 microg m(-3) and showed a significant exposure-response trend over the exposure range from 0.002 to 1152 microg m(-3) (p <0.0001). Multiple linear regression analyses indicated that age and cigarette smoke were not associated with Cr in RBC. However, female subjects had lower Cr in RBC compared with their male counterparts for about the same exposure levels (p <0.05). The genotypes of band III, which encodes for anion transport protein and may regulate CrO4(-2) across cell membranes, were also identified and included for analysis. The ratios of Cr in RBC to CrVI exposure were higher in subjects with a wild genotype than in those who had heterozygous or homozygous variant alleles. However, the difference was not statistically significant probably due to the limited number of participating subjects. In addition, 15 of the 141 workers were selected for multiple exposure monitoring and blood sample collections to evaluate the inter- and intraindividual variations of Cr in RBC. Compared with the personal exposure levels, Cr in RBC had small intraindividual variations with a reliability coefficient of 0.88. The study suggests that Cr in RBC may serve as a sensitive and reliable biomarker for long-term exposure to CrVI
— id: 93384, year: 2008, vol: 13, page: 467, stat: Journal Article,

Polo-like kinase 3 functions as a tumor suppressor and is a negative regulator of hypoxia-inducible factor-1 alpha under hypoxic conditions
Yang, Yali; Bai, Jingxiang; Shen, Rulong; Brown, Sharron A N; Komissarova, Elena; Huang, Ying; Jiang, Ning; Alberts, Gregory F; Costa, Max; Lu, Luo; Winkles, Jeffrey A; Dai, Wei
2008 Jun 1;68(11):4077-4085, Cancer research
Polo-like kinase 3 (Plk3) is an important mediator of the cellular responses to genotoxic stresses. In this study, we examined the physiologic function of Plk3 by generating Plk3-deficient mice. Plk3(-/-) mice displayed an increase in weight and developed tumors in various organs at advanced age. Many tumors in Plk3(-/-) mice were large in size, exhibiting enhanced angiogenesis. Plk3(-/-) mouse embryonic fibroblasts were hypersensitive to the induction of hypoxia-inducible factor-1 alpha (HIF-1 alpha) under hypoxic conditions or by nickel and cobalt ion treatments. Ectopic expression of the Plk3-kinase domain (Plk3-KD), but not its Polo-box domain or a Plk3-KD mutant, suppressed the nuclear accumulation of HIF-1 alpha induced by nickel or cobalt ions. Moreover, hypoxia-induced HIF-1 alpha expression was tightly associated with a significant down-regulation of Plk3 expression in HeLa cells. Given the importance of HIF-1 alpha in mediating the activation of the 'survival machinery' in cancer cells, these studies strongly suggest that enhanced tumorigenesis in Plk3-null mice is at least partially mediated by a deregulated HIF-1 pathway
— id: 81058, year: 2008, vol: 68, page: 4077, stat: Journal Article,

Arsenite alters global histone H3 methylation
Zhou, Xue; Sun, Hong; Ellen, Thomas P; Chen, Haobin; Costa, Max
2008 Sep;29(9):1831-1836, Carcinogenesis
Arsenic (As) is a well-characterized human carcinogen but is generally not mutagenic. The evidence that As induces both loss of global DNA methylation and gene promoter DNA hypermethylation has suggested that epigenetic mechanisms may play an important role in As-induced carcinogenesis. In the present study, we examined the change in histone methylation by As exposure. In human lung carcinoma A549 cells, exposure to inorganic trivalent As (arsenite) increased H3K9 dimethylation (H3K9me2) and decreased H3K27 trimethylation (H3K27me3), both of which represent gene silencing marks, while increasing the global levels of the H3K4 trimethylation (H3K4me3), a gene-activating mark. The increase in H3K9me2 was mediated by an increase in the histone methyltransferase G9a protein and messenger RNA levels. We also observed strikingly significant altered histone modifications induced by very low-dose (0.1 microM) arsenite. Taken together, these results suggest a potential mechanism by which As induces carcinogenesis through the alteration of specific histone methylations that represent both gene silencing and activating marks. Furthermore, these marks are known to affect DNA methylation, and it is likely that arsenic's effect is not limited to histone modifications alone, but extends, perhaps by them, to DNA methylations as well. Future studies in our laboratory will address the genomic location of these silencing and activating marks using ChIP-on-chip technology
— id: 93297, year: 2008, vol: 29, page: 1831, stat: Journal Article,

Nickel compounds
Cohen M; Klein C; Costa M
Environmental and occupational medicine Philadelphia : Wolters Kluwer/Lippincott Williams & Wilkins, 2007,
— id: 4440, year: 2007, vol: , page: ?, stat: Chapter,

Chromium compounds
Cohen MD; Costa M
Environmental and occupational medicine Philadelphia : Wolters Kluwer/Lippincott Williams & Wilkins, 2007,
— id: 4439, year: 2007, vol: , page: ?, stat: Chapter,

Molecular mechanisms of metal toxicity and carcinogenicity
Davidson T; Ke Q; Costa M
Handbook on the toxicology of metals Burlington MA : Academic Press, 2007,
— id: 4438, year: 2007, vol: , page: 79, stat: Chapter,

Overview of chromium (III) toxicology
Ke Q; Costa M
Nutritional biochemisty of chromium (III) Amsterdam : Elsevier, 2007,
— id: 4441, year: 2007, vol: , page: 257, stat: Chapter,

Carcinogenicity of metal compounds
Ke Q; Costa M; Kazantzis G
Handbook on the toxicology of metals Burlington MA : Academic Press, 2007,
— id: 4436, year: 2007, vol: , page: 177, stat: Chapter,

Fluorescent tracking of nickel ions in human cultured cells
Ke, Qingdong; Davidson, Todd; Kluz, Thomas; Oller, Adriana; Costa, Max
2007 Feb 15;219(1):18-23, Toxicology & applied pharmacology
The carcinogenic activity of various nickel (Ni) compounds is likely dependent upon their ability to enter cells and elevate intracellular levels of Ni ions. Water-insoluble Ni compounds such as NiS and Ni(3)S(2) were shown in vitro to enter cells by phagocytosis and potently induce tumors in experimental animals at the site of exposure. These water-insoluble nickel compounds are generally considered to be more potent carcinogens than the water-soluble forms. However, recent in vitro studies have shown similar effects for insoluble and soluble Ni compounds. Using a dye that fluoresces when intracellular Ni ion binds to it, we showed that both soluble and insoluble Ni compounds were able to elevate the levels of Ni ions in the cytoplasmic and nuclear compartments. However, when the source of Ni ions was removed from the culture dish, the intracellular Ni ions derived from soluble Ni compound were lost from the cells at a significantly faster rate than those derived from the insoluble Ni compound. Within 10 h after NiCl(2) removal from the culture medium, Ni ions disappeared from the nucleus and were not detected in the cells by 16 h, while insoluble Ni(3)S(2) yielded Ni ions that persisted in the nucleus after 16 h and were detected in the cytoplasm even after 24 h following Ni removal. These effects are discussed in terms of whole body exposure to water-soluble and -insoluble Ni compounds and consistency with animal carcinogenicity studies
— id: 71340, year: 2007, vol: 219, page: 18, stat: Journal Article,

Nickel
Klein CB; Costa M
Handbook on the toxicology of metals Burlington MA : Academic Press, 2007,
— id: 4437, year: 2007, vol: , page: 743, stat: Chapter,

Correlation of N-myc downstream-regulated gene 1 expression with clinical outcomes of colorectal cancer patients of different race/ethnicity
Koshiji, Minori; Kumamoto, Kensuke; Morimura, Keiichirou; Utsumi, Yasufumi; Aizawa, Michiko; Hoshino, Masami; Ohki, Shinji; Takenoshita, Seiichi; Costa, Max; Commes, Therese; Piquemal, David; Harris, Curtis-C; Tchou-Wong, Kam-Meng
2007 May 28;13(20):2803-2810, World journal of gastroenterology : WJG
AIM: To evaluate the role of N-myc downstream-regulated gene 1 (NDRG1) expression in prognosis and survival of colorectal cancer patients with different ethnic backgrounds. METHODS: Because NDRG1 is a downstream target of p53 and hypoxia inducible factor-1 alpha (HIF-1 alpha), we examined NDRG1 expression together with p53 and HIF-1 alpha by immunohistochemistry. A total of 157 colorectal cancer specimens including 80 from Japanese patients and 77 from US patients were examined. The correlation between protein expression with clinicopathological features and survival after surgery was analyzed. RESULTS: NDRG1 protein was significantly increased in colorectal tumor compared with normal epithelium in both Japanese and US patient groups. Expression of NDRG1 protein was significantly correlated with lymphatic invasion, venous invasion, depth of invasion, histopathological type, and Dukes' stage in Japanese colorectal cancer patients. NDRG1 expression was correlated to histopathological type, Dukes' stage and HIF-1 alpha expression in US-Caucasian patients but not in US-African American patients. Interestingly, Kaplan-Meier survival analysis demonstrated that NDRG1 expression correlated significantly with poorer survival in US-African American patients but not in other patient groups. However, in p53-positive US cases, NDRG1 positivity correlated significantly with better survival. In addition, NDRG1 expression also correlated significantly with improved survival in US patients with stages III and IV tumors without chemotherapy. In Japanese patients with stages II and III tumors, strong NDRG1 staining in p53-positive tumors correlated significantly with improved survival but negatively in patients without chemotherapy. CONCLUSION: NDRG1 expression was correlated with various clinicopathological features and clinical outcomes in colorectal cancer depending on the race/ethnicity of the patients. NDRG1 may serve as a biological basis for the disparity of clinical outcomes of colorectal cancer patients with different ethnic backgrounds.
— id: 73067, year: 2007, vol: 13, page: 2803, stat: Journal Article,

Chromium
Langard S; Costa M
Handbook on the toxicology of metals Burlington MA : Academic Press, 2007,
— id: 4435, year: 2007, vol: , page: 487, stat: Chapter,

Dietary chromium and nickel enhance UV-carcinogenesis in skin of hairless mice
Uddin, Ahmed N; Burns, Fredric J; Rossman, Toby G; Chen, Haobin; Kluz, Thomas; Costa, Max
2007 Jun 15;221(3):329-338, Toxicology & applied pharmacology
The skin cancer enhancing effect of chromium (in male mice) and nickel in UVR-irradiated female Skh1 mice was investigated. The dietary vitamin E and selenomethionine were tested for prevention of chromium-enhanced skin carcinogenesis. The mice were exposed to UVR (1.0 kJ/m(2) 3x weekly) for 26 weeks either alone, or combined with 2.5 or 5.0 ppm potassium chromate, or with 20, 100 or 500 ppm nickel chloride in drinking water. Vitamin E or selenomethionine was added to the lab chow for 29 weeks beginning 3 weeks before the start of UVR exposure. Both chromium and nickel significantly increased the UVR-induced skin cancer yield in mice. In male Skh1 mice, UVR alone induced 1.9+/-0.4 cancers/mouse, and 2.5 or 5.0 ppm potassium chromate added to drinking water increased the yields to 5.9+/-0.8 and 8.6+/-0.9 cancers/mouse, respectively. In female Skh1 mice, UVR alone induced 1.7+/-0.4 cancers/mouse, and the addition of 20, 100 or 500 ppm nickel chloride increased the yields to 2.8+/-0.9, 5.6+/-0.7 and 4.2+/-1.0 cancers/mouse, respectively. Neither vitamin E nor selenomethionine reduced the cancer yield enhancement by chromium. These results confirm that chromium and nickel, while not good skin carcinogens per se, are enhancers of UVR-induced skin cancers in Skh1 mice. Data also suggest that the enhancement of UVR-induced skin cancers by chromate may not be oxidatively mediated since the antioxidant vitamin E as well as selenomethionine, found to prevent arsenite-enhanced skin carcinogenesis, failed to suppress enhancement by chromate
— id: 72149, year: 2007, vol: 221, page: 329, stat: Journal Article,

JNK1, but not JNK2, is required for COX-2 induction by nickel compounds
Zhang, Dongyun; Li, Jingxia; Wu, Kangjian; Ouyang, Weiming; Ding, Jin; Liu, Zheng-gang; Costa, Max; Huang, Chuanshu
2007 Apr;28(4):883-891, Carcinogenesis
Activation of the signaling pathways leading to gene expression regulation, is critical in the carcinogenic effects of nickel exposure. In the present study, we found nickel exposure could induce cyclooxygenase-2 (COX-2) expression at transcriptional and protein levels in both human bronchoepithelial cells (Beas-2B) and murine embryonic fibroblasts (MEFs). We further provided direct evidence for the specific involvement of the JNK1 signaling pathway in the COX-2 induction using specific gene knockout approaches. Our results demonstrated that COX2 induction by nickel was impaired in JNK1(-/-) MEFs, but not in JNK2(-/-) MEFs. Moreover, re-constitutional expression of JNK1 restored COX-2 induction, confirming the specific requirement of JNK1 in COX-2 induction. Further investigation revealed that JNK1 mediated the nickel-induced COX-2 expression in a c-Jun/AP-1-dependent manner. Ectopic expression of TAM67, a c-Jun dominant negative mutant, also suppressed the COX-2 induction. Our results demonstrate that the JNK1/c-Jun/AP-1 pathway, but not the JNK2 pathway, plays a critical role in nickel-induced COX-2 expression
— id: 68994, year: 2007, vol: 28, page: 883, stat: Journal Article,

Egr-1 mediates hypoxia-inducible transcription of the NDRG1 gene through an overlapping Egr-1/Sp1 binding site in the promoter
Zhang, Ping; Tchou-Wong, Kam-Meng; Costa, Max
2007 Oct 1;67(19):9125-9133, Cancer research
N-myc down-regulated gene 1 (NDRG1/Cap43) is inducible by a variety of environmental stressors, including hypoxia. The present study identified a cis-acting element mediating the transactivation of the NDRG1 gene in murine RAW264.7 macrophage cells treated with hypoxia or deferoxamine, an iron chelator mimicking hypoxia. Through a series of deletions of the promoter of NDRG1 luciferase constructs, a minimal cis-acting element conferring inducibility by hypoxia and deferoxamine was localized to an early growth response 1 (Egr-1) and Sp1 overlapping binding site. Electrophoretic mobility shift assay, antibody supershift assay, and mutations of the Egr-1 binding site confirmed the specific binding of Egr-1 protein to this Egr-1/Sp1 motif. In addition, hypoxia increased the level of Egr-1 protein that correlated with induction of NDRG1 expression at both RNA and protein levels. Transient transfection of the Egr-1 gene into HeLa cells also resulted in up-regulation of the NDRG1 mRNA. The role of Egr-1 was further verified by mutations in the Egr-1 binding site, which reduced promoter inducibility by hypoxia and deferoxamine. Furthermore, the induction of NDRG1 expression by hypoxia and deferoxamine was diminished by RNA interference knockdown of Egr-1 gene expression and in Egr-1-/- mouse embryonic fibroblasts (MEF) compared with Egr-1+/- MEFs. These results showed for the first time that Egr-1 regulates NDRG1 transcription through an overlapping Egr-1/Sp1 binding site that acts as a major site of positive regulation of the NDRG1 promoter by hypoxia signaling
— id: 74580, year: 2007, vol: 67, page: 9125, stat: Journal Article,

Sequence specificity of Cr(III)-DNA adduct formation in the p53 gene: NGG sequences are preferential adduct-forming sites
Arakawa, Hirohumi; Wu, Feng; Costa, Max; Rom, William; Tang, Moon-Shong
2006 Mar;27(3):639-645, Carcinogenesis
Hexavalent chromium [Cr(VI)] is a known etiological factor in human lung cancer. Cr(VI) exposure-related lung cancer has a high mutation incidence in the p53 gene. Upon intake in human cells Cr(VI) is reduced to Cr(III), which is able to conjugate with amino acids and consequently form either binary Cr(III)-DNA or ternary Cr(III)-amino acid-DNA adducts. Both binary and ternary Cr(III)-DNA adducts are mutagenic. We have found that the Escherichia coli nucleotide excision enzyme UvrABC nuclease is able to incise Cr(III)- and Cr(III)-histidine-modified plasmid DNA and the extent of incision is proportional to the amount of Cr(III)-DNA adducts in the plasmid. In order to determine the role of Cr(III)-DNA adducts in the mutagenesis of the p53 gene in human cancer using the UvrABC nuclease incision method, we have mapped the Cr(III)-DNA distribution in PCR DNA fragments amplified from exons 5, 7 and 8 of the p53 gene. We have found that the sequence specificities of Cr(III)-DNA and Cr(III)-histidine-DNA adducts in the p53 gene sequence are identical and that both types of adducts are preferentially formed at -NGG- sequences, including codons 245, 248 and 249, the mutational hotspots in human lung cancer. It has been found that Cr(III)-DNA adducts induce mainly G to T mutations. Therefore, these results suggest that Cr(III)-DNA adduct formation contributes to the p53 gene mutations in lung carcinogenesis
— id: 63597, year: 2006, vol: 27, page: 639, stat: Journal Article,

Molecular mechanisms of nickel toxicity and carcinogenicity
Chen H; Davidson TL; Li Q; Ke Q; Costa M
2006 ;9:391-395, Metal ions in biology & medicine = Les ions metalliques en biologie et en medicine
— id: 72796, year: 2006, vol: 9, page: 391, stat: Journal Article,

Effect of soluble nickel on cellular energy metabolism in A549 cells
Chen, Haobin; Costa, Max
2006 Oct;231(9):1474-1480, Experimental biology & medicine
Iron is an essential nutrient to most organisms, and is actively involved in oxygen delivery, electron transport, DNA synthesis, and many other biochemical reactions important for cell survival. We previously reported that nickel (Ni) ion exposure decreases cellular iron level and converts cytosolic aconitase (c-aconitase) to iron-regulatory protein-1 in A549 cells (Chen H, Davidson T, Singleton S, Garrick MD, Costa M. Toxicol Appl Pharmacol 206:275-287, 2005). Here, we further investigated the effect of Ni ion exposure on the activity of mitochondrial iron-sulfur (Fe-S) enzymes and cellular energy metabolism. We found that acute Ni ion treatment up to 1 mM exhibits minimal toxicity in A549 cells. Ni ion treatment decreases the activity of several Fe-S enzymes related to cellular energy metabolism, including mitochondrial aconitase (m-aconitase), succinate dehydrogenase (SDH), and NADH:ubiquinone oxidoreductase (complex I). Low doses of Ni ion for 4 weeks resulted in an increased cellular glycolysis and NADH to NAD+ (NADH/NAD+) ratio, although glycolysis was inhibited at higher levels. Collectively, our results show that Ni ions decrease the activity of cellular iron (Fe)-containing enzymes, inhibit oxidative phosphorylation (OxPhos), and increase cellular glycolytic activity. Since increased glycolysis is one of the fundamental alterations of energy metabolism in cancer cells (the Warburg effect), the inhibition of Fe-S enzymes and subsequent changes in cellular energy metabolism caused by Ni ions may play an important role in Ni carcinogenesis
— id: 68893, year: 2006, vol: 231, page: 1474, stat: Journal Article,

Nickel ions increase histone H3 lysine 9 dimethylation and induce transgene silencing
Chen, Haobin; Ke, Qingdong; Kluz, Thomas; Yan, Yan; Costa, Max
2006 May;26(10):3728-3737, Molecular & cellular biology
We have previously reported that carcinogenic nickel compounds decreased global histone H4 acetylation and silenced the gpt transgene in G12 Chinese hamster cells. However, the nature of this silencing is still not clear. Here, we report that nickel ion exposure increases global H3K9 mono- and dimethylation, both of which are critical marks for DNA methylation and long-term gene silencing. In contrast to the up-regulation of global H3K9 dimethylation, nickel ions decreased the expression and activity of histone H3K9 specific methyltransferase G9a. Further investigation demonstrated that nickel ions interfered with the removal of histone methylation in vivo and directly decreased the activity of a Fe(II)-2-oxoglutarate-dependent histone H3K9 demethylase in nuclear extract in vitro. These results are the first to show a histone H3K9 demethylase activity dependent on both iron and 2-oxoglutarate. Exposure to nickel ions also increased H3K9 dimethylation at the gpt locus in G12 cells and repressed the expression of the gpt transgene. An extended nickel ion exposure led to increased frequency of the gpt transgene silencing, which was readily reversed by treatment with DNA-demethylating agent 5-aza-2'-deoxycytidine. Collectively, our data strongly indicate that nickel ions induce transgene silencing by increasing histone H3K9 dimethylation, and this effect is mediated by the inhibition of H3K9 demethylation
— id: 64479, year: 2006, vol: 26, page: 3728, stat: Journal Article,

Hypoxic stress induces dimethylated histone H3 lysine 9 through histone methyltransferase G9a in mammalian cells
Chen, Haobin; Yan, Yan; Davidson, Todd L; Shinkai, Yoichi; Costa, Max
2006 Sep 15;66(18):9009-9016, Cancer research
Dimethylated histone H3 lysine 9 (H3K9me2) is a critical epigenetic mark for gene repression and silencing and plays an essential role in embryogenesis and carcinogenesis. Here, we investigated the effects of hypoxic stress on H3K9me2 at both global and gene-specific level. We found that hypoxia increased global H3K9me2 in several mammalian cell lines. This hypoxia-induced H3K9me2 was temporally correlated with an increase in histone methyltransferase G9a protein and enzyme activity. The increase in H3K9me2 was significantly mitigated in G9a-/- mouse embryonic stem cells following hypoxia challenge, indicating that G9a was involved in the hypoxia-induced H3K9me2. In addition to the activation of G9a, our results also indicated that hypoxia increased H3K9me2 by inhibiting H3K9 demethylation processes. Hypoxic mimetics, such as deferoxamine and dimethyloxalylglycine, were also found to increase H3K9me2 as well as G9a protein and activity. Finally, hypoxia increased H3K9me2 in the promoter regions of the Mlh1 and Dhfr genes, and these increases temporally correlated with the repression of these genes. Collectively, these results indicate that G9a plays an important role in the hypoxia-induced H3K9me2, which would inhibit the expression of several genes that would likely lead to solid tumor progression
— id: 68894, year: 2006, vol: 66, page: 9009, stat: Journal Article,

Response to comments by post and stern on article "Toxicity and carcinogenicity of chromium compounds in humans"
Costa, M; Klein, C
2006 OCT ;36(9):779-779, Critical reviews in toxicology
— id: 68959, year: 2006, vol: 36, page: 779, stat: Journal Article,

Toxicity and carcinogenicity of chromium compounds in humans
Costa, Max; Klein, Catherine B
2006 Feb;36(2):155-163, Critical reviews in toxicology
Chromium is a human carcinogen primarily by inhalation exposure in occupational settings. Although lung cancer has been established as a consequence of hexavalent chromium exposure in smokers and nonsmokers, some cancers of other tissues of the gastrointestinal and central nervous systems have also been noted. Except for a few reports from China, little is known about the health risks of environmental exposures to chromium. Likewise, there has been a lack of epidemiological studies of human exposure to hexavalent Cr by drinking water or ingestion, and it has been suggested that humans can perhaps tolerate hexavalent Cr at higher levels than the current drinking water standard of 50 ppb. This review highlights the most recent data on the induction of skin tumors in mice by chronic drinking-water exposure to hexavalent chromium in combination with solar ultraviolet light. This experimental system represents an important new animal model for chromate-induced cancers by ingestion of drinking water, and it suggests by extrapolation that chromate can likely be considered a human carcinogen by ingestion as well. The potential use of this animal model for future risk assessment is discussed
— id: 64671, year: 2006, vol: 36, page: 155, stat: Journal Article,

Soluble nickel inhibits HIF-prolyl-hydroxylases creating persistent hypoxic signaling in A549 cells
Davidson, Todd L; Chen, Haobin; Di Toro, Dominic M; D'Angelo, Gisela; Costa, Max
2006 Jul;45(7):479-489, Molecular carcinogenesis
Soluble nickel compounds are carcinogenic to humans although the mechanism by which they cause cancer remains unclear. One major consequence of exposure to nickel is the stabilization of hypoxia inducible factor-1alpha (HIF-1alpha), a protein known to be overexpressed in a variety of cancers. In this study, we report a persistent stabilization of HIF-1alpha by nickel chloride up to 72 h after the removal of nickel from the culture media. In addition, we show that the HIF-prolyl hydroxylases (PHD's) are inhibited when cells are exposed to nickel and that they remain repressed for up to 72 h after nickel is removed. We then show that nickel can inhibit purified HIF-PHD's 2 in vitro, through direct interference with the enzyme. Through theoretical calculations, we also demonstrate that nickel may be able to replace the iron in the active site of this enzyme, providing a plausible mechanism for the persistent inhibition of HIF-PHD's by nickel. The data presented suggest that nickel can interfere with HIF-PHD directly and does not inhibit the enzyme by simply depleting cellular factors, such as iron or ascorbic acid. Understanding the mechanisms by which nickel can inhibit HIF-PHD's and stabilize HIF-1alpha may be important in the treatment of cancer and ischemic diseases
— id: 67001, year: 2006, vol: 45, page: 479, stat: Journal Article,

Nickel compounds render anti-apoptotic effect to human bronchial epithelial Beas-2B cells by induction of COX-2 through IKKbeta /p65-dependent, and IKKalpha - and p50-independent pathway
Ding, Jin; Zhang, Xinhai; Li, Jingxia; Song, Lun; Ouyang, Weiming; Zhang, Dongyun; Xue, Caifang; Costa, Max; Melendez, J Andres; Huang, Chuanshu
2006 Dec 22;281(51):39022-39032, Journal of biological chemistry
The carcinogenicity of nickel compounds has been well documented both in vivo and in vivo, however the molecular mechanisms by which nickel compounds cause cancers are far from understood yet. Since suppression of apoptosis is thought to contribute to carcinogenesis, current studies investigated the mechanisms implicated in nickel-induced anti-apoptotic effect in human bronchial epithelial (Beas-2B) cells. We found that exposure of Beas-2B cells to nickel compounds resulted in an increased COX-2 expression, and knockdown of COX-2 expression with its small interfering RNA (siCOX-2) resulted in the cell more sensitivity to nickel-triggered cell apoptosis, demonstrating that COX-2 induction renders the anti-apoptotic effect to Beas-2B cells. Overexpression of IKKb-KM, a kinase inactive mutant of IKKb, blocked NF-kB activation and COX-2 induction by nickel compounds, indicating that activated NF-kB may be a mediator for COX-2 induction. To further explore the implication of the NF-kB pathway in COX-2 induction and anti-apoptotic effect upon nickel exposure, mouse embryonic fibroblasts (MEFs) deficient with IKKb, IKKa, p65 and p50, were employed. Abortion of IKKb in IKKb-/- cells impaired COX-2 induction by nickel exposure, whereas knockout of IKKa only showed a marginal effect. Moreover, p65, and not p50 of NF-kB subunit, was critical for nickel-induced COX-2 expression. In addition, a deficiency of IKKb or p65 renders cells more sensitive to nickel-induced apoptosis as compared to those in wild type cells. Finally, it was shown that reactive oxygen species (ROS) were involved in both NFkB activation and COX-2 expression. Collectively, our results demonstrate that COX-2 induction by nickel compounds via IKKb/p65 NF-kB-dependent, and IKKa- and p50-independent pathway, plays a crucial role in antagonizing nickel-induced cell apoptosis in Beas-2B cells
— id: 68995, year: 2006, vol: 281, page: 39022, stat: Journal Article,

DMT1: which metals does it transport?
Garrick, Michael D; Singleton, Steven T; Vargas, Farida; Kuo, H-C; Zhao, Lin; Knopfel, Martin; Davidson, Todd; Costa, Max; Paradkar, Prasad; Roth, Jerome A; Garrick, Laura M
2006 ;39(1):79-85, Biological research
DMT1-Divalent Metal (Ion) Transporter 1 or SLC11A2/DCT1/Nramp2 - transports Fe2+ into the duodenum and out of the endosome during the transferrin cycle. DMTI also is important in non-transferrin bound iron uptake. It plays similar roles in Mn2+ trafficking. Voltage clamping showed that six other metals evoked currents, but it is unclear if these metals are substrates for DMT1. This report summarizes progress on which metals DMT1 transports, focusing on results from the authors' labs. We recently cloned 1A/+IRE and 2/-IRE DMT1 isoforms to generate HEK293 cell lines that express them in a tetracycline-inducible fashion, then compared induced expression to uninduced expression and to endogenous DMT1 expression. Induced expression increases approximately 50x over endogenous expression and approximately 10x over uninduced levels. Fe2+, Mn2+, Ni2+ and Cu1+ or Cu2+ are transported. We also explored competition between metal ions using this system because incorporation essentially represents DMT1 transport and find this order for transport affinity: Mn>?Cd>?Fe>Pb-Co-Ni>Zn. The effects of decreased DMT1 also could be examined. The Belgrade rat has diminished DMT1 function and thus provides ways of testing. A series of DNA constructs that generate siRNAs specific for DMT1 or certain DMT1 isoforms yield another way to test DMT1-based transport
— id: 68997, year: 2006, vol: 39, page: 79, stat: Journal Article,

Effect of metal ions on HIF-1alpha and Fe homeostasis in human A549 cells
Kang, Gi Soo; Li, Qin; Chen, Haobin; Costa, Max
2006 Nov 7;610(1-2):48-55, Mutation research
Several metals are carcinogenic but little is known about the mechanisms by which they cause cancer. A pathway that may contribute to metal ion induced carcinogenesis is by hypoxia signaling, which involves a disruption of cellular iron homeostasis by competition with iron transporters or iron-regulated enzymes. To examine the involvement of iron in the hypoxia signaling activity of these metal ions we investigated HIF-1alpha protein stabilization, IRP-1 activity, and ferritin protein levels in human lung carcinoma A459 cells exposed to various agents in serum- and iron-free salt-glucose medium (SGM) or in normal complete medium. We also studied the effects of excess exogenous iron on these responses induced by nickel ion exposure. Our results show the following: (1) SGM enhanced metals-induced HIF-1alpha stabilization and IRP-1 activation (e.g., nickel and cobalt ions). (2) If SGM was reconstituted with a slight excess level (25muM of FeSO(4)) of iron, this enhancing ability was significantly decreased. (3) The effect of a high level of exogenous iron (500muM of FeSO(4)) on metal-induced hypoxia and iron metabolism was highly dependent on the order of addition. If treatment with the Fe and metal ions was simultaneous (co-treatment), the effects of nickel ion exposure were overwhelmed, since the added Fe reversed HIF-1alpha stabilization, decreased IRP-1 activity, and increased ferritin level. Pre-treatment with iron was not able to reverse the responses caused by nickel ion exposure. These results imply that it is important to consider the available iron concentration and suitable exposure design when studying metal-induced hypoxia or metal-induced disruption of Fe homeostasis
— id: 68895, year: 2006, vol: 610, page: 48, stat: Journal Article,

Hypoxia-Inducible Factor-1 (HIF-1)
Ke, Qingdong; Costa, Max
2006 Nov;70(5):1469-1480, Molecular pharmacology
Adaptation to low oxygen tension (hypoxia) in cells and tissues leads to the transcriptional induction of a series of genes that participate in angiogenesis, iron metabolism, glucose metabolism, and cell proliferation/survival. The primary factor mediating this response is the hypoxia-inducible factor-1 (HIF-1), an oxygen-sensitive transcriptional activator. HIF-1 consists of a constitutively expressed subunit HIF-1beta and an oxygen-regulated subunit HIF-1alpha (or its paralogs HIF-2alpha and HIF-3alpha). The stability and activity of the alpha subunit of HIF are regulated by its post-translational modifications such as hydroxylation, ubiquitination, acetylation, and phosphorylation. In normoxia, hydroxylation of two proline residues and acetylation of a lysine residue at the oxygen-dependent degradation domain (ODDD) of HIF-1alpha trigger its association with pVHL E3 ligase complex, leading to HIF-1alpha degradation via ubiquitin-proteasome pathway. In hypoxia, the HIF-1alpha subunit becomes stable and interacts with coactivators such as cAMP response element-binding protein binding protein/p300 and regulates the expression of target genes. Overexpression of HIF-1 has been found in various cancers, and targeting HIF-1 could represent a novel approach to cancer therapy
— id: 68996, year: 2006, vol: 70, page: 1469, stat: Journal Article,

Alterations of histone modifications and transgene silencing by nickel chloride
Ke, Qingdong; Davidson, Todd; Chen, Haobin; Kluz, Thomas; Costa, Max
2006 Jul;27(7):1481-1488, Carcinogenesis
Although it has been well established that insoluble nickel compounds are potent carcinogens and soluble nickel compounds are less potent, the mechanisms remain unclear. Nickel compounds are weakly mutagenic, but cause epigenetic effects in cells. Previous studies have shown that insoluble nickel compounds enter cells by phagocytosis and silence gene expression, but the entry of soluble nickel compounds and their effects on gene silencing have not been well studied. Here, we have demonstrated, using a dye that fluoresces when nickel ions bind, that soluble nickel compounds were taken up by cells. Nickel ions localized initially in the cytoplasm, but later entered the nucleus and eventually silenced a transgene. In addition, we described three major changes in histone modification of cells exposed to soluble nickel compounds: (i) loss of acetylation of H2A, H2B, H3 and H4; (ii) increases of H3K9 dimethylation; and (iii) substantial increases of the ubiquitination of H2A and H2B. These effects were observed at nickel exposure conditions that had minimum effects on cell cytotoxicity. Moreover, we demonstrated that nickel-induced transgene silencing was associated with similar changes of histone modifications in their nuclesomes. This study is the first to show that nickel compounds increase histone ubiquitination in cells. These new findings will further our understanding of the epigenetic mechanisms of nickel-mediated carcinogenesis
— id: 67527, year: 2006, vol: 27, page: 1481, stat: Journal Article,

Essential role of ROS-mediated NFAT activation in TNF-alpha induction by crystalline silica exposure
Ke, Qingdong; Li, Jingxia; Ding, Jin; Ding, Min; Wang, Liying; Liu, Bingci; Costa, Max; Huang, Chuanshu
2006 Aug;291(2):L257-L264, American journal of physiology. Lung cellular & molecular physiology
Occupational exposure to crystalline silica has been associated with progressive pulmonary silicosis and lung cancer, but the underlying molecular mechanisms are not well understood. Previous studies have shown that crystalline silica exposure can generate reactive oxygen species (ROS) and induce the expression of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in cells. TNF-alpha is believed to be critical in the development of silica-related diseases. Thus it will be of significance to understand the mechanisms of TNF-alpha induction by silica exposure. Given the fact that the transcription factor nuclear factor of activated T cells (NFAT) plays an important role in the regulation of TNF-alpha and can also be activated by ROS, in this study we investigated the potential role of ROS in silica-induced NFAT activity as well as TNF-alpha expression in Cl41 cells. The results showed that exposure of cells to silica led to NFAT transactivation and TNF-alpha induction, where superoxide anion radical (O(2)(-).), but not H(2)O(2), was involved. The knockdown of NFAT3 by its specific small interfering RNA significantly attenuated the silica-induced TNF-alpha transcription. This study demonstrated that silica was able to activate NFAT in an O(2)(-).-dependent manner, which was required for TNF-alpha induction
— id: 68743, year: 2006, vol: 291, page: L257, stat: Journal Article,

Effects of 12 metal ions on iron regulatory protein 1 (IRP-1) and hypoxia-inducible factor-1 alpha (HIF-1alpha) and HIF-regulated genes
Li, Qin; Chen, Haobin; Huang, Xi; Costa, Max
2006 Jun 15;213(3):245-255, Toxicology & applied pharmacology
Several metal ions that are carcinogenic affect cellular iron homeostasis by competing with iron transporters or iron-regulated enzymes. Some metal ions can mimic a hypoxia response in cells under normal oxygen tension, and induce expression of HIF-1alpha-regulated genes. This study investigated whether 12 metal ions altered iron homeostasis in human lung carcinoma A549 cells as measured by an activation of IRP-1 and ferritin level. We also studied hypoxia signaling by measuring HIF-1alpha protein levels, hypoxia response element (HRE)-driven luciferase reporter activity, and Cap43 protein level (an HIF-1alpha responsive gene). Our results show the following: (i) Ni(II), Co(II), V(V), Mn(II), and to a lesser extent As(III) and Cu(II) activated the binding of IRP-1 to IRE after 24 h, while the other metal ions had no effect; (ii) 10 of 12 metal ions induced HIF-1alpha protein but to strikingly different degrees. Two of these metal ions, Al(III) and Cd(II), did not induce HIF-1alpha protein; however, as indicated below, only Ni(II), Co (II), and to lesser extent Mn(II) and V(V) activated HIF-1alpha-dependent transcription. The combined effects of both [Ni(II) + As(III)] and [Ni(II) + Cr(VI)] on HIF-1alpha protein were synergistic; (iii) Addition of Fe(II) with Ni(II), Co(II), and Cr(VI) attenuated the induction of HIF-1alpha after 4 h treatment; (iv) Ni(II), Co(II), and Mn(II) significantly decrease ferritin level after 24 h exposure; (v) Ni(II), Co(II), V(V), and Mn(II) activated HRE reporter gene after 20 h treatment; (vi) Ni(II), Co(II), V(V), and Mn(II) increased the HIF-1-dependent Cap43 protein level after 24 h treatment. In conclusion, only Ni (II), Co (II), and to a lesser extent Mn(II) and V(V) significantly stabilized HIF-1alpha protein, activated IRP, decreased the levels of ferritin, induced the transcription of HIF-dependent reporter, and increased the expression of Cap43 protein levels (HIF-dependent gene). The mechanism for the significant stabilization and elevation of HIF-1alpha protein which drives these other parameters was previously shown by us and others to involve a loss of cellular Fe as well as inhibition of HIF-1alpha-dependent prolyl hydroxylases which target the binding of VHL ubiquitin ligase and degrade HIF-1alpha. Even though there were small effects of some of the other metals on IRP and HIF-1alpha, downstream effects of HIF-1alpha activation and therefore robust hypoxia signaling were only observed with Ni(II), Co(II), and to much lesser extents with Mn(II) and V(V) in human A549 lung cells. It is of interest that the metal ions that were most effective in activating hypoxia signaling were the ones that were poor inducers of metallothionein protein and also decreased Ferritin levels, since both of these proteins can bind metal ions and protect the cell against toxicity in human lung cells. It is important to study effects of these metals in human lung cells since this represents a major route of human environmental and occupational exposure to these metal ions
— id: 65794, year: 2006, vol: 213, page: 245, stat: Journal Article,

Altered iron homeostasis involvement in arsenite-mediated cell transformation
Wu, Jing; Eckard, Jonathan; Chen, Haobin; Costa, Max; Frenkel, Krystyna; Huang, Xi
2006 Feb 1;40(3):444-452, Free radical biology & medicine
Chronic exposure to low doses of arsenite causes transformation of human osteogenic sarcoma (HOS) cells. Although oxidative stress is considered important in arsenite-induced cell transformation, the molecular and cellular mechanisms by which arsenite transforms human cells are still unknown. In the present study, we investigated whether altered iron homeostasis, known to affect cellular oxidative stress, can contribute to the arsenite-mediated cell transformation. Using arsenite-induced HOS cell transformation as a model, it was found that total iron levels are significantly higher in transformed HOS cells in comparison to parental control HOS cells. Under normal iron metabolism conditions, iron homeostasis is tightly controlled by inverse regulation of ferritin and transferrin receptor (TfR) through iron regulatory proteins (IRP). Increased iron levels in arsenite transformed cells should theoretically lead to higher ferritin and lower TfR in these cells than in controls. However, the results showed that both ferritin and TfR are decreased, apparently through two different mechanisms. A lower ferritin level in cytoplasm was due to the decreased mRNA in the arsenite-transformed HOS cells, while the decline in TfR was due to a lowered IRP-binding activity. By challenging cells with iron, it was further established that arsenite-transformed HOS cells are less responsive to iron treatment than control HOS cells, which allows accumulation of iron in the transformed cells, as exemplified by significantly lower ferritin induction. On the other hand, caffeic acid phenethyl ester (CAPE), an antioxidant previously shown to suppress As-mediated cell transformation, prevents As-mediated ferritin depletion. In conclusion, our results suggest that altered iron homeostasis contributes to arsenite-induced oxidative stress and, thus, may be involved in arsenite-mediated cell transformation
— id: 64134, year: 2006, vol: 40, page: 444, stat: Journal Article,

Nickel decreases cellular iron level and converts cytosolic aconitase to iron-regulatory protein 1 in A549 cells
Chen, Haobin; Davidson, Todd; Singleton, Steven; Garrick, Michael D; Costa, Max
2005 Aug 15;206(3):275-287, Toxicology & applied pharmacology
Nickel (Ni) compounds are well-established carcinogens and are known to initiate a hypoxic response in cells via the stabilization and transactivation of hypoxia-inducible factor-1 alpha (HIF-1alpha). This change may be the consequence of nickel's interference with the function of several Fe(II)-dependent enzymes. In this study, the effects of soluble nickel exposure on cellular iron homeostasis were investigated. Nickel treatment decreased both mitochondrial and cytosolic aconitase (c-aconitase) activity in A549 cells. Cytosolic aconitase was converted to iron-regulatory protein 1, a form critical for the regulation of cellular iron homeostasis. The increased activity of iron-regulatory protein 1 after nickel exposure stabilized and increased transferrin receptor (Tfr) mRNA and antagonized the iron-induced ferritin light chain protein synthesis. The decrease of aconitase activity after nickel treatment reflected neither direct interference with aconitase function nor obstruction of [4Fe-4S] cluster reconstitution by nickel. Exposure of A549 cells to soluble nickel decreased total cellular iron by about 40%, a decrease that likely caused the observed decrease in aconitase activity and the increase of iron-regulatory protein 1 activity. Iron treatment reversed the effect of nickel on cytosolic aconitase and iron-regulatory protein 1. To assess the mechanism for the observed effects, human embryonic kidney (HEK) cells over expressing divalent metal transporter-1 (DMT1) were compared to A549 cells expressing only endogenous transporters for inhibition of iron uptake by nickel. The inhibition data suggest that nickel can enter via DMT1 and compete with iron for entry into the cell. This disturbance of cellular iron homeostasis by nickel may have a great impact on the ability of the cell to regulate a variety of cell functions, as well as create a state of hypoxia in cells under normal oxygen tension. These effects may be very important in how nickel exerts phenotypic selection pressure to convert a normal initiated cell into a cancer cell
— id: 57867, year: 2005, vol: 206, page: 275, stat: Journal Article,

Nickel carcinogenesis: epigenetics and hypoxia signaling
Costa, Max; Davidson, Todd L; Chen, Haobin; Ke, Qingdong; Zhang, Ping; Yan, Yan; Huang, Chuanshu; Kluz, Thomas
2005 Dec 30;592(1-2):79-88, Mutation research
Both water soluble and insoluble nickel compounds have been implicated in the etiology of human lung and nasal cancers. Water insoluble nickel compounds have been shown to enter cells by phagocytosis and are contained in cytoplasmic vacuoles, which are acidified thus accelerating the dissolution of soluble nickel from the particles. Using Newport Green, a dye that fluoresces when ionic nickel is bound, we have shown that following exposure (48-72 h) of human lung (A549) cells to NiS particles, most of the nickel is contained in the nucleus, while cells exposed to soluble NiCl2 exhibit most of the ions localized in the cytoplasm. This effect is consistent with previously published reports showing that short-term exposure of cells to crystalline nickel particles (1-3 days) is able to epigenetically silence target genes placed near heterochromatin, while similar short-term exposure to soluble nickel compounds are not able to induce silencing of genes placed near heterochromatin. However, a 3 week exposure of cells to soluble NiCl2 is also able to induce gene silencing. A similar effect was found in yeast cells where nickel was able to silence the URA-3 gene placed near (1.3 kb) a telomere silencing element, but not when the gene was placed farther away from the silencing element (2.0 kb). In addition to epigenetic effects, nickel compounds activate hypoxia signaling pathways. The mechanism of this effect involves the ability of either soluble or insoluble nickel compounds to block iron uptake leading to cellular iron depletion, directly affect iron containing enzymes, or both. This results in the inhibition of a variety of iron-dependent enzymes, such as aconitase and the HIF proline hydroxylases (PHD1-3). The inhibition of the HIF proline hydroxylases stabilizes the HIF protein and activates hypoxic signaling. Additional studies have shown that nickel and hypoxia decrease histone acetylation and increase the methylation of H3 lysine 9. These events are involved in gene silencing and hypoxia can also cause these effects in human cells. It is hypothesised that the state of hypoxia either by low oxygen tension or as a result of agents that signal hypoxia under normal oxygen tension (iron chelation, nickel and cobalt) results in low levels of acetyl CoA, which is a substrate for histone and other protein acetylation. This effect may in part be responsible for the gene silencing following nickel exposure and during hypoxia
— id: 67383, year: 2005, vol: 592, page: 79, stat: Journal Article,

Soluble nickel interferes with cellular iron homeostasis
Davidson, Todd; Chen, Haobin; Garrick, Michael D; D'Angelo, Gisela; Costa, Max
2005 Nov;279(1-2):157-162, Molecular & cellular biochemistry
Soluble nickel compounds are likely human carcinogens. The mechanism by which soluble nickel may contribute to carcinogenesis is unclear, though several hypotheses have been proposed. Here we verify the ability of nickel to enter the cell via the divalent metal ion transporter 1 (DMT1) and disturb cellular iron homeostasis. Nickel may interfere with iron at both an extracellular level, by preventing iron from being transported into the cell, and at an intracellular level, by competing for iron sites on enzymes like the prolyl hydroxylases that modify hypoxia inducible factor-1alpha (HIF-1alpha). Nickel was able to decrease the binding of the Von Hippel-Lindau (VHL) protein to HIF-1alpha, indicating a decrease in prolyl hydroxylase activity. The ability of nickel to affect various iron dependent processes may be an important step in nickel dependent carcinogenesis. In addition, understanding the mechanisms by which nickel activates the HIF-1alpha pathway may lead to new molecular targets in fighting cancer
— id: 68896, year: 2005, vol: 279, page: 157, stat: Journal Article,

Down-regulation of the expression of the FIH-1 and ARD-1 genes at the transcriptional level by nickel and cobalt in the human lung adenocarcinoma A549 cell line
Ke, Qingdong; Kluz, Thomas; Costa, Max
2005 Apr;2(1):10-13, International Journal of Environmental Research & Public Health
Although nickel and cobalt compounds have been known to cause induction of the transcription factor hypoxia-inducible factor 1 (HIF-1) and activation of a battery of hypoxia-inducible genes in the cell, the molecular mechanisms of this induction remain unclear. The post-translational modification of HIF-1a, the oxygen-sensitive subunit of HIF-1, regulates stabilization, nuclear translocation, DNA binding activity, and transcriptional activity of the protein. Among the enzymes regulating the post-translational modification of HIF-la, the factor inhibiting HIF-1 (FIH-1) hydroxylates the protein at asparagine 803, suppressing the interaction of HIF-1a with transcription coactivators p300/CBP and reducing the transcriptional activity of the protein. ARD-1, the acetyltransferase, acetylates HIF-1a at lysine 532, which enhances the interaction of HIF-1a with pVHL. Therefore, FIH-1 and ARD-1 negatively regulate the transcriptional activity and the stability of HIF-1a. We examined the mRNA levels of FIH-l and ARD-1 genes after exposure nickel (II) or cobalt (II) to the cell and found that both genes were down-regulated by the chemical treatment, which may lead to reduced levels of both proteins and result in increased level of HIF-1 a and its transcriptional activity
— id: 66459, year: 2005, vol: 2, page: 10, stat: Journal Article,

ERKs activation and calcium signaling are both required for VEGF induction by vanadium in mouse epidermal Cl41 cells
Li, Jingxia; Tong, Qiangsong; Shi, Xianglin; Costa, Max; Huang, Chuanshu
2005 Nov;279(1-2):25-33, Molecular & cellular biochemistry
The previous studies have demonstrated that vanadium exposure can cause a variety of biological effects. However, the mechanisms involved in the biological effects caused by vanadium are not well understood. Our previous studies have shown that exposure of mouse epidermal Cl 41 cells to vanadate stimulated the phosphorylation of both ERKs and p38K, and calcium signaling leading NFAT activation. In view of the evidence that ERKs and p38 kinase contribute to VEGF induction, we investigated in the present study the potential roles of ERKs, p38K, and calcium signaling in VEGF induction caused by vanadium exposure. Exposure of Cl 41 cells to vanadium led to VEGF induction in both time- and dose-dependent manners. Pre-treatment of Cl 41 cells with PD98059, an inhibitor of MEK1/2-ERKs pathway, but not SB202190, an inhibitor for p38K pathway, resulted in a dramatic inhibition of VEGF induction by vanadium. More interesting, pre-treatment of Cl 41 cells with intracellular calcium chelator, but not calcium channel blocker, resulted in a dramatic decrease in VEGF induction by vanadium. However, both PI-3K inhibitors and overexpression of Deltap85, a dominant negative PI-3K mutant, resulted in only a marginal decrease in VEGF induction by vanadium. Moreover, mTOR, as a downstream molecule of PI-3K, did not attribute to VEGF induction by vanadium because rapamycin pre-treatment did not show any inhibitory effect on VEGF induction. These results indicate that ERKs and intracellular stored calcium release play a critical role in VEGF induction by vanadium. PI-3K is partially involved in VEGF induction by vanadium, while p38K and mTOR are not involved. Those results will help us to understand the molecular mechanisms involved in vanadium-induced biological effects
— id: 70870, year: 2005, vol: 279, page: 25, stat: Journal Article,

Carcinogenic effect of nickel compounds
Lu, Haitian; Shi, Xianglin; Costa, Max; Huang, Chuanshu
2005 Nov;279(1-2):45-67, Molecular & cellular biochemistry
Nickel is a widely distributed metal that is industrially applied in many forms. Accumulated epidemiological evidence confirms that exposures to nickel compounds are associated with increased nasal and lung cancer incidence, both in mostly occupational exposures. Although the molecular mechanisms by which nickel compounds cause cancer are still under intense investigation, the carcinogenic actions of nickel compounds are thought to involve oxidative stress, genomic DNA damage, epigenetic effects, and the regulation of gene expression by activation of certain transcription factors related to corresponding signal transduction pathways. The present review summarizes our current knowledge on the molecular mechanisms of nickel carcinogenesis, with special emphasis on the role of nickel induced reactive oxygen species (ROS) and signal transduction pathways
— id: 70872, year: 2005, vol: 279, page: 45, stat: Journal Article,

Essential role of PI-3K, ERKs and calcium signal pathways in nickel-induced VEGF expression
Ouyang, Weiming; Li, Jingxia; Shi, Xianglin; Costa, Max; Huang, Chuanshu
2005 Nov;279(1-2):35-43, Molecular & cellular biochemistry
Exposure to a highly nickel-polluted environment has the potential to cause a variety of adverse health effects, such as the respiratory tract cancers. Since numerous studies have demonstrated that nickel generally has weak mutagenic activity, research focus had turned to cell signalling activation leading to gene modulation and epigenetic changes as a plausible mechanism of carcinogenesis. Previous studies have revealed that nickel compounds can induce the expression of vascular endothelial growth factor (VEGF), which is a key mediator of angiogenesis both in physiological and pathologic conditions. In the present study, we investigated the potential roles of PI-3K, ERKs, p38 kinase and calcium signalling in VEGF induction by nickel in Cl 41 cells. Exposure of Cl 41 cells to nickel compounds led to VEGF induction in both time- and dose-dependent manners. Pre-treatment of Cl 41 cells with PI-3K inhibitor, wortmannin or Ly294002, resulted in a striking inhibition of VEGF induction by nickel compounds, implicating the role of PI-3K in the induction. However, mTOR, one of downstream molecules of PI-3K, may not contribute to the induction because pre-treatment of Cl 41 cells with its inhibitor, rapamycin, did not show obvious decrease in nickel-induced VEGF expression. Furthermore, pre-treatment of Cl 41 cells with MEK1/2-ERKs pathway inhibitor, PD98059, significantly inhibited VEGF induction by both NiCl2 and Ni3S2, whereas p38 kinase inhibitor, SB202190, did not impair the induction. Pre-treatment of Cl 41 cells with intracellular calcium chelator, but not calcium channel blocker, inhibited VEGF induction by nickel. Collectively these data demonstrate that PI-3K, ERKs and cytosolic calcium, but not p38 kinase, play essential roles in VEGF induction by nickel compounds
— id: 70871, year: 2005, vol: 279, page: 35, stat: Journal Article,

Altered N-myc downstream-regulated gene 1 protein expression in African-American compared with caucasian prostate cancer patients
Caruso, Robert P; Levinson, Benjamin; Melamed, Jonathan; Wieczorek, Rosemary; Taneja, Samir; Polsky, David; Chang, Caroline; Zeleniuch-Jacquotte, Anne; Salnikow, Konstantin; Yee, Herman; Costa, Max; Osman, Iman
2004 Jan 1;10(1 Pt 1):222-227, Clinical cancer research
PURPOSE: The protein encoded by N-myc downstream-regulated gene 1 (NDRG1) is a recently discovered protein whose transcription is induced by androgens and hypoxia. We hypothesized that NDRG1 expression patterns might reveal a biological basis for the disparity of clinical outcome of prostate cancer patients with different ethnic backgrounds. EXPERIMENTAL DESIGN: Patients who underwent radical prostatectomy between 1990 and 2000 at Veterans Administration Medical Center of New York were examined. We studied 223 cases, including 157 African Americans and 66 Caucasians (T2, n = 144; >/=T3, n = 79; Gleason <7, n = 122; >/=7, n = 101). Three patterns of NDRG1 expression were identified in prostate cancer: (a) intense, predominately membranous staining similar to benign prostatic epithelium; (b) intense, nucleocytoplasmic localization; and (c) low or undetectable expression. We then examined the correlations between patients' clinicopathological parameters and different NDRG1 expression patterns. RESULTS: In this study of patients with equal access to care, African-American ethnic origin was an independent predictor of prostate-specific antigen recurrence (P < 0.05). We also observed a significant correlation between different patterns of NDRG1 expression and ethnic origin. Pattern 2 was less frequent in African Americans (21% versus 38%), whereas the reverse was observed for pattern 3 (60% in African Americans versus 44% in Caucasians; P = 0.03). This association remained significant after controlling for both grade and stage simultaneously (P = 0.02). CONCLUSIONS: Our data suggest that different NDRG1 expression patterns reflect differences in the response of prostatic epithelium to hypoxia and androgens in African-American compared with Caucasian patients. Further studies are needed to determine the contribution of NDRG1 to the disparity in clinical outcome observed between the two groups
— id: 44771, year: 2004, vol: 10, page: 222, stat: Journal Article,

Research profile. Max Costa
Costa, Max
2004 May;6(5):57N-59N, Journal of environmental monitoring. JEM
— id: 134097, year: 2004, vol: 6, page: 57N, stat: Journal Article,

Exposure to chromium (VI) in the drinking water increases susceptibility to UV-induced skin tumors in hairless mice
Davidson, Todd; Kluz, Thomas; Burns, Fredric; Rossman, Toby; Zhang, Qunwei; Uddin, Ahmed; Nadas, Arthur; Costa, Max
2004 May 1;196(3):431-437, Toxicology & applied pharmacology
Hexavalent chromium (Cr (VI)) is a well known-human carcinogen with exposures occurring in both occupational and environmental settings. Although lung carcinogenicity has been well documented for occupational exposure via inhalation, the carcinogenic hazard of drinking water exposure to Cr (VI) has yet to be established. We used a hairless mouse model to study the effects of K(2)CrO(4) in the drinking water on ultraviolet radiation (UVR)-induced skin tumors. Hairless mice were unexposed or exposed to UVR alone (1.2 kJ/m(2)), K(2)CrO(4) alone at 2.5 and 5.0 ppm, or the combination of UVR and K(2)CrO(4) at 0.5, 2.5, and 5.0 ppm. Mice were observed on a weekly basis for the appearance of skin tumors larger than 2 mm. All the mice were euthanized on day 182. The skin tumors were excised and subsequently analyzed microscopically for malignancy by histopathology. There was a total absence of observable skin tumors in untreated mice and in mice exposed to chromate alone. However, there was a dose-dependent increase in the number of skin tumors greater than 2 mm in mice exposed to K(2)CrO(4) and UV compared with mice exposed to UV alone. The increase in tumors larger than 2 mm was statistically significant (P < 0.05) for UV and K(2)CrO(4) at the two highest K(2)CrO(4) doses (2.5 and 5.0 ppm), and there was a statistically significant increase in the numbers of malignant tumors per mouse in the UVR plus K(2)CrO(4) (5 ppm) group compared with UV alone. The data presented here indicate that K(2)CrO(4) increases the number of UV-induced skin tumors in a dose-dependent manner, and these results support the concern that regulatory agencies have relative to the carcinogenic health hazards of widespread human exposure to Cr (VI) in drinking water
— id: 43216, year: 2004, vol: 196, page: 431, stat: Journal Article,

Molecular mechanisms of arsenic carcinogenesis
Huang, Chuanshu; Ke, Qingdong; Costa, Max; Shi, Xianglin
2004 Jan;255(1-2):57-66, Molecular & cellular biochemistry
Arsenic is a metalloid compound that is widely distributed in the environment. Human exposure of this compound has been associated with increased cancer incidence. Although the exact mechanisms remain to be investigated, numerous carcinogenic pathways have been proposed. Potential carcinogenic actions for arsenic include oxidative stress, genotoxic damage, DNA repair inhibition, epigenetic events, and activation of certain signal transduction pathways leading to abberrant gene expression. In this article, we summarize current knowledge on the molecular mechanisms of arsenic carcinogenesis with an emphasis on ROS and signal transduction pathways
— id: 42693, year: 2004, vol: 255, page: 57, stat: Journal Article,

Differential effects of polycyclic aromatic hydrocarbons on transactivation of AP-1 and NF-kappaB in mouse epidermal cl41 cells
Li, Jingxia; Chen, Haobin; Ke, Qingdong; Feng, Zhaohui; Tang, Moon-Shong; Liu, Bingci; Amin, Shantu; Costa, Max; Huang, Chuanshu
2004 Jun;40(2):104-115, Molecular carcinogenesis
Polycyclic aromatic hydrocarbons (PAHs) and their derivatives, such as benzo[a]pyrene (B[a]P), (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), and 5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE), are complete carcinogens. However, the tumor promotion effects of PAHs remain unclear. We therefore investigated the possible activation of activator protein-1 (AP-1) and nuclear factor-kappaB (NFkappaB) in mouse epidermal Cl41 cells after different PAHs treatments, including B[a]P, B[a]PDE, chrysene-1,2-diol-3,4-epoxid (CDE), and 5-MCDE. The results showed that B[a]PDE and 5-MCDE were able to activate AP-1 and NF-kappaB, whereas B[a]P showed only marginal effect on AP-1 activation, and B[a]P and CDE had no effect on NF-kappaB activation. Treatment with either B[a]PDE or 5-MCDE also resulted in mitogen-activated protein kinases (MAPKs) activation as well as inhibitory subunit kappa-B (IkappaBalpha) phosphorylation and degradation, whereas B[a]P and CDE had no effect. Pretreatment with PD98059, a specific inhibitor for extracellular signal-regulated protein kinases (ERKs) upstream kinase MEK1/2, or SB202190, a p38 kinase inhibitor, resulted in a dramatic inhibition of B[a]PDE-induced AP-1 transactivation. In addition, B[a]PDE-induced AP-1 activation was also inhibited by overexpressing a dominant negative mutant of JNK1 in the cells. All these suggest ERKs, c-jun N-terminal kinases (JNKs), and p38 kinase signal transduction pathways are required for AP-1 induction by B[a]PDE. Taken together, B[a]PDE and 5-MCDE are the active compounds of PAHs to initiate signaling pathways. Considering the important roles of AP-1 and NF-kappaB in tumor promotion, we speculated the activation of AP-1 and NF-kappaB by B[a]PDE and 5-MCDE may involve in their or their parent compounds' tumor promotion effects. This study may help in better understanding the tumor promotion effects of PAHs
— id: 42690, year: 2004, vol: 40, page: 104, stat: Journal Article,

PI-3K and Akt are mediators of AP-1 induction by 5-MCDE in mouse epidermal Cl41 cells
Li, Jingxia; Chen, Haobin; Tang, Moon-Shong; Shi, Xianglin; Amin, Shantu; Desai, Dhimant; Costa, Max; Huang, Chuanshu
2004 Apr 12;165(1):77-86, Journal of cell biology
5-Methylchrysene has been found to be a complete carcinogen in laboratory animals. However, the tumor promotion effects of (+/-)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) remain unclear. In the present work, we found that 5-MCDE induced marked activator protein-1 (AP-1) activation in Cl41 cells. 5-MCDE also induced a marked activation of phosphatidylinositol 3-kinase (PI-3K). Inhibition of PI-3K impaired 5-MCDE-induced AP-1 transactivation, suggesting that PI-3K is an upstream kinase involved in AP-1 activation by 5-MCDE. Furthermore, we found that Akt is a PI-3K downstream mediator for 5-MCDE-induced AP-1 transactivation, whereas another PI-3K downstream kinase, p70(S6K), was not involved in AP-1 activation by 5-MCDE. Moreover, inhibition of Akt activation blocked 5-MCDE-induced activation of extracellular signal-regulated protein kinases (ERKs) and c-Jun NH(2)-terminal kinases (JNKs), whereas it did not affect p38K activation. Consistently, overexpression of a dominant-negative mutant of ERK2 or JNK1 blocked the AP-1 activation by 5-MCDE. These results demonstrate that 5-MCDE is able to induce AP-1 activation, and the AP-1 induction is specifically through a PI-3K/Akt-dependent and p70(S6K)-independent pathway
— id: 42691, year: 2004, vol: 165, page: 77, stat: Journal Article,

Nickel compounds act through phosphatidylinositol-3-kinase/Akt-dependent, p70(S6k)-independent pathway to induce hypoxia inducible factor transactivation and Cap43 expression in mouse epidermal Cl41 cells
Li, Jingxia; Davidson, Gerard; Huang, Yi; Jiang, Bing-Hua; Shi, Xianglin; Costa, Max; Huang, Chuanshu
2004 Jan 1;64(1):94-101, Cancer research
Nickel compounds are a somewhat unique class of carcinogens. Previous studies have demonstrated that NiCl(2) exposure leads to marked induction of hypoxia inducible factor 1 (HIF-1) in human osteosarcoma and BALB/c 3T3 cells, a transcription factor that has been considered to play an important role in tumor promotion and progression. However, the signal transduction pathways leading to HIF-1 induction are not well understood. The present study indicated that exposure of mouse epidermal Cl41 cells to either Ni(3)S(2) or NiCl(2) resulted in activation of phosphatidylinositol 3-kinase (PI-3K), Akt, and p70 S6 kinase (p70(S6k)). Inhibition of PI-3K, Akt, and p70(S6k) by overexpression of a dominant-negative mutant of PI-3K (Deltap85) impaired nickel-induced HIF-1 transactivation. Furthermore, an overexpression of the dominant-negative Akt mutant (Akt-T308A/S473A) blocked nickel-induced Akt phosphorylation and HIF-1 transactivation, whereas inhibition of p70(S6k) activation by pretreatment of cells with rapamycin did not show significant inhibitory effects on HIF-1 transactivation induced by nickel compounds. Consistent with HIF-1 transactivation, inhibition of the PI-3K/Akt pathway by either overexpression of Deltap85 or Akt-T308A/S473A caused dramatic inhibition of Cap43 protein expression induced by nickel compounds, whereas pretreatment of cells with rapamycin did not exhibit inhibition of Cap43 induction. These results demonstrated that nickel compounds induce HIF-1 transactivation and Cap43 protein expression through a PI-3K/Akt-dependent and p70(S6k)-independent pathway. This study should help us understand the signal transduction pathways involved in the carcinogenic effects of nickel compounds
— id: 42615, year: 2004, vol: 64, page: 94, stat: Journal Article,

Activation of aPKC is required for vanadate-induced phosphorylation of protein kinase B (Akt), but not p70S6k in mouse epidermal JB6 cells
Li, Jingxia; Dokka, Sujatha; Wang, Liying; Shi, Xianglin; Castranova, Vincent; Yan, Yan; Costa, Max; Huang, Chuanshu
2004 Jan;255(1-2):217-225, Molecular & cellular biochemistry
Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms by which vanadate mediates adverse effects are not well understood. The present study investigated the vanadate-induced phosphorylation of Akt and p70S6K, two kinases known to be vital for cell survival, growth, transformation, and transition of the cell cycle in mammals. Exposure of mouse epidermal JB6 cells to vanadium led to phosphorylation of Akt and p70S6K in a time- and dose-dependent manner. Vanadium exposure also caused translocation of atypical isoforms of PKC (lambda, zeta) from the cytosol to the membrane, but had no effect on PKCalpha translocation, suggesting that the atypical PKCs (aPKC) were specifically involved in vanadium-induced cellular response. Importantly, overexpression of a dominant negative mutant PKClambda blocked Akt phosphorylation at Ser473 and Thr308, whereas it did not inhibit p70S6k phosphorylation at Thr389 and Thr421/Ser424, suggesting that aPKC activation is specifically involved in vanadium-induced activation of Akt, but not in activation of p70S6k. Furthermore, vanadium-induced p70S6k phosphorylation at Thr389 and Thr421/Ser424 and Akt phosphorylation at Thr308 occurred through a PI-3K-dependent pathway because a PI-3K dominant negative mutant inhibited induction as compared with vector control cells. These results indicate that there was a differential role of aPKC in vanadate-induced phosphorylation of Akt and p70S6k, suggesting that signal transduction pathways leading to the activation of Akt and p70S6k were different
— id: 42692, year: 2004, vol: 255, page: 217, stat: Journal Article,

Chromatin immunoprecipitation assays
Yan, Yan; Chen, Haobin; Costa, Max
2004 ;287(8):9-19, Methods in molecular biology
Chromatin immunoprecipitation (ChIP) is a powerful tool to study protein-DNA interaction and is widely used in many fields to study proteins associated with chromatin, such as histone and its isoforms and transcription factors, across a defined DNA domain. Here, we show the step-by-step methods currently used in our lab to immunoprecipitate the formaldehyde crosslinked chromatin and further analyze the immuprecipitated DNA by semiquantitative PCR
— id: 46102, year: 2004, vol: 287, page: 9, stat: Journal Article,

Nickel-induced 1,4-alpha-glucan branching enzyme 1 up-regulation via the hypoxic signaling pathway
Zhao, Jianhua; Chen, Haobin; Davidson, Todd; Kluz, Thomas; Zhang, Qunwei; Costa, Max
2004 Jun 1;196(3):404-409, Toxicology & applied pharmacology
Using the mouse Affymetrix gene chip, we found that 1,4-alpha-glucan branching enzyme 1 (GBE1) was one of the most up-regulated genes following nickel exposure. This result was confirmed by Northern blot in two mouse cell lines, four mouse tissues, and three human cell lines. We further found that this gene was also up-regulated by cobalt, hypoxia, the iron chelator (deferoxamine, or DFO), and the prolyl hydroxylase (PH) inhibitor (dimethyloxalyglycine, DMOG), suggesting that hypoxia inducible factor-1alpha (HIF-1alpha) was involved in the up-regulation of this gene. Experiments using HIF-1alpha +/+ and HIF-1alpha -/- mouse cells demonstrated this gene was up-regulated through a HIF-1alpha-dependent hypoxic signaling pathway. Because the hypoxic signaling pathway is believed to be important in the initiation and progression of carcinogenesis, it is important to study genes regulated by this pathway
— id: 46203, year: 2004, vol: 196, page: 404, stat: Journal Article,

Nickel-induced down-regulation of serpin by hypoxic signaling
Zhao, Jianhua; Yan, Yan; Salnikow, Konstantin; Kluz, Thomas; Costa, Max
2004 Jan 1;194(1):60-68, Toxicology & applied pharmacology
Nickel (Ni) carcinogenesis is thought to involve gene chip silencing by epigenetic mechanisms. Serpina3g, a member of the mouse serpin family, was among the most down-regulated genes (32-fold) in response to Ni exposure of mouse cells based on the Affymetrix gene chip. Serpina3g down-regulation was controlled by a hypoxia inducible factor (HIF) mechanism. The exposure of cells to cobalt (Co), hypoxia, the iron chelator deferoxamine, and the proline hydroxylase inhibitor dimethyloxalylglycine (DMOG) also down-regulated serpina3g transcription to similar extents as soluble Ni exposure. These results support the mounting experimental evidence that water-soluble Ni compounds have a predominant effect on hypoxia signaling because of their ability to interfere with Fe homeostasis in the cell. Trichostatin A (TSA) and 5-azacytidine (5-AzaC) reactivated the Ni-silenced serpina3g gene, indicating that its silencing by Ni involved either a direct or indirect epigenetic mechanism. Analysis of the chromatin state of the serpina3g promoter by the ChIP assay revealed that exposure of mouse fibroblast cells to Ni resulted in the methylation of H3 lysine 9 within its promoter, as well as a decrease in the phosphorylation of serine 10 of H3 and a marked decrease in the acetylation of H3 and H4. Serpina3g gene expression returned to basal levels following Ni removal, suggesting that the observed silencing was a dynamic and reversible process
— id: 42617, year: 2004, vol: 194, page: 60, stat: Journal Article,

Nickel(II) binding to Cap43 protein fragments
Zoroddu, M A; Peana, M; Kowalik-Jankowska, T; Kozlowski, H; Costa, M
2004 Jun;98(6):931-939, Journal of inorganic biochemistry
Cap43 protein has been tested for metal binding domains. The protein, specifically induced by nickel compounds in cultured human cells, had a new mono-histidinic motif consisting of 10 amino acids repeated three times in the C-terminus. The 20-Ac-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His (356)-Thr-Ser-Glu-Gly(360) - peptide 1) and the 30-Ac-TRSRSHTSEG-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His (356)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(366)-Thr-Ser-Glu-Gly(370) - peptide 2) amino acids sequence has been analyzed as a site for Ni(II) binding. A combined pH-metric and spectroscopic (UV-visible, CD, NMR) studies of Ni(II) binding to both fragments were performed. The 20-amino acid peptide can bind one and two metal ions while the 30-amino acid fragment one, two and three metal ions. At physiological pH, depending on the metal to ligand molar ratio, peptide 1 forms the Ni(2)L species while peptide 2 the NiL, Ni(2)L and Ni(3)L complexes where each metal ion is coordinated to the imidazole nitrogen atom of the histidine residue of the 10-amino acid fragment. Octahedral complexes at pH 8-9 and planar 4N complexes with (N(Im), 3N(-)) bonding mode at pH above 9, are formed. This work supports the existence of an interesting binding site at the COOH-terminal domain of the Cap43 protein
— id: 141435, year: 2004, vol: 98, page: 931, stat: Journal Article,

Different mechanisms in suppressing MTH expression by nickel chloride and cobalt chloride
Chen, H; Yan, Y; Kluz, T; Costa, M
2003 MAR ;72(5):1050-419, Toxicological sciences
— id: 98229, year: 2003, vol: 72, page: 1050, stat: Journal Article,

Potential hazards of hexavalent chromate in our drinking water
Costa, Max
2003 Apr 1;188(1):1-5, Toxicology & applied pharmacology
A consideration of the consequences of human exposure to hexavalent Cr in the drinking water has been compiled. Since there is an absence of adequate human data on this subject the problem has been analyzed not only from human and animal studies but also from a mechanistic point of view. This treatise has been inspired by recent reviews and speculations that suggest that we may safely drink hexavalent Cr in great excess of the current EPA and states drinking water standards of 50-100 ppb
— id: 111733, year: 2003, vol: 188, page: 1, stat: Journal Article,

Molecular mechanisms of nickel carcinogenesis: gene silencing by nickel delivery to the nucleus and gene activation/inactivation by nickel-induced cell signaling
Costa, Max; Yan, Yan; Zhao, Daoji; Salnikow, Konstantin
2003 Apr;5(2):222-223, Journal of environmental monitoring. JEM
We have summarized the molecular and cellular events involved in nickel (Ni) compound induced carcinogenesis. The major hypothesis for nickel carcinogenic action has involved the ability of the Ni compound to deliver high concentrations of Ni intracellularly, enter the nucleus and interact with chromatin. Ni has been found to selectively damage heterochromatin, and a major action of Ni is its ability to silence the expression of genes located near heterochromatin by inducing a loss of histone H4 and H3 acetylation and DNA hypermethylation. When Ni silences critical genes, such as tumor suppressor genes, the cell is altered to a greater state of neoplastic transformation. The carcinogenic hazard of Ni compounds has been directly related to the ability of that Ni compound to raise the intracellular Ni ions. The mechanisms of Ni-induced gene silencing will be discussed. However, recently it has been found that soluble Ni ions can interact with the cell surface receptors and activate cell signaling resulting in the induction of a variety of cellular genes. In particular, the Ca and hypoxia inducible factor pathway is activated in all cells exposed to soluble Ni ions. In the case of HIF-1 induction, a cell is now equipped with the expression of a variety of genes that will allow the cell to survive the lack of oxygen and thus should enable a previously initiated cancer cell to progress into a full malignant state and metastasize. These new findings support the view that soluble Ni ions exhibit carcinogenic potential by activating cell promotion and lend strength to the epidemiological data showing soluble Ni to be associated with cancer risk in Ni refinery workers
— id: 34642, year: 2003, vol: 5, page: 222, stat: Journal Article,

Regulation of HIF transactivation and CAP43 expression by nickel compounds through PI-3K/AKT dependent, P70S6K-independent pathway
Davidson, G; Li, J; Huang, Y; Costa, M; Huang, C
2003 MAR ;72(1):106-106, Toxicological sciences
— id: 38494, year: 2003, vol: 72, page: 106, stat: Journal Article,

Inhibition of phase I and phase II metabolic enzymes in vitro by nickel chloride
Davidson, TL; Salnikow, K; Costa, M
2003 MAR ;72(5):1058-419, Toxicological sciences
— id: 98230, year: 2003, vol: 72, page: 1058, stat: Journal Article,

Hypoxia inducible factor-1 alpha-independent suppression of aryl hydrocarbon receptor-regulated genes by nickel
Davidson, Todd; Salnikow, Konstantin; Costa, Max
2003 Dec;64(6):1485-1493, Molecular pharmacology
Aryl hydrocarbon receptor (AhR)-dependent enzymes are involved in the biotransformation of harmful xenobiotics into more easily excretable metabolites. Cross-talk between the AhR pathway and the hypoxia inducible factor-1alpha (HIF-1alpha) pathway has been demonstrated previously, although the mechanism remains unclear and quite controversial. Because nickel is known to mimic hypoxia, we investigated the effects of short-term nickel exposure on AhR-dependent gene expression. Gene-chip analysis identified several AhR-dependent genes that are suppressed by exposure to nickel. Using Northern blots, we then confirmed that nickel can down-regulate both the basal and benzo[a]pyrene-inducible expression of AhR-dependent genes in mouse and human cell lines. Using a HIF-1alpha knockout cell line and 3-[2-[4-(bis-(4-fluorophenyl) methylene]-1-piperidinyl)ethyl]-2,3-dihydro-2-thioxo-4(1H)quinazolinone (R59949), which blocks HIF-1alpha protein accumulation, we show HIF-1alpha-independent suppression of AhR-dependent genes by nickel. Desferrioxamine and hypoxia were also able to suppress the basal and inducible expression levels of AhR-regulated genes. Finally, dimethyloxalylglycine, an inhibitor of Fe(II)- and 2-oxoglutarate-dependent dioxygenases also inhibited AhR-dependent expression in a HIF-1alpha-independent manner. Our data suggest that an Fe(II)-, oxoglutarate-, and oxygen-dependent enzyme may directly or indirectly be involved in the regulation of AhR-dependent transcriptional activity by nickel and other hypoxia-mimicking agents
— id: 44774, year: 2003, vol: 64, page: 1485, stat: Journal Article,

Chromium(VI) exposure enhances polycyclic aromatic hydrocarbon-DNA binding at the p53 gene in human lung cells
Feng, Zhaohui; Hu, Wenwei; Rom, William N; Costa, Max; Tang, Moon-Shong
2003 Apr;24(4):771-778, Carcinogenesis
Chromium(VI) [Cr(VI)] is a ubiquitous environmental and industrial contaminant. Cr(VI) exposure is strongly associated with a higher incidence of human lung cancer, but the mechanism of Cr(VI) carcinogenicity remains unclear. Cigarette smoking has been known as the prominent cause of lung cancer, and polycyclic aromatic hydrocarbons (PAHs), the major carcinogens in cigarette smoke, have been suggested as being responsible for the initiation and development of lung cancer. It has been reported that lung cancer from workers exposed to Cr(VI) has a high percentage of G to T transversion mutations in the non-transcribed strand of the p53 gene, a hallmark of PAH-induced mutation. Cr(VI) is a weak mutagen although it can induce a high percentage of G to T transversion mutations. These results raise the possibility that Cr(VI) may enhance PAH binding at the p53 gene in lung tissue. To test this possibility, we have determined the effect of Cr(VI) exposure on benzo[a]pyrene diol epoxides (BPDE)-DNA binding at total genomic DNA level and at the p53 gene in normal human lung fibroblast cells. We found that in lung cells Cr(VI) pre-exposure does not affect the BPDE-DNA binding at the total genomic DNA level or at exons 5, 6 and 9 of the p53 gene; however, it greatly enhances BPDE-DNA binding at exons 7 and 8 of the p53 gene, especially at mutational hotspots of lung cancer: codons 248, 273 and 282 of the p53 gene. No enhancement of BPDE-DNA binding in the p53 was observed when naked genomic DNA isolated from Cr(VI)-exposed cells was modified with BPDE in vitro. These results suggest that Cr(VI) exposure may enhance chromatin structure-dependent carcinogen-DNA binding. This effect may contribute to the synergism of Cr(VI) and BPDE on mutagenesis and cell transformation, and may also contribute to the higher incidence of lung cancer in Cr(VI)-exposed populations
— id: 39234, year: 2003, vol: 24, page: 771, stat: Journal Article,

Inhibition and reversal of cellular transformation by the histone deacetylase inhibitor trichostatin A
Kluz, T; Zhang, Q; Salnikow, K; Costa, M
2003 MAR ;72(1):212-212, Toxicological sciences
— id: 38496, year: 2003, vol: 72, page: 212, stat: Journal Article,

The analysis of transcriptional regulation of nickel-inducible gene Cap43
Salnikow, K; Zhang, P; Costa, M
2003 MAR ;72(1):234-234, Toxicological sciences
— id: 38500, year: 2003, vol: 72, page: 234, stat: Journal Article,

GeneChip analysis of signaling pathways effected by nickel
Salnikow, Konstantin; Davidson, Todd; Kluz, Thomas; Chen, Haobin; Zhou, Daoji; Costa, Max
2003 Apr;5(2):206-209, Journal of environmental monitoring. JEM
The carcinogenicity of nickel compounds has been shown in numerous epidemiological and animal studies. Carcinogenesis is generally considered as a multistep accumulation of genetic alterations. Nickel, however, being highly carcinogenic is only a weak mutagen. We hypothesize that nickel may act by modulating signaling pathways, and subsequently by reprogramming transcription factors. Insoluble nickel is considered to be more carcinogenic than soluble. In this study using GeneChip technology we compared changes in gene expression caused by soluble and insoluble nickel compounds. We found that both soluble and insoluble nickel compounds induce similar signaling pathways following 20 h of in vitro exposure. For example, both nickel compounds activated a number of transcription factors including hypoxia-inducible factor I (HIF-1) and p53. The induction of these important transcription factors exerts potent selective pressure leading to cell transformation. The obtained data are in agreement with our previous observations that acute nickel exposure activates HIF-1 and p53 transcription factors and in nickel-transformed cells, the ratio of HIF-I activity to p53 activity was shifted towards high HIF-I activity. The activation of the same signaling pathways by soluble and insoluble nickel compounds suggested that both nickel compounds have similar carcinogenic potential in vitro
— id: 34643, year: 2003, vol: 5, page: 206, stat: Journal Article,

The involvement of hypoxia-inducible transcription factor-1-dependent pathway in nickel carcinogenesis
Salnikow, Konstantin; Davidson, Todd; Zhang, Qunwei; Chen, Lung Chi; Su, Weichen; Costa, Max
2003 Jul 1;63(13):3524-3530, Cancer research
Nickel is a potent environmental pollutant in industrial countries. Because nickel compounds are carcinogenic, exposure to nickel represents a serious hazard to human health. The understanding of how nickel exerts its toxic and carcinogenic effects at a molecular level may be important in risk assessment, as well as in the treatment and prevention of occupational diseases. Previously, using human and rodent cells in vitro, we showed that hypoxia-inducible signaling pathway was activated by carcinogenic nickel compounds. Acute exposure to nickel resulted in the accumulation of hypoxia-inducible transcription factor (HIF)-1, which strongly activated hypoxia-inducible genes, including the recently discovered tumor marker NDRG1 (Cap43). To further identify HIF-1-dependent nickel-inducible genes and to understand the role of the HIF-dependent signaling pathway in nickel-induced transformation, we used the Affymetrix GeneChip to compare the gene expression profiles in wild-type cells or in cells from HIF-1 alpha knockout mouse embryos exposed to nickel chloride. As expected, when we examined 12,000 genes for expression changes, we found that genes coding for glycolytic enzymes and glucose transporters, known to be regulated by HIF-1 transcription factor, were induced by nickel only in HIF-1 alpha-proficient cells. In addition, we found a number of other hypoxia-inducible genes up-regulated by nickel in a HIF-dependent manner including BCL-2-binding protein Nip3, EGLN1, hypoxia-inducible gene 1 (HIG1), and prolyl 4-hydroxylase. Additionally, we found a number of genes induced by nickel in a HIF-independent manner, suggesting that Ni activated other signaling pathways besides HIF-1. Finally, we found that in HIF-1 alpha knockout cells, nickel strongly induced the expression of the whole group of genes that were not expressed in the presence of HIF-1. Because the majority of modulated genes were induced or suppressed by nickel in a HIF-1-dependent manner, we elucidated the role of HIF-1 transcription factor in cell transformation. In HIF-1 alpha-proficient cells, nickel exposure increased soft agar growth, whereas it decreased soft agar growth in HIF-1 alpha-deficient cells. We hypothesize that the induction of HIF-1 transcription factor by nickel may be important during the nickel-induced carcinogenic process
— id: 39165, year: 2003, vol: 63, page: 3524, stat: Journal Article,

Epigenetics and the environment
Sutherland, Jessica E; Costa, Max
2003 Mar;983:151-160, Annals of the New York Academy of Sciences
DNA methylation and histone modification promote changes in chromatin structure that may affect gene expression in a heritable manner without directly altering the genome. As such, these phenomena are considered to be epigenetic in nature and are believed to contribute to the normal processes of human development but also to aberrant disease states such as cancer. Epigenetic processes probably contribute mechanistically to toxicant-induced changes in gene expression and cancer. Nickel is a potent human carcinogen that has been shown to alter DNA methylation patterns and affect histone acetylation status. Both of these changes are associated with the proximity of the affected regions to heterochromatin. The two processes probably occur in concert in mammalian cells. However, in yeast cells, DNA methylation is absent, and nickel is capable of regulating gene expression through changes in acetylation of the lysine residues in the N terminal tail of histone H4. Arsenic is another important environmental carcinogen, and it is methylated during its metabolism. Hence, it has been proposed that arsenic metabolism may deplete intracellular methyl group stores and thereby lead to changes in DNA methylation that may be involved in carcinogenesis. However, the data concerning DNA methylation changes following arsenic exposure are equivocal, leading researchers to propose that DNA hypo- and hypermethylation are both important in the development of arsenic-induced cancers. Heightened awareness by toxicologists of the importance of epigenetics in normal human development and in carcinogenesis should lead to the identification of other toxicants that manifest their effects, at least in part, via epigenetic mechanisms
— id: 134098, year: 2003, vol: 983, page: 151, stat: Journal Article,

Analysis of chromatin structure in nickel silenced G12 Chinese hamster cells
Yan, Y; Kluz, T; Zhang, P; Chen, H; Costa, M
2003 MAR ;72(1):216-216, Toxicological sciences
— id: 38497, year: 2003, vol: 72, page: 216, stat: Journal Article,

Analysis of specific lysine histone H3 and H4 acetylation and methylation status in clones of cells with a gene silenced by nickel exposure
Yan, Yan; Kluz, Thomas; Zhang, Ping; Chen, Hao-bin; Costa, Max
2003 Aug 1;190(3):272-277, Toxicology & applied pharmacology
We have previously reported that the gpt transgene in G12 Chinese hamster cells could be silenced by water-insoluble nickel compounds nickel sulfide (NiS) or nickel subsulfide (Ni(3)S(2)) and showed that the transgene was silenced by de novo DNA methylation and chromatin condensation. To further understand the nature of this silencing, we used the chromatin immunoprecipitation assay to elucidate the chromatin structure in nickel-induced silenced G12 clones. We also analyzed the effects of the DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) and a histone deacetylase inhibitor trichostatin A (TSA) on histone H3 and H4 acetylation and gpt gene expression in selected nickel-silenced clones. We observed that both histone H3 and H4 were hypoacetylated and a methyl DNA-binding protein MeCP2 was targeted to the gpt gene locus, resulting in a localized inactive chromatin configuration in nickel-silenced cell clones. The histone H3K9 was also found methylated in three of four nickel- silenced cell clones, whereas the histone H3K9 was deacetylated in all four cell clones, indicating that the H3K9 methylation was involved in nickel-induced gene silencing. The acetylation of the gpt gene could be increased by a combination of 5-AzaC and TSA treatment, but not by either 5-AzaC or TSA alone. The gpt transcript was studied by either Northern blot or by semiquantitative RT-PCR following treatment of the silenced clones with TSA or 5-AzaC. An increase in gpt mRNA could be detected by RT-PCR in the clones that regained acetylation of H3 and H4. These data show that gene silencing induced by nickel in the gpt transgenic cell line involved a loss of histone acetylation and an activation of histone methylation. Both H4 and H3 histone acetylation were lost in the silenced clones and these clones exhibited an increase in the methylation of the lysine 9 in histone H3
— id: 39116, year: 2003, vol: 190, page: 272, stat: Journal Article,

Downregulation of a zinc finger protein-ZFP61 by Ni in mouse cells in vitro and in vivo
Zhang, P; Costa, M
2003 MAR ;72(1):234-234, Toxicological sciences
— id: 38501, year: 2003, vol: 72, page: 234, stat: Journal Article,

Chromate induces the HGPRT gene silencing by DNA methylation: A new epigenetic mechanism for chromate carcinogenesis
Zhang, Q; Kluz, T; Salnikow, K; Costa, M
2003 MAR ;72(1):234-234, Toxicological sciences
— id: 38499, year: 2003, vol: 72, page: 234, stat: Journal Article,

Inhibition and reversal of nickel-induced transformation by the histone deacetylase inhibitor trichostatin A
Zhang, Qunwei; Salnikow, Konstantin; Kluz, Thomas; Chen, Lung Chi; Su, Wei Cheng; Costa, Max
2003 Nov 1;192(3):201-211, Toxicology & applied pharmacology
The carcinogenic process initiated by nongenotoxic carcinogens involves modulation of gene expression. Nickel compounds have low mutagenic activity, but are highly carcinogenic. In vitro both mouse and human cells can be efficiently transformed by soluble and insoluble nickel compounds to anchorage-independent growth. Because previous studies have shown that carcinogenic nickel compounds silence genes by inhibiting histone acetylation and enhancing DNA methylation, we investigated the effect of enhancing histone acetylation on cell transformation. The exposure of nickel-transformed cells to the histone deacetylase inhibitor trichostatin A (TSA) resulted in the appearance of significant number of revertants measured by their inability to grow in soft agar. Using the Affymetrix GeneChip we found that the level of expression of a significant number of genes was changed (suppressed or upregulated) in nickel-transformed clones but returned to a normal level in revertants obtained following TSA treatment. Moreover, we found that treatment of cells with TSA inhibited the ability of nickel to transform mouse PW cells to anchorage-independent growth. Treatment with TSA also inhibited the ability of nickel to transform human HOS cells, although to a lesser extent. In contrast, treatment with TSA was not able to revert established cancer cell lines as readily as the nickel-transformed cells. These data indicated that modulation of gene expression is important for nickel-induced transformation
— id: 44775, year: 2003, vol: 192, page: 201, stat: Journal Article,

Down regulation of a serine proteinase inhibitor by Ni and Co is dependent on hypoxia inducible factor signalling
Zhao, J; Yan, Y; Costa, M
2003 MAR ;72(1):234-234, Toxicological sciences
— id: 38502, year: 2003, vol: 72, page: 234, stat: Journal Article,

Molecular mechanisms of nickel carcinogenesis
Cangul, H; Broday, L; Salnikow, K; Sutherland, J; Peng, W; Zhang, Q; Poltaratsky, V; Yee, H; Zoroddu, M A; Costa, M
2002 Feb 28;127(1-3):69-75, Toxicology letters
Humans are exposed to carcinogenic nickel (Ni) compounds both occupationally and environmentally. In this paper, molecular mechanisms of nickel carcinogenesis are considered from the point-of-view of the uptake of nickel sulfide particles in cells, their dissolution and their effects on heterochromatin. Molecular mechanisms by which nickel induces gene silencing, DNA hypermethylation and inhibition of histone acetylation, will be discussed
— id: 34648, year: 2002, vol: 127, page: 69, stat: Journal Article,

Enhanced expression of a novel protein in human cancer cells: a potential aid to cancer diagnosis
Cangul, H; Salnikow, K; Yee, H; Zagzag, D; Commes, T; Costa, M
2002 ;18(2):87-96, Cell biology & toxicology
Cap43 is a protein whose RNA is induced under conditions of severe hypoxia or prolonged elevations of intracellular calcium. Cap43 protein is expressed at low levels in normal tissues; however, in a variety of cancers, including lung, brain, melanoma, liver, prostate, breast, and renal cancers, Cap43 protein is overexpressed in cancer cells. The low level of expression of Cap43 in some normal tissues compared to their cancerous counterparts combined with the high stability of Cap43 protein and mRNA makes the Cap43 gene a new, important cancer marker. We hypothesize that the mechanism of Cap43 overexpression in cancer cells involves a state of hypoxia characteristic of cancer cells where the Cap43 protein becomes a signature for this hypoxic state
— id: 34649, year: 2002, vol: 18, page: 87, stat: Journal Article,

Enhanced overexpression of an HIF-1/hypoxia-related protein in cancer cells
Cangul, Hakan; Salnikow, Konstantin; Yee, Herman; Zagzag, David; Commes, Therese; Costa, Max
2002 Oct;110 Suppl 5(2):783-788, Environmental health perspectives
Cap43 is a protein whose RNA is induced under conditions of severe hypoxia or prolonged elevations of intracellular calcium. Additionally, Ni and Co also induce Cap43 because they produce a state of hypoxia in cells. Cap43 protein is expressed at low levels in normal tissues; however, in a variety of cancers, including lung, brain, melanoma, liver, prostate, breast, and renal cancers, Cap43 protein is overexpressed in cancer cells. The low level of expression of Cap43 in some normal tissues compared with their cancerous counterparts, combined with the high stability of Cap43 protein and mRNA, makes the Cap43 gene a new, important cancer marker. We hypothesize that the mechanism of Cap43 overexpression in cancer cells involves a state of hypoxia characteristic of cancer cells where the Cap43 protein becomes a signature for this hypoxic state
— id: 34644, year: 2002, vol: 110 Suppl 5, page: 783, stat: Journal Article,

Use of XAS for the elucidation of metal structure and function: applications to nickel biochemistry, molecular toxicology, and carcinogenesis
Carrington, Paul E; Al-Mjeni, Faizah; Zoroddu, Maria A; Costa, Max; Maroney, Michael J
2002 Oct;110 Suppl 5:705-708, Environmental health perspectives
Nickel has been shown to be an essential trace element involved in the metabolism of several species of bacteria, archea, and plants. In these organisms, nickel is involved in enzymes that catalyze both non-redox (e.g., urease, glyoxalase I) and redox (e.g., hydrogenase, carbon monoxide dehydrogenase, superoxide dismutase) reactions, and proteins involved in the transport, storage, metallocenter assembly, and regulation of nickel concentration have evolved. Studies of structure/function relationships in nickel biochemistry reveal that cysteine ligands are used to stabilize the Ni(III/II) redox couple. Certain nickel compounds have also been shown to be potent human carcinogens. A likely target for carcinogenic nickel is nuclear histone proteins. Here we present X-ray absorption spectroscopic studies of a model Ni peptide designed to help characterize the structure of the nickel complexes formed with histones and place them in the context of nickel structure/function relationships, to gain insights into the molecular mechanism of nickel carcinogenesis
— id: 134099, year: 2002, vol: 110 Suppl 5, page: 705, stat: Journal Article,

Molecular responses of mammalian cells to nickel and chromate exposure
Costa M; Salnikow K; Sutherland JE; Klutz T; Peng W; Tang M-S
Biomarkers of environmentally associated disease : technologies, concepts, and perspectives Boca Raton FL : Lewis Publishers, 2002,
— id: 4434, year: 2002, vol: , page: 261, stat: Chapter,

Preface - Third International Meeting on the Molecular Mechanisms of Metal Toxicity and Carcinogenicity
Costa, M; Zoroddu, MA; Suk, WA; Thompson, C; Rossman, T
2002 OCT ;110(3):687-687, Environmental health perspectives
— id: 33048, year: 2002, vol: 110, page: 687, stat: Journal Article,

Molecular mechanisms of nickel carcinogenesis
Costa, Max
2002 Jun;383(6):961-967, Biological chemistry
A brief review of the molecular mechanisms of nickel carcinogenesis is presented. Molecular mechanisms of nickel carcinogenesis are considered from the point-of-view of nickel-induced gene silencing by DNA hypermethylation in mammalian cells and by its ability to inhibit histone acetylation. Model systems designed to study the molecular mechanism of gene silencing are discussed
— id: 39406, year: 2002, vol: 383, page: 961, stat: Journal Article,

The role of oxidative stress in nickel and chromate genotoxicity
Costa, Max; Salnikow, Konstantin; Sutherland, Jessica E; Broday, Limor; Peng, Wu; Zhang, Qunwei; Kluz, Thomas
2002 May-Jun;234-235(1-2):265-275, Molecular & cellular biochemistry
Some general principles regarding oxidative stress and molecular responses to toxic metals are presented in this manuscript. The remainder of the manuscript, however, will focus on the role of oxidative stress in particulate nickel-induced genetic damage and mutations. The phagocytosis of particulate nickel compounds and the dissolution of the particles inside the cell and the resulting oxidative stress produced in the nucleus is a key component of the nickel carcinogenic mechanism. The crosslinking of amino acids to DNA by nickel that does not involve direct participation of nickel in a ternary complex but nickel-induced oxidative stress will be discussed as well. The selective ability of particulate nickel compounds to silence the expression of genes located near heterochromatin and the effect of vitamin E on the genotoxicity and mutations induced by particulate and soluble nickel compounds will also be discussed. Particulate nickel compounds have been shown to produce more oxidative stress than water-soluble nickel compounds. In addition to nickel, the role of oxidative stress in chromate-induced genotoxicity will also be discussed with particular attention directed to the effects of vitamin E on mutations and chromosomal aberrations inducedby chromate
— id: 34647, year: 2002, vol: 234-235, page: 265, stat: Journal Article,

Differential role of hydrogen peroxide in UV-induced signal transduction
Ding, Min; Li, Jingxia; Leonard, Stephen S; Shi, Xianglin; Costa, Max; Castranova, Vincent; Vallyathan, Val; Huang, Chuanshu
2002 May-Jun;234-235(1-2):81-90, Molecular & cellular biochemistry
The present study investigated the differential requirement of ROS in UV-induced activation of these pathways. Exposure of the mouse epidermal C141 cells to UV radiation led to generation of ROS as measured by electron spin resonance (ESR) and by H2O2 and O2. fluorescence staining assay. Treatment of cells with UV radiation or H2O2 also markedly activated Erks, JNKs, p38 kinase and led to increases in phosphorylation of Akt and p70(S6k) in mouse epidermal JB6 cells. The scavenging of UV-generated H2O2 by N-acety-L-cyteine (NAC, a general antioxidant) or catalase (a specific H2O2 inhibitor) inhibited UV-induced activation of JNKs, p38 kinase, Akt and p70(S6k), while it did not show any inhibitory effects on Erks activation. Further, pretreatment of cells with sodium formate (an .OH radical scavenger) or superoxide dismutase (O2-. radical scavenger) did not inhibit any of these pathways. These results demonstrate that H2O2 generation is required for UV-induced phosphorylation of Akt and p70(S6k), and involved in activation of JNKs and p38 kinase, but not Erks
— id: 38402, year: 2002, vol: 234-235, page: 81, stat: Journal Article,

Ultraviolet-induced phosphorylation of p70(S6K) at Thr(389) and Thr(421)/Ser(424) involves hydrogen peroxide and mammalian target of rapamycin but not Akt and atypical protein kinase C
Huang, Chuanshu; Li, Jingxia; Ke, Qingdong; Leonard, Stephen S; Jiang, Bing-Hua; Zhong, Xiao-Song; Costa, Max; Castranova, Vincent; Shi, Xianglin
2002 Oct 15;62(20):5689-5697, Cancer research
The p70 S6 kinase (p70(S6k)) is a Ser/Thr kinase that plays an important role in cell growth, transformation, and the transition of the cell cycle in mammalian cells. Because UV radiation has been reported to induce activation of p70(S6k), which is believed to play some role in the carcinogenic effects of sun exposure, the present study investigated the signaling pathways involved in this activation induced by UV radiation in mouse epidermal JB6 Cl41 cells. Exposure of cells to UV radiation led to marked increases in p70(S6k) activity and phosphorylation at Thr(389) and Thr(421)/Ser(424). UV radiation also generated reactive oxygen species as measured by electron spin resonance and by H(2)O(2) and O( minus sign, dot below )(2) fluorescence staining assays in JB6 Cl 41 cells. The scavenging of UV-generated H(2)O(2) by N-acety-L-cyteine (a general antioxidant) or catalase (a specific H(2)O(2) scavenger) inhibited p70(S6k) phosphorylation at Thr(389) and Thr(421)/Ser(424), whereas pretreatment of cells with sodium formate (an.OH radical scavenger) or superoxide dismutase (an O( minus sign, dot below )(2) radical scavenger) did not show any inhibitory effects. Importantly, UV-induced increases in p70(S6k) phosphorylation at Thr(389) and Thr(421)/Ser(424) were dramatically inhibited by pretreatment of cells with rapamycin, LY294002, or PD98059, whereas overexpression of dominant-negative mutants of PKClambda/iota and Akt1 did not inhibit p70(S6k) phosphorylation at Thr(389) and Thr(421)/Ser(424). These results demonstrated that H(2)O(2), phosphatidylinositol 3-kinase, and mammalian target of rapamycin were important players for UV-induced p70(S6k) phosphorylation at Thr(389) and Thr(421)/Ser(424), whereas Akt and atypical protein kinase C were not involved in this activation. The role of H(2)O(2) in p70(S6k) phosphorylation at Thr(389) and Thr(421)/Ser(424) was further supported by the findings that treatment of cells with H(2)O(2) also caused p70(S6k) phosphorylation at Thr(389) and Thr(421)/Ser(424)
— id: 38401, year: 2002, vol: 62, page: 5689, stat: Journal Article,

Activation of nuclear factor-kappaB and not activator protein-1 in cellular response to nickel compounds
Huang, Yi; Davidson, Gerard; Li, Jingxia; Yan, Yan; Chen, Fei; Costa, Max; Chen, Lung Chi; Huang, Chuanshu
2002 Oct;110 Suppl 5(4):835-839, Environmental health perspectives
The predominant exposure route for nickel compounds is by inhalation, and several studies have indicated the correlation between nickel exposure and respiratory cancers. The tumor-promoting effects of nickel compounds are thought to be associated with their transactivation of transcription factors. We have investigated the possible activation of activator protein-1 (AP-1) and nuclear factor KB (NF-kappaB) in mouse C141 epidermal cells and fibroblasts 3T3 and B82, and human bronchoepithelial BEAS-2B cells in response to nickel compound exposure. Our results show that NF-kappaB activity is induced by nickel exposure in 3T3 and BEAS-2B cells. Conversely, similar nickel treatment of these cells did not induce AP-1 activity, suggesting that nickel tumorigenesis occurs through NF-kappaB and not AP-1. We also investigated the role of NF-kappaB in the induction of Cap43 by nickel compounds using dominant negative mutant Ikappabeta kinase b-KM BEAS-2B transfectants
— id: 38400, year: 2002, vol: 110 Suppl 5, page: 835, stat: Journal Article,

The role of hypoxia-inducible signaling pathway in nickel carcinogenesis
Salnikow, Konstantin; Davidson, Todd; Costa, Max
2002 Oct;110 Suppl 5(2):831-834, Environmental health perspectives
Using human and rodent cells in vitro, we characterized a hypoxia-inducible signaling pathway as one of the pathways affected by carcinogenic nickel compounds. Acute exposure to nickel activates hypoxia-inducible transcription factor-1 (HIF-1), which strongly induces hypoxia-inducible genes, including the recently discovered tumor marker Cap43. This gene has been cloned based on its nickel inducibility and was found to be highly inducible by hypoxia. To identify other HIF-1-dependent/independent nickel-inducible genes, we used cells obtained from HIF-1 alpha null mouse embryos and analyzed gene expression changes using the microarray technique. We found that genes coding for glycolytic enzymes, known to be regulated by HIF-1, were also induced in nickel-exposed cells. In addition, we identified a number of new genes highly induced by nickel in an HIF-dependent manner. Elevated HIF-1 activity after acute nickel exposure might be selectively advantageous because nickel-transformed rodent and human cells possess increased HIF-1 transcriptional activity. Hypoxia plays an important role in tumor progression. It selects for cells with enhanced glycolytic activity, causing production of large amounts of lactic acid, one of the most common features of tumor cells (Warburg effect). Here, we hypothesize that exposure to nickel activates the hypoxia-inducible pathway and facilitates selection of cells with increased transcriptional activity of hypoxia-inducible genes, which may be important in the nickel-induced carcinogenic process
— id: 34645, year: 2002, vol: 110 Suppl 5, page: 831, stat: Journal Article,

The regulation of hypoxic genes by calcium involves c-Jun/AP-1, which cooperates with hypoxia-inducible factor 1 in response to hypoxia
Salnikow, Konstantin; Kluz, Thomas; Costa, Max; Piquemal, David; Demidenko, Zoya N; Xie, Keping; Blagosklonny, Mikhail V
2002 Mar;22(6):1734-1741, Molecular & cellular biology
Hypoxia causes the accumulation of the transcription factor hypoxia-inducible factor 1 (HIF-1), culminating in the expression of hypoxia-inducible genes such as those for vascular endothelial growth factor (VEGF) and NDRG-1/Cap43. Previously, we have demonstrated that intracellular calcium (Ca(2+)) is required for the expression of hypoxia-inducible genes. Here we found that, unlike with hypoxia or hypoxia-mimicking conditions, the elevation of intracellular Ca(2+) neither induced the HIF-1alpha protein nor stimulated HIF-1-dependent transcription. Furthermore, the elevation of intracellular Ca(2+) induced NDRG-1/Cap43 mRNA in HIF-1alpha-deficient cells. It also increased levels of c-Jun protein, causing its phosphorylation. The protein kinase inhibitor K252a abolished c-Jun induction and activator protein 1 (AP-1)-dependent reporter expression caused by Ca(2+) ionophore or hypoxia. K252a also significantly decreased hypoxia-induced VEGF and NDRG-1/Cap43 gene expression in both human and mouse cells. Using a set of deletion VEGF-Luc promoter constructs, we found that both HIF-1 and two AP-1 sites contribute to hypoxia-mediated induction of transcription. In contrast, only AP-1 sites contributed to Ca(2+)-mediated VEGF-Luc induction. A dominant-negative AP-1 prevented Ca(2+)-dependent transcription and partially impaired hypoxia-mediated transcription. In addition, dominant-negative AP-1 diminished the expression of the NDRG-1/Cap43 gene following hypoxia. We conclude that during hypoxia, an increase in intracellular Ca(2+) activates a HIF-1-independent signaling pathway that involves AP-1-dependent transcription. Cooperation between the HIF-1 and AP-1 pathways allows fine regulation of gene expression during hypoxia
— id: 26987, year: 2002, vol: 22, page: 1734, stat: Journal Article,

DNA methylation and gene silencing
Sutherland JE; Costa M
Comprehensive toxicology : volume 14, Cellular and molecular toxicology New York : Elsevier, 2002,
— id: 4432, year: 2002, vol: , page: 299, stat: Chapter,

Nickel
Sutherland JE; Costa M
Heavy metals in the environment New York : Dekker, 2002,
— id: 4433, year: 2002, vol: , page: ?, stat: Chapter,

Comparison of the cytotoxicity, cellular uptake, and DNA-protein crosslinks induced by potassium chromate in lymphoblast cell lines derived from three different individuals
Zhang, Qunwei; Kluz, Thomas; Salnikow, Konstantin; Costa, Max
2002 Apr;86(1):11-22, Biological trace element research
We are trying to understand individual differences in susceptibility to chromate toxicity by comparing three different lymphoblastic cell lines derived from three different individuals. We have compared the uptake of CrO4(2-), the release of LDH from cells, the proliferation ability of the cells, and the DNA-protein crosslinks in these lymphoblastic cell lines exposed to chromate. We report here that one lymphoblastic cell line, GM0922B, appears to be considerably less sensitive than the other two cells lines to the cytotoxic effects of hexavalent chromium. The diminished sensitivity is almost twofold and can be accounted for by the decreased uptake of hexavalent chromium, which results in less lactate dehydrogenase release, and greater tolerance to chromate inhibition of cell proliferation and less DNA-protein crosslinking. This lower uptake of chromate combined with interindividual differences in extracellular Cr(VI) reducing capacity are probably the two most important determinants of genetic susceptibility to chromate toxicity
— id: 34650, year: 2002, vol: 86, page: 11, stat: Journal Article,

Ni(II) and Cu(II) binding with a 14-aminoacid sequence of Cap43 protein, TRSRSHTSEGTRSR (vol 84, pg 47, 2001)
Zoroddu, MA; Kowalik-Jankowska, T; Kozlowski, H; Salnikow, K; Costa, M
2002 AUG 15 ;91(2):421-421, Journal of inorganic biochemistry
— id: 55290, year: 2002, vol: 91, page: 421, stat: Journal Article,

The binding of Ni(II) and Cu(II) with the N-terminal tail of the histone H4
Zoroddu, MA; Peana, M; Kowalik-Jankowska, T; Kozlowski, H; Costa, M
2002 Jan 20;18(3):458-465, Journal of the Chemical Society. Dalton transactions
We have analyzed, for Ni(II) and Cu(II) binding, the sequence of the N-terminal tail of the histone H4, the 22-amino acid peptide Ac-SGRGKGGKGLGKGGAKRHRKVL-Am and, in addition, the 7- and 11-amino acid peptides Ac-AK(Ac)RHRK(Ac)V-Am, Ac- GK(Ac)GGAK(Ac)RHRK(Ac)V-Am where all side chains of lysines were blocked by acetylation. Potentiometric and spectroscopic studies (UV-Vis, CD, EPR, NMR) showed that histidine 18 acted as an anchoring binding site for metal ions in all the peptides investigated. The stability constants of the 3N and 4N complexes are higher than those obtained for simple peptides with glycine instead of arginine and lysine residues in the metal binding site. The coordination was not significantly affected by the acetylation of lysines. The behavior of the 'tail' suggested a possible bent structure with organized side- chain orientation promoted by Ni(II)
— id: 28193, year: 2002, vol: 18, page: 458, stat: Journal Article,

Molecular mechanisms in nickel carcinogenesis: modeling Ni(II) binding site in histone H4
Zoroddu, Maria Antonietta; Schinocca, Laura; Kowalik-Jankowska, Teresa; Kozlowski, Henryk; Salnikow, Konstantin; Costa, Max
2002 Oct;110 Suppl 5(2):719-723, Environmental health perspectives
Ni(II) compounds are well known as human carcinogens, though the molecular events which are responsible for this are not yet fully understood. It has been proposed that the binding of N(II) ions within the cell nucleus is a crucial element in the mechanism of carcinogenesis. The most abundant proteins in the cell nucleus are histones, and this makes them the prime candidates for this role. This article is a review of our recent studies of histone H4 models of Ni(II) binding. We analyzed the sequence of the N-terminal tail of the histone H4, Ac-SGRGKGGKGLGKGGAKRH(18)RKVL-Am, for Ni(II) binding. This site has been proposed mainly because of the potent inhibitory effect of Ni(II) on the acetylation of lysine residues near the histidine H(18), and also because of the accessibility of the H4 tail in the histone octamer. Combined potentiometric and spectroscopic studies showed that the histidine 18 acted as an anchoring binding site for metal ions in the peptide investigated. Comparison with the results for Cu(II) binding are also reported. The results allowed us to propose that the binding of Ni(II) is able to promote a secondary structure with organized side-chain orientation on the N-terminal tail of histone H4
— id: 34646, year: 2002, vol: 110 Suppl 5, page: 719, stat: Journal Article,

Molecular biology of nickel carcinogenesis
Costa M; Sutherland JE; Peng W; Salnikow K; Broday L; Kluz T
2001 Jun;222(1-2):205-211, Molecular & cellular biochemistry
A review of the molecular mechanisms of nickel carcinogenesis has been compiled. This work is based upon approximately 20 years of research conducted in my laboratory. Molecular mechanisms of nickel carcinogenesis are considered from the point-of-view of the uptake of nickel, both soluble and insoluble particles in cells, its dissolution and its effects on heterochromatin. Molecular mechanisms by which nickel induces gene silencing in cells by DNA hypermethylation in mammalian cells and by inhibiting histone acetylation in yeast cells are also discussed
— id: 26988, year: 2001, vol: 222, page: 205, stat: Journal Article,

Hydrogen peroxide mediates activation of nuclear factor of activated T cells (NFAT) by nickel subsulfide
Huang C; Li J; Costa M; Zhang Z; Leonard SS; Castranova V; Vallyathan V; Ju G; Shi X
2001 Nov 15;61(22):8051-8057, Cancer research
Nickel compounds induce cell transformation in cell culture models and tumor formation in experimental animals. However, the molecular mechanisms by which nickel compounds induce tumors are not yet well understood. The present study found that exposure of cells to either Ni(3)S(2) or NiCl(2) could result in specific transactivation of nuclear factor of activated T cells (NFAT), although it did not show any activation of p53 or AP-1. Furthermore, nickel compounds were also able to cause generation of reactive oxygen species (ROS). The scavenging of nickel-induced H(2)O(2) with N-acety-L-cyteine (a general antioxidant) or catalase, or the chelation of nickel with deferoxamine, resulted in inhibition of NFAT activation. In contrast, pretreatment of cells with sodium formate (an .OH radical scavenger) or superoxide dismutase (an O(-.)(2) radical scavenger) did not show any inhibitory effects. These results demonstrate that nickel compounds are able to induce NFAT activation, and that the mechanism of NFAT activation seems to be mediated by the generation of H(2)O(2) by these metal compounds. This study should help us understand the signal transduction pathways involved in carcinogenic effects of these nickel compounds
— id: 26560, year: 2001, vol: 61, page: 8051, stat: Journal Article,

Transactivation of RARE and GRE in the cellular response to arsenic
Huang C; Li J; Ding M; Costa M; Castranova V; Vallyathan V; Ju G; Shi X
2001 Jun;222(1-2):119-125, Molecular & cellular biochemistry
Arsenic compounds are a somewhat unique class of metals, which have been considered as both carcinogens and chemotherapeutic agents for cancers. Tumor promotion effects of arsenic are believed to be associated with its transactivational activities on transcription factors, such as AP-1 and NFkappaB, while the induction of cell apoptosis and differentiation by arsenic is considered to be a mechanism for the chemotherapeutic effects of arsenic. Here, we found that exposure of cells to arsenite and arsenate leads to transactivation of retinoic acid response elements (RARE) and glucocorticoid response elements (GRE) in mouse epidermal JB6 cells. These inductions occur in a time-dependent manner. Furthermore, induction of RARE activity by arsenic was synergistically enhanced by co-treatment of cells with retinoic acid, while GRE activation by arsenic was not affected by combined treatment of cells with fluocinolone acetonide (FA). In consideration of the important role of RARE and GRE in induction of cell differentiation, we speculate that transactivation of RARE and GRE by arsenic may be involved in its induction of cell differentiation and anti-cancer activities in addition to its induction of apoptosis
— id: 38405, year: 2001, vol: 222, page: 119, stat: Journal Article,

Arsenic-induced NFkappaB transactivation through Erks- and JNKs-dependent pathways in mouse epidermal JB6 cells
Huang C; Li J; Ding M; Wang L; Shi X; Castranova V; Vallyathan V; Ju G; Costa M
2001 Jun;222(1-2):29-34, Molecular & cellular biochemistry
Tumor promoting effects of arsenic are believed to be associated with its transactivation activity on transcription factors, such as AP-1 and NFkappaB. However, the results from different groups studying the effects of arsenic on NFkappaB activation are contradictory in different cell models. Since arsenic is a strong skin carcinogen, we have investigated the activation of NFkappaB by arsenic in a mouse skin epidermal cell line, JB6 cells. Exposure of cells to arsenite or arsenate led to NFkappaB transactivation in mouse epidermal JB6 NFkappaB-luciferase reporter stable transfectants, C141 NFkappaB mass1. This induction of NFkappaB activity by arsenic was dose- and time-dependent. The transactivation of NFkappaB by arsenic appeared to be through activation of Erks and JNKs pathways because increased NFkappaB activity by arsenic could be dramatically inhibited by either pre-treatment of cells with PD98059 or overexpression of dominant negative JNK1. That Erks activation is required for arsenic-induced NFkappaB transactivation was further supported by the findings that arsenic-induced NFkappaB transactivation was impaired in JB6 30.7b cells, which were deficient in Erks
— id: 38403, year: 2001, vol: 222, page: 29, stat: Journal Article,

Involvement of Erks activation in cadmium-induced AP-1 transactivation in vitro and in vivo
Huang C; Zhang Q; Li J; Shi X; Castranova V; Ju G; Costa M; Dong Z
2001 Jun;222(1-2):141-147, Molecular & cellular biochemistry
Cadmium is a potent and effective carcinogen in rodents and has recently been accepted by IARC (International Agency for Research on Cancer) as a category I carcinogen. Cadmium-induced up-regulation of intracellular signaling pathways leading to increased mitogenesis is thought to be a major mechanism for the carcinogenic activity following chronic cadmium exposure. In the present study, we found that exposure of cells to cadmium induced significant activation of AP-1 and all three members of the MAP kinase family in mouse epidermal JB6 cells. The induction of AP-1 activity by cadmium appears to involve activation of Erks, since the induction of AP-1 activity by cadmium was blocked by pretreatment of cells with PD98058. Interestingly, the induction of AP-1 by cadmium was greatly enhanced by the chemical tumor promoter, TPA and the growth factor EGF, but not by ultraviolet C radiation. In vivo studies demonstrated that cadmium could also induce transactivation of AP-1 in AP-1-luciferase report transgenic mice. Considering the role of AP-1 activation in tumor promotion, the results presented in this study provide a possible molecular mechanism for cadmium-induced carcinogenesis
— id: 38404, year: 2001, vol: 222, page: 141, stat: Journal Article,

Mutagenesis assays in Mammalian cells
Klein, C B; Broday, L; Costa, M
2001 May;Chapter 3:Unit3.3-Unit3.3, Current protocols in toxicology
Mutagenesis assays in mammalian cells are frequently used to complement bacterial mutagenesis assays. This unit describes a mutagenesis assay using either Chinese hamster V79 cells or V79-derivative gpt transgenic cell line to assess the effects of chemical agents on mammalian cells
— id: 113815, year: 2001, vol: Chapter 3, page: Unit3.3, stat: Journal Article,

The histone deacetylase inhibitor trichostatin A reduces nickel-induced gene silencing in yeast and mammalian cells
Sutherland JE; Peng W; Zhang Q; Costa M
2001 Aug 8;479(1-2):225-233, Mutation research
We have previously reported that nickel (Ni)-silenced expression of the URA3 gene in yeast (Saccharomyces cerevisiae) and gpt transgene in G12 Chinese hamster cells. In both cases, close proximity to a heterochromatic region was required for gene silencing. Yeast exposed to Ni exhibited reduced acetylation of the lysine residues in the N-terminal tail of histone H4. Ni-induced silencing of the gpt gene in mammalian cells involved hypermethylation of promoter region DNA. Yeast do not employ DNA methylation to silence gene expression. To determine if histone deacetylation participates in Ni-induced silencing of the URA3 and gpt genes, we exposed yeast and G12 hamster cells to the histone deacetylase inhibitor trichostatin A (TSA) prior to and concurrently with Ni. Treatment of yeast cells with 0.2-0.6mM NiCl(2) resulted in reduced expression of the URA3 gene as assessed by increased resistance to 1g/l 5-fluorotic acid (5-FOA). This effect was lessened when yeast were pre-treated with 50 microg TSA/ml. Similarly, treatment of G12 cells with 5 ng/ml TSA during and after exposure to 0.3 microg Ni(3)S(2)/cm(2) reduced silencing of the gpt gene as gauged by resistance to 10 microg/ml 6-thioguanine (6-TG). The ability of TSA alone and in combination with the DNA-demethylating agent (5-AzaC) to reactivate the gpt gene in Ni-silenced variants was also assessed. Although treatment with 100 ng/ml TSA for 48 h was partially effective in reactivating the gpt gene, treatment with 5 microM 5-AzaC was more efficacious. The greatest gpt gene reversion frequencies were observed following a sequential 5-AzaC/TSA treatment. Taken all together, our data from mammalian cells suggests that both DNA methylation and histone deacetylation participate in Ni-induced silencing of the gpt gene with DNA hypermethylation playing the more dominant role in maintaining the silenced state
— id: 26632, year: 2001, vol: 479, page: 225, stat: Journal Article,

Ni(II) and Cu(II) binding with a 14-aminoacid sequence of Cap43 protein, TRSRSHTSEGTRSR
Zoroddu MA; Kowalik-Jankowska T; Kozlowski H; Salnikow K; Costa M
2001 Mar;84(1-2):47-54, Journal of inorganic biochemistry
The tetradecapeptide containing the 10 aminoacid repeated sequence on the C-terminus of the Ni(II)-induced Cap43 protein, was analyzed for Ni(II) and Cu(II) binding. A combined pH-metric and spectroscopic UV-VIS, EPR, CD and NMR study of Ni(II) and Cu(II) binding to the blocked CH3CO-Thr-Arg-Ser-Arg-Ser-His-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-NH2 (Ac-TRSRSHTSEGTRSR-Am) peptide, modeling a part of the C-terminal sequence of the Cap43 protein, revealed the formation of octahedral complexes involving imidazole nitrogen of histidine, at pH 5.5 and pH 7 for Cu(II) and Ni(II), respectively; a major square planar 4N-Ni(II) complex (about 100% at pH 9, log K* = -28.16) involving imidazole nitrogen of histidine and three deprotonated amide nitrogens of the backbone of the peptide was revealed; a 3N-Cu(II) complex (maximum about 70% at pH 7, log K*=-13.91) and a series of 4N-Cu(II) complexes starting at pH 5.5 (maximum about 90% at pH 8.7, log K* = -21.39 for CuH(-3)L), were revealed. This work supports the existence of a metal binding site at the COOH-terminal part of the Cap43 peptide
— id: 34652, year: 2001, vol: 84, page: 47, stat: Journal Article,

Nickel compounds are novel inhibitors of histone H4 acetylation
Broday L; Peng W; Kuo MH; Salnikow K; Zoroddu M; Costa M
2000 Jan 15;60(2):238-241, Cancer research
Environmental factors influence carcinogenesis by interfering with a variety of cellular targets. Carcinogenic nickel compounds, although generally inactive in most gene mutation assays, induce chromosomal damage in heterochromatic regions and cause silencing of reporter genes when they are located near telomere or heterochromatin in either yeast or mammalian cells. We studied the effects of nickel on the lysine acetylation status of the NH2-terminal region of histone H4. At nontoxic levels, nickel decreased the levels of histone H4 acetylation in vivo in both yeast and mammalian cells, affecting only lysine 12 in mammalian cells and all of the four lysine residues in yeast. In yeast, lysine 12 and 16 were more greatly affected than lysine 5 and 8. Interestingly, a histidine Ni2+ anchoring site is found at position 18 from the NH2-terminal tail of H4. Nickel was also found to inhibit the acetylation of H4 in vitro using purified recombinant histone acetyltransferase. To our knowledge, this is the first agent shown to decrease histone H4 acetylation at nontoxic levels
— id: 11839, year: 2000, vol: 60, page: 238, stat: Journal Article,

Chromium
Cohen M; Costa M
Environmental toxicants : human exposures and their health effects New York : Wiley, 2000,
— id: 4430, year: 2000, vol: , page: 173, stat: Chapter,

Chromium
Costa M
Molecular biology and toxicology of metals New York : Taylor & Francis, 2000,
— id: 4429, year: 2000, vol: , page: 113, stat: Chapter,

Trace elements: alumninum, arsenic, cadmium, and nickel
Costa M
Environmental toxicants : human exposures and their health effects New York : Wiley, 2000,
— id: 4431, year: 2000, vol: , page: 811, stat: Chapter,

Transcriptional inactivation of genes by nickel compounds involves inhibition of histone H$ acetylation
Costa M; Salnikow K; Broday L; Peng W; Sutherland JE; Zoroddu M
2000 ;6:95-97, Metal ions in biology & medicine = Les ions metalliques en biologie et en medicine
— id: 72793, year: 2000, vol: 6, page: 95, stat: Journal Article,

Vanadate induces p53 transactivation through hydrogen peroxide and causes apoptosis
Huang C; Zhang Z; Ding M; Li J; Ye J; Leonard SS; Shen HM; Butterworth L; Lu Y; Costa M; Rojanasakul Y; Castranova V; Vallyathan V; Shi X
2000 Oct 20;275(42):32516-32522, Journal of biological chemistry
Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms controlling vanadate-induced adverse effects remain to be elucidated. The present study investigated the vanadate-induced p53 activation and involvement of reactive oxygen species (ROS) in p53 activation as well as the role of p53 in apoptosis induction by vanadate. Exposure of mouse epidermal JB6 cells to vanadate led to transactivation of p53 activity in a time- and dose-dependent manner. It also caused mitochondrial damage, apoptosis, and generated ROS. Scavenging of vanadate-induced H(2)O(2) by N-acetyl-l-cysteine (a general antioxidant) or catalase (a specific H(2)O(2) inhibitor), or the chelation of vanadate by deferoxamine, resulted in inhibition of p53 activation and cell mitochondrial damage. In contract, an increase in H(2)O(2) generation in response to superoxide dismutase or NADPH enhanced these effects caused by vanadate. Furthermore, vanadate-induced apoptosis occurred in cells expressing wild-type p53 (p53+/+) but was very weak in p53-deficient (p53-/-) cells. These results demonstrate that vanadate induces p53 activation mainly through H(2)O(2) generation, and this activation is required for vanadate-induced apoptosis
— id: 34185, year: 2000, vol: 275, page: 32516, stat: Journal Article,

Carcinogenic nickel induces genes involved with hypoxic stress
Salnikow K; Blagosklonny MV; Ryan H; Johnson R; Costa M
2000 Jan 1;60(1):38-41, Cancer research
Carcinogenic nickel compounds alter the program of gene expression in normal cells and induce a pattern of gene expression similar to that found in nickel-induced cancers. Here we have demonstrated that nickel exposure induced hypoxic signaling pathways by inducing hypoxia-inducible transcription factor-1 (HIF-1), which mediated the induction of genes required by cells to survive hypoxia. We also show that a new gene, Cap43, is dependent upon HIF-1 because only HIF-1-proficient cells induced Cap43 when exposed to either hypoxia or nickel. We also show that glyceraldehyde-3-phosphate dehydrogenase, a gene induced by hypoxia through HIF-1, was similar to Cap43 in that it required HIF-1-proficient cells to be induced by either nickel or hypoxia. These data demonstrate that nickel exposure turns on signaling for hypoxic stress, which may be important in its carcinogenesis
— id: 10355, year: 2000, vol: 60, page: 38, stat: Journal Article,

Epigenetic mechanisms of nickel carcinogenesis
Salnikow K; Costa M
2000 ;19(3):307-318, Journal of environmental pathology, toxicology & oncology
This article considers the mechanism of nickel carcinogenesis, focusing primarily on the epigenetic changes associated with exposure of cells to carcinogenic nickel compounds. We discuss the delivery of nickel in the cell and contrast the genetic and epigenetic changes that have occurred. Within the epigenetic effects, alteration in the levels of transcription factors, such as ATF-1, p53, HIF-1, HIF-1alpha, and NFkappaB, are considered. The relationship between nickel and calcium metabolism and the role it plays in nickel carcinogenesis is also considered, as are reactive oxygen species and the interactions of nickel with proteins. We discuss these epigenetic discussions in light of the effects that nickel has on inducing DNA methylation in cells. It is of interest that nickel induces both a variety of signaling pathways as well as genes that seem to be important for the survival of cancer cells. It is also interesting that the same genes induced or repressed by nickel are similarly overexpressed or not expressed in nickel-transformed cells. It is suggested that this may represent a selection process crucial to the nickel carcinogenesis process
— id: 10343, year: 2000, vol: 19, page: 307, stat: Journal Article,

Hyperinducibility of hypoxia-responsive genes without p53/p21-dependent checkpoint in aggressive prostate cancer
Salnikow K; Costa M; Figg WD; Blagosklonny MV
2000 Oct 15;60(20):5630-5634, Cancer research
Hypoxia limits tumor growth but selects for higher metastatic potential. We tested the functional activity of hypoxia-inducible factor-1 (HIF-1) in prostate cell lines ranging from normal epithelial cells (PrEC), hormone-dependent LNCaP, hormone-independent DU145, PC-3 to highly metastatic PC-3M cancer cell lines. We found that HIF-1-stimulated transcription was the lowest in PrEC and LNCaP cells and the highest in PC-3M cells. The induction by hypoxia of the HIF-1 dependent genes Cap43 and GAPDH was the highest in the most aggressive PC-3M cancer cells. Because these advanced prostate cancer cell lines have lost p53 function, this further shifts a balance from p53 to HIF-1 transcriptional regulation, and a high ratio of HIF-1-dependent:p53-dependent transcription was a marker of the advanced malignant phenotype. Transient transfection of HIF-1alpha expression vector induced transcription from p21 promoter construct in prostate cancer cell lines. Furthermore, hypoxia slightly induced p21 mRNA in these cells. However, neither expression of p21 nor hypoxia caused growth arrest in PC-3M cells. Therefore, high inducibility of HIF-1-dependent genes, loss of p53 functions with high ratio of HIF-1-dependent:p53-dependent transcription, and loss of sensitivity to p21 inhibition is a part of hypoxic phenotype associated with aggressive cancer behavior
— id: 15999, year: 2000, vol: 60, page: 5630, stat: Journal Article,

Identification of signaling pathways affected by nickel compounds
Salnikow K; Costa M; Klutz T; Zoroddu M
2000 ;6:98-100, Metal ions in biology & medicine = Les ions metalliques en biologie et en medicine
— id: 72794, year: 2000, vol: 6, page: 98, stat: Journal Article,

Carcinogenic metals induce hypoxia-inducible factor-stimulated transcription by reactive oxygen species-independent mechanism
Salnikow K; Su W; Blagosklonny MV; Costa M
2000 Jul 1;60(13):3375-3378, Cancer research
Nickel (Ni2+) and cobalt (Co2+) mimic hypoxia and were used as a tool to study the role of oxygen sensing and signaling cascades in the regulation of hypoxia-inducible gene expression. These metals can produce oxidative stress; therefore, it was conceivable that reactive oxygen species (ROS) may trigger signaling pathways resulting in the activation of the hypoxia-inducible factor (HIF)-1 transcription factor and up-regulation of hypoxia-related genes. We found that the exposure of A549 cells to Co2+ or Ni2+ produced oxidative stress, and although Co2+ was a more potent producer of ROS than Ni2+, both metals equally increased the expression of Cap43, a hypoxia-regulated gene. The coadministration of hydrogen peroxide with metals induced more ROS; however, this did not further increase the expression of Cap43 mRNA. The free radical scavenger 2-mercaptoethanol completely suppressed ROS generation by CoCl2 and NiCl2 but did not diminish the induced Cap43 gene expression. The activity of the HIF-1 transcription factor as assessed in transient transfection assays was stimulated by Ni2+, hypoxia, and desferrioxamine, but this activation was not diminished when oxidative stress was attenuated nor was HIF-dependent transcription enhanced by hydrogen peroxide. We conclude that ROS are produced during the exposure of cells to metals that mimic hypoxia, but the formation of ROS was not involved in the activation of HIF-1-dependent genes
— id: 10345, year: 2000, vol: 60, page: 3375, stat: Journal Article,

Rats retain chromium in tissues following chronic ingestion of drinking water containing hexavalent chromium
Sutherland JE; Zhitkovich A; Kluz T; Costa M
2000 Apr;74(1):41-53, Biological trace element research
Humans have sometimes been exposed to as much as 10 ppm Cr(VI) in drinking water from contaminated wells. The risks to these individuals are not well understood because the digestive tract reduces some of the Cr(VI) to the less bioavailable Cr(III) prior to absorption, and the disposition of the remaining Cr(VI) has not been well studied. We determined tissue Cr concentrations in rats after chronic ingestion of Cr(VI) in drinking water at concentrations relevant to human exposure levels. Adult male and female Fischer 344 rats consumed ad libitum 0, 0.5, 3, or 10 ppm Cr(VI) as K2CrO4 in drinking water for 44 wk. Rats then were given deionized water 4-6 d prior to sample collection. Females given 3 or 10 ppm Cr(VI) consumed more Cr(VI) per unit of body weight than did males. Bone Cr concentrations were significantly elevated in rats that drank 10 ppm Cr(VI). Renal Cr concentrations were significantly elevated in male rats that drank 3 or 10 ppm Cr(VI) and in female rats dosed with 10 ppm Cr(VI). Female rats had elevated liver Cr concentrations after drinking 3 or 10 ppm Cr(VI). Testicular Cr concentrations were slightly elevated in rats that drank 10 ppm Cr(VI). Brain, ovarian, and whole-blood Cr concentrations were below detection limits in all exposure groups. Although tissue Cr accumulation may have resulted from absorption of Cr(III), it is poorly absorbed. Therefore, the increased tissue retention may also have resulted, in part, from increased absorption of Cr(VI) and its subsequent uptake from the systemic circulation
— id: 39531, year: 2000, vol: 74, page: 41, stat: Journal Article,

Interaction of Ni(II) and Cu(II) with metal binding sequences of histone H4
Zoroddu M; Kowalik-Jankowska T; Kozlowski H; Salnikow K; Broady L; Costa M
2000 ;6:101-103, Metal ions in biology & medicine = Les ions metalliques en biologie et en medicine
— id: 72795, year: 2000, vol: 6, page: 101, stat: Journal Article,

Interaction of Ni(II) and Cu(II) with a metal binding sequence of histone H4: AKRHRK, a model of the H4 tail
Zoroddu MA; Kowalik-Jankowska T; Kozlowski H; Molinari H; Salnikow K; Broday L; Costa M
2000 Jul 3;1475(2):163-168, Biochimica & biophysica acta
Chromatin proteins are believed to represent reactive sites for nickel binding. The unique structure of the N-terminal tail of histone H4 contains sites for post-translational modification close to a histidine residue capable of anchoring binding sites for metal ions. We have analyzed as a minimal model for the H4 tail, the blocked peptide CH(3)CO-AKRHRK-CONH(2) for nickel and copper binding. Ultraviolet-visible, circular dichroism, electron paramagnetic resonance and nuclear magnetic resonance spectroscopic analysis showed that histidine acts as an anchoring metal binding site. A 1N complex is formed between pH=5-7 and 4-6 for Ni(II) and Cu(II), respectively, while at a higher pH a series of 4N complexes are formed. Above pH 8, the 2N high-spin octahedral resulted in a 4N low-spin planar Ni(II) complex. The stability constants of the Cu(II) (3N, 4N) and Ni(II) (4N) complexes with the peptide model of the H4 were distinctly higher than those for a similar blocked peptide with a histidine in the fourth position. Significant shifts in the alphaproton region in the 1H NMR spectrum of the 4N Ni-complex showed that the conformation of the peptide had been dramatically affected following Ni(II) complexation
— id: 16000, year: 2000, vol: 1475, page: 163, stat: Journal Article,

Nickel enhances telomeric silencing in Saccharomyces cerevisiae
Broday L; Cai J; Costa M
1999 Apr 6;440(2):121-130, Mutation research
Certain nickel compounds including crystalline nickel sulfide (NiS) and subsulfide (Ni3S2) are potent human and animal carcinogens. In Chinese hamster embryo cells, an X-linked senescence gene was inactivated following nickel-induced DNA methylation. Nickel also induced the inactivation of the gpt reporter gene by chromatin condensation and a DNA methylation process in a transgenic gpt+ Chinese hamster cell line (G12), which is located near a heterochromatic region. To determine if nickel can cause gene silencing independently of DNA methylation, based only on the induction of changes in chromatin structure, we measured its effect on gene silencing in Saccharomyces cerevisiae. Growth of yeast in the presence of nickel chloride repressed a telomeric marker gene (URA3) and resulted in a stable epigenetic switch. This phenomenon was dependent on the number of cell doubling prior to selection and also on the distance of the marker gene from the end of the chromosome. The level of TPE (telomeric position effect) increased linearly with elevations of nickel concentration. Addition of magnesium inhibited this effect, but magnesium did not silence the reporter gene by itself. The level of silencing was also assessed following treatment with other transition metals: cobalt, copper and cadmium. In the sublethal range, cobalt induced similar effects as nickel, while copper and cadmium did not change the basal level of gene expression. Silencing by copper and cadmium were evident only at concentrations of those metals where the viability was very low.
— id: 6096, year: 1999, vol: 440, page: 121, stat: Journal Article,

5-azacytidine induces transgene silencing by DNA methylation in Chinese hamster cells
Broday L; Lee YW; Costa M
1999 Apr;19(4):3198-3204, Molecular & cellular biology
The cytosine analog 5-azacytidine (5-AzaC) is a demethylating agent that is also known to induce mutagenesis in mammalian cells. In this study, the mutagenic potential of this drug was tested in the G10 and G12 transgenic Chinese hamster cell lines, which have a single bacterial gpt gene integrated into the genome at different sites, with its expression driven by a simian virus 40 (SV40) promoter. We show that the mutation frequencies following a 48-h exposure to different concentrations of 5-AzaC were 10 to 20 times higher than those of any of the other numerous mutagens that have been tested in the G10-G12 system. Moreover, the mutation frequencies were much higher in the G10 cell line than in the G12 cells. Detailed molecular analysis of the 6-thioguanine (6-TG)-resistant variants demonstrated that transgene silencing by de novo DNA methylation and increased chromatin condensation in the SV40 promoter was the major factor responsible for this high level of 6-TG resistance. As would be expected, exposure to 5-AzaC lowered the overall genomic DNA methylation levels, but it unexpectedly caused hypermethylation and increased chromatin condensation of the transgene in both the G10 and G12 cell lines. These results provide the first evidence that 5-AzaC may also induce transgene-specific DNA methylation, a phenomenon that can further be used for the elucidation of the mechanism that controls silencing of foreign DNA
— id: 6060, year: 1999, vol: 19, page: 3198, stat: Journal Article,

Cell transformation assays
Costa M; Sutherland JE
1999 ;1:3.4.1-3.4.9, Current protocols in toxicology
— id: 72792, year: 1999, vol: 1, page: 3.4.1, stat: Journal Article,

Nickel carcinogenesis, mutation, epigenetics, or selection
Costa, M; Klein, CB
1999 SEP ;107(9):A438-A439, Environmental health perspectives
— id: 53956, year: 1999, vol: 107, page: A438, stat: Journal Article,

Mutagenesis assays in mammalian cells
Klein CB; Broday L; Costa M
1999 ;1:3.3.1-3.3.7, Current protocols in toxicology
— id: 72791, year: 1999, vol: 1, page: 3.3.1, stat: Journal Article,

Nickel-induced transformation shifts the balance between HIF-1 and p53 transcription factors
Salnikow K; An WG; Melillo G; Blagosklonny MV; Costa M
1999 Sep;20(9):1819-1823, Carcinogenesis
Nickel (Ni) compounds are potent carcinogens and can induce malignant transformation of rodent and human cells. In an attempt to unravel the molecular mechanisms of Ni-induced transformation we investigated transcriptional activity of hypoxia-inducible factor (HIF-1) and p53 tumor suppressor protein in Ni-transformed cells. We demonstrated that the activity of HIF-1-responsive promoters was increased in Ni-transformed rodent cells resulting in the increased ratio between HIF-1- and p53-stimulated transcription. To further elucidate the roles of HIF-1 and p53 in Ni-induced transformation we used human osteosarcoma (HOS) cells and a Ni-transformed derivative, SA-8 cells. Since non-functional p53 was expressed in both HOS and SA-8 cells, acute Ni treatment induced HIF-1alpha protein and HIF-1-dependent transcription without affecting p53. In MCF-7 and A549, human cancer cells with the wild-type p53, both functional p53 and HIF-1alpha proteins accumulated following exposure to Ni. The induction of HIF-1alpha and wild-type p53 by Ni was detected after 6 h and was most pronounced by 24 h. These results suggest that acute Ni treatment causes accumulation of HIF-1alpha protein and simultaneous accumulation of wild-type, but not mutant, p53. We suggest that the induction of hypoxia-like conditions in Ni-treated cells with subsequent selection for increased HIF-1-dependent transcription is involved in Ni-induced carcinogenesis
— id: 6193, year: 1999, vol: 20, page: 1819, stat: Journal Article,

Role of Ca(2+) in the regulation of nickel-inducible Cap43 gene expression
Salnikow K; Kluz T; Costa M
1999 Oct 15;160(2):127-132, Toxicology & applied pharmacology
We have recently cloned a gene, Cap43, that was significantly induced by exposure to nontoxic levels of both water-soluble and -insoluble Ni(2+) compounds. In this paper, we utilized the expression levels of this gene as a tool to identify second messengers involved in nickel-inducible transcription. We report here that the Ca(2+) ionophore A23187 substantially stimulated Cap43 gene expression. In addition, we found that BAPTA-AM, a specific chelator of free intracellular Ca(2+), consistently attenuated the induction of Cap43, indicating that elevation of intracellular Ca(2+) was essential for this response. TPEN, a chelator of heavy metals, such as Ni(2+) with a very low affinity for Ca(2+), did not attenuate Cap43 induced by Ni or calcium ionophore, suggesting that elevations of Ca(2+) but probably not elevations of other metal ions were involved in the induction of Cap43. A direct measurement of Ca(2+) levels using the fluorescent probe Fluo-3 AM showed elevations of free intracellular Ca(2+) in Ni-treated cells. A strong induction of Cap43 by okadaic acid suggested the involvement of a serine/threonine phosphorylation in a signaling pathway that was presumably activated by Ni and that led to enhanced Cap43 gene expression. However, calcium-dependent protein kinase(s) involved in the nickel-activated signaling pathway remains to be identified.
— id: 6223, year: 1999, vol: 160, page: 127, stat: Journal Article,

Chromium compounds
Cohen MD; Costa M
Environmental and occupational medicine Philadelphia : Lippincott-Raven, 1998,
— id: 4427, year: 1998, vol: , page: 1045, stat: Chapter,

Carcinogenic metals
Costa M
1998 ;81 ( Pt 4):329-339, Science progress
— id: 6069, year: 1998, vol: 81 ( Pt 4), page: 329, stat: Journal Article,

Molecular biology of nickel carcinogenesis
Costa, M
1998 JUN ;361(4):381-385, Fresenius journal of analytical chemistry
The molecular mechanisms of nickel carcinogenesis are discussed and reviewed with emphasis on work done in my laboratory. The most important determinant of nickel carcinogenesis is the ability of the nickel ion to reach relevant targets for carcinogenesis, which involves chromatin and depends on the ability of the nickel compound to enter cells. The mechanisms that regulate the phagocytosis and intracellular dissolution of the highly carcinogenic particulate nickel compounds are discussed, as is the ability of these nickel compounds to enhance the DNA methylation pattern and turn off the expression of critical tumor suppressor genes. These findings show these nickel compounds to be a somewhat unique class of carcinogens
— id: 53384, year: 1998, vol: 361, page: 381, stat: Journal Article,

Effects of nickel on DNA methyltransferase activity and genomic DNA methylation levels
Lee YW; Broday L; Costa M
1998 Jul 31;415(3):213-218, Mutation research
Methylation of DNA plays an important role in organizing the genome into transcriptionally active and inactive zones. Nickel compounds cause chromatin condensation and DNA methylation in the transgenic gpt+ Chinese hamster cell line (G12). Here we show that nickel is an inhibitor of cytosine 5-methyltransferase activity in vivo and in vitro. In living cells, this inhibition is transient and following a recovery period after nickel treatment, Mtase activity slightly rebounds. Genomic DNA methylation levels are also somewhat decreased following nickel treatment, but with time, there is an elevation of total DNA methylation above basal levels and before any rebound of methyltransferase activity. These results suggest that nickel exposure can elevate total genomic DNA methylation levels even when DNA methyltransferase activity is depressed. These findings may explain the hypermethylation of senescence and tumor suppressor genes found during nickel carcinogenesis and support the model of a direct effect of Ni2+ on chromatin leading to de novo DNA methylation
— id: 7646, year: 1998, vol: 415, page: 213, stat: Journal Article,

Nickel toxicity and nickel carcinogenesis
Snow ET; Costa M
Environmental and occupational medicine Philadelphia : Lippincott-Raven, 1998,
— id: 4428, year: 1998, vol: , page: 1057, stat: Chapter,

Cr(III)-mediated crosslinks of glutathione or amino acids to the DNA phosphate backbone are mutagenic in human cells
Voitkun V; Zhitkovich A; Costa M
1998 Apr 15;26(8):2024-2030, Nucleic acids research
Carcinogenic Cr(VI) compounds were previously found to induce amino acid/glutathione-Cr(III)-DNA crosslinks with the site of adduction on the phosphate backbone. Utilizing the pSP189 shuttle vector plasmid we found that these ternary DNA adducts were mutagenic in human fibroblasts. The Cr(III)-glutathione adduct was the most potent in this assay, followed by Cr(III)-His and Cr(III)-Cys adducts. Binary Cr(III)-DNA complexes were only weakly mutagenic, inducing a significant response only at a 10 times higher number of adducts compared with Cr(III)-glutathione. Single base substitutions at the G:C base pairs were the predominant type of mutations for all Cr(III) adducts. Cr(III), Cr(III)-Cys and Cr(III)-His adducts induced G:C-->A:T transitions and G:C-->T:A transversions with almost equal frequency, whereas the Cr(III)-glutathione mutational spectrum was dominated by G:C-->T:A transversions. Adduct-induced mutations were targeted toward G:C base pairs with either A or G in the 3' position to the mutated G, while spontaneous mutations occurred mostly at G:C base pairs with a 3' A. No correlation was found between the sites of DNA adduction and positions of base substitution, as adducts were formed randomly on DNA with no base specificity. The observed mutagenicity of Cr(III)-induced phosphotriesters demonstrates the importance of a Cr(III)-dependent pathway in Cr(VI) carcinogenicity
— id: 8085, year: 1998, vol: 26, page: 2024, stat: Journal Article,

Utilization of DNA-protein cross-links as a biomarker of chromium exposure
Zhitkovich A; Voitkun V; Kluz T; Costa M
1998 Aug;106 Suppl 4:969-974, Environmental health perspectives
Human exposure to carcinogenic Cr(VI) compounds is found among workers in a large number of professional groups, and it can also occur through environmental pollution. A significant number of toxic waste sites contain Cr as a major contaminant. In this paper we summarize our efforts to apply measurements of DNA-protein cross-links (DPC) as test for biologically active doses of Cr(VI). DPC were found at elevated levels in lymphocytes in several human populations with low to medium Cr exposures. At high exposure to Cr(VI), exemplified by a group of Bulgarian chromeplaters, DPC plateaued and adducts' levels were similar to those found in environmentally exposed individuals. Lymphocytic DPC correlated strongly with Cr levels in erythrocytes that are indicative of Cr(VI) exposure. DPC in lymphocytes were not confounded by such variables as smoking, age, body weight, gender, or ethnicity. A new version of the cross-link assay offers improved sensitivity and requires a small amount of biologic material. Preliminary results indicate that the ability of DPC to reach detectable levels at low levels of Cr exposure could be related to a lack of repair of these lesions in lymphoid cells. Cr(III)-mediated cross-links of DNA with peptide glutathione or single amino acids were mutagenic in human cells, with a relationship of higher molecular weight of the peptide/amino acid correlating with a more potent mutagenic response. We speculate that bulky DPC could also have a significant promutagenic effect. The current methodology does not allow specific determination of Cr-induced DPC; however, demonstrated sensitivity of DPC measurements and the assay's large sample capacity may allow this assay to be used as the initial screening test for the occurrence of DNA damage in Cr(VI)-exposed populations
— id: 7869, year: 1998, vol: 106 Suppl 4, page: 969, stat: Journal Article,

Biologic markers
Zhitkovitch A; Costa M
Environmental and occupational medicine Philadelphia : Lippincott-Raven, 1998,
— id: 4426, year: 1998, vol: , page: 177, stat: Chapter,

Cap43, a novel gene specifically induced by Ni2+ compounds
Zhou D; Salnikow K; Costa M
1998 May 15;58(10):2182-2189, Cancer research
To better understand the molecular mechanism(s) involved in the essentiality, toxicity, and/or carcinogenicity of nickel compounds, a mRNA differential display technique was used to identify gene(s) that were specifically induced by these carcinogens. Differential expression of several genes was observed in human lung A549 cells exposed to nickel subsulfide. One gene, Cap43, which expressed a 3.0-kb mRNA encoding a Mr 43,000 protein, was found to be induced within 4-6 h by either Ni3S2 or NiCl2 in A549 cells and attained a level as high as 30-fold within 24-36 h of treatment. Twelve other tested metal compounds failed to induce Cap43 expression, leading to the conclusion that, with regard to metals, the induction of this gene was nickel-specific. Oxidative stress that is often caused by metals and heat shock did not induce Cap43 further, suggesting a specific nature in the signaling pathway involved in Cap43 induction. Activation of signaling pathways with vanadate did not induce Cap43 nor did trifluoperazine block its induction by nickel; however, okadaic acid, a serine/threonine phosphatase inhibitor, induced Cap43 to a greater extent than any nickel compound tested. Homocysteine did not induce Cap43 in a number of cell lines, with the exception of human endothelial cells. The Cap43 gene was found to be induced by nickel not only in all tested human and rodent cell lines in vitro but also in several rat organs after oral exposure to NiCl2. We have found that the primary signal for Cap43 induction was an elevation of free intracellular Ca2+ caused by Ni2+ exposure because Cap43 was induced by calcium ionophores and its induction was attenuated by bis-(O-aminophenyl)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)-ester, a chelator of intracellular Ca2+. We found that the Cap43 gene was evolutionarily conserved and similarly regulated in humans, mice, and rats. Recent studies have shown that Cap43 is expressed at lower levels in colon cancer. Further studies of Cap43 regulation by Ca2+ should enhance our understanding of the role of Cap43 in cell function and cancer pathogenesis
— id: 7870, year: 1998, vol: 58, page: 2182, stat: Journal Article,

Chromatin condensation and DNA methylation during nickel carcinogenesis
Costa M
1997 ;5:359-366, Metal ions in biology & medicine = Les ions metalliques en biologie et en medicine
— id: 72782, year: 1997, vol: 5, page: 359, stat: Journal Article,

Epigenetic mechanisms of nickel carcinogenesis
Costa M
Trace elements in man and animals -- 9 Ottawa : NRC Research Press, 1997,
— id: 4424, year: 1997, vol: , page: 534, stat: Chapter,

Nonmutagenic mechanisms of nickel carcinogenesis : inactivation of critical genes by nickel-induced DNA methylation and chromatin condensation
Costa M
Correlations between in vitro and in vivo investigations in inhalation toxicology Washington DC : ILSI Press, 1997,
— id: 4423, year: 1997, vol: , page: 359, stat: Chapter,

Toxicity and carcinogenicity of Cr(VI) in animal models and humans
Costa M
1997 Sep;27(5):431-442, Critical reviews in toxicology
The toxicity and carcinogenicity of hexavalent chromium (Cr) in animal and human models are reviewed. The focus of this review is not on the well-established fact that hexavalent Cr compounds of low and high water solubility can induce respiratory cancers, but rather this review addresses other types of cancers induced by exposure to hexavalent Cr compounds. Additionally, non-cancer endpoints are also discussed with documentation of human and animal studies showing non-cancer health effects of hexavalent Cr exposure on the respiratory system, GI system, immune system, liver, and kidney. There is an emerging understanding that because hexavalent chromate is isostructural with phosphate and sulfate, it is readily taken up by the G.I. tract and penetrates to many tissues and organs throughout the body. This is supported by animal studies and experiments using human volunteers. From the epidemiological studies, there is suggestive evidence that hexavalent Cr causes increased risk of bone, prostate, lymphomas, Hodgkins, leukemia, stomach, genital, renal, and bladder cancer, reflecting the ability of hexavalent chromate to penetrate all tissues in the body. A high accumulation of Cr(III) in all tissues and organs is a strong indication of the wide toxic potential of exposure to soluble hexavalent Cr in the drinking water and in the ambient environment
— id: 10361, year: 1997, vol: 27, page: 431, stat: Journal Article,

DNA-protein cross-links produced by various chemicals in cultured human lymphoma cells
Costa M; Zhitkovich A; Harris M; Paustenbach D; Gargas M
1997 Apr 11;50(5):433-449, Journal of toxicology & environmental health
Chemicals such as cis-platinum, formaldehyde, chromate, copper, and certain arsenic compounds have been shown to produce DNA-protein cross-links in human in vitro cell systems at high doses, such as those in the cytotoxic range. Thus far there have only been a limited number of other chemicals evaluated for their ability to produce cross-links. The purpose of the work described here was to evaluate whether select industrial chemicals can form DNA-protein cross-links in human cells in vitro. We evaluated acetaldehyde, acrolein, diepoxybutane, paraformaldehyde, 2-furaldehyde, propionaldehyde, chloroacetaldehyde, sodium arsenite, and a deodorant tablet [Mega Blue; hazardous component listed as tris(hydroxymethyl)nitromethane]. Short- and long-term cytotoxicity was evaluated and used to select appropriate doses for in vitro testing. DNA-protein cross-linking was evaluated at no fewer than three doses and two cell lysate washing temperatures (45 and 65 degrees C) in Epstein-Barr virus (EBV) human Burkitt's lymphoma cells. The two washing temperatures were used to assess the heat stability of the DNA-protein cross-link, 2-Furaldehyde, acetaldehyde, and propionaldehyde produced statistically significant increases in DNA-protein cross-links at washing temperatures of 45 degrees C, but not 65 degrees C, and at or above concentrations of 5, 17.5, and 75 mM, respectively. Acrolein, diepoxybutane, paraformaldehyde, and Mega Blue produced statistically significant increases in DNA-protein cross-links washed at 45 and 65 degrees C at or above concentrations of 0.15 mM, 12.5 mM, 0.003%, and 0.1%, respectively. Sodium arsenite and chloroacetaldehyde did not produce significantly increased DNA-protein cross-links at either temperature nor at any dose tested. Excluding paraformaldehyde and 2-furaldehyde treatments, significant increases in DNA-protein cross-links were observed only at doses that resulted in complete cell death within 4 d following dosing. This work demonstrates that DNA-protein cross-links can be formed in vitro following exposure to a variety of industrial compounds and that most cross-links are formed at cytotoxic concentrations
— id: 12327, year: 1997, vol: 50, page: 433, stat: Journal Article,

Carcinogenic inorganic chemicals
Farrell RP; Costa M
Comprehensive toxicology New York : Pergamon Press, 1997,
Volume title: "Chemical carcinogens and anticarcinogens"
— id: 4425, year: 1997, vol: 12, page: 225, stat: Chapter,

DNA methylation and gene expression: introduction and overview
Klein CB; Costa M
1997 Apr;386(2):103-105, Mutation research
— id: 12337, year: 1997, vol: 386, page: 103, stat: Journal Article,

DNA methylation, heterochromatin and epigenetic carcinogens
Klein CB; Costa M
1997 Apr;386(2):163-180, Mutation research
This paper will explore emerging concepts related to alternative carcinogenic mechanisms of 'non-mutagenic,' and hence epigenetic, carcinogens that may heritably alter DNA methylation without changing the underlying DNA sequence. In this review, we will touch on the basic concepts of DNA methylation, and will elaborate in greater detail on related topics including chromatin condensation, and heterochromatin spreading that is well known to induce gene silencing by position effect variegation in Drosophila and other species. Data from our model transgenic G12 cell system will be presented to support our hypothesis that certain carcinogens, such as nickel, may be carcinogenic not primarily because of their overt mutability, but rather as the result of their ability to promote DNA hypermethylation of important cancer-related genes. We will conclude with a discussion of the broader relevance of our findings and its application to other so-called 'epigenetic' carcinogens
— id: 8210, year: 1997, vol: 386, page: 163, stat: Journal Article,

Carcinogenicity assessment of selected nickel compounds
Oller, A R; Costa, M; Oberdorster, G
1997 Mar;143(1):152-166, Toxicology & applied pharmacology
The early epidemiological data indicated different carcinogenic risks from inhalation of different nickel compounds, but it was not clear what characteristics governed the intrinsic carcinogenic hazard of the various nickel compounds. Based on the earlier results, all soluble and insoluble nickel compounds were assumed to have the same carcinogenic mechanism albeit different potencies. Recent in vivo and in vitro studies challenged this assumption. In this paper an attempt is made to integrate the most relevant human, animal, and in vitro data into a general model that can help understand the different carcinogenic potentials of the various nickel compounds. In this perspective, it is recognized that there are two main components that could contribute to the development of lung cancer via exposure to certain nickel compounds. The first component corresponds to the heritable changes (genetic or epigenetic) derived from the direct or indirect actions of nickel compounds. The second component may be the promotion of cell proliferation elicited by certain nickel compounds. The different contributions of three nickel compounds to these two components are presented. This paper emphasizes the importance of recognizing the individuality of the different nickel species in reaching regulatory decisions and the fact that different risk assessment considerations may apply for compounds that appear to produce immortality and cancer by genetic/epigenetic mechanisms (like nickel subsulfide), compounds that may present a threshold for the induction of tumors in rats (like high-temperature nickel oxide), or compounds that may only have an enhancing effect on carcinogenicity (like nickel sulfate)
— id: 141436, year: 1997, vol: 143, page: 152, stat: Journal Article,

Induction of activating transcription factor 1 by nickel and its role as a negative regulator of thrombospondin I gene expression
Salnikow K; Wang S; Costa M
1997 Nov 15;57(22):5060-5066, Cancer research
Thrombospondin I (TSP I) is an extracellular matrix glycoprotein that influences cell adhesion, motility, and growth. On the basis of its effects on endothelial cell proliferation, TSP I has attracted interest as a potential regulator of solid tumor growth through modulation of tumor blood supply. The regulation of TSP I expression is of critical importance for designing new approaches in tumor therapy. Recently, we have shown that TSP I expression is lost in nickel-transformed cells. In this paper, we identified an activating transcription factor (ATF)/cAMP-responsive element-binding protein binding site as a negative regulatory site in the 5'-flanking sequence of mouse TSP I promoter. We identified ATF-1 as a major component of the ATF/cAMP-responsive element-binding protein binding complex. This Mr 35,000 nuclear ATF-1 protein was shown to be present in higher amounts in nickel-transformed 3T3 cells that do not express TSP 1. Acute treatment of 3T3 cells with NiCl2 resulted in the induction of this transcription factor, and this induction was correlated temporally with the suppression of TSP I expression in the same cells. These results show that nickel exposure causes accumulation of the ATF-1 transcription factor, which is responsible for the down-regulation of transcription of TSP I, and possibly other tumor suppressor genes during nickel-induced cellular transformation
— id: 12220, year: 1997, vol: 57, page: 5060, stat: Journal Article,

Ternary DNA adducts formed by carcinogenic Cr compounds: In vitro formation and mutagenic potential
Zhitkovich A; Voitkun V; Costa M
1997 ;38:A271-A271, Proceedings of the annual meeting of the American Association for Cancer Research
We have recently found that exposure of cultured cells to carcinogenic Cr(VI) compounds led to the formation of numerous amino acids/glutathione-DNA crosslinks mediated by Cr(III). In order to elucidate a possible crosslinking mechanism adduction of Cys and His by Cr(III) and Cr(VI) was studied in vitro. Neither of the amino acids reacted with Cr(III)-DNA however extensive DNA adduction occurred when binary Cys-Cr(III) or His-Cr(III) complexes were added to DNA. Crosslinking of amino acids by Cr(VI)/reducer mixtures revealed that the DNA adduction continued even after complete conversion of Cr(VI) into Cr(III) suggesting a primary role of Cr(III) in the formation of DNA-reactive species. Additional experiments showed that the major site of DNA adduction was on the phosphate group. To assess mutagenic potential of individual CT-DNA crosslinks an SV40-based shuttle vector (pSP189 plasmid) was adducted in vitro followed by its replication in human fibroblasts and mutagenic response was estimated in an indicator bacterial strain. Binary Cr(III)-DNA crosslinks exhibited the weakest response whereas ternary glutathione adducts were the most potent premutagenic lesions. Sequencing results revealed that majority of mutations were G:C to A:T or G:C to T:A base substitutions. There was a noticeable effect of adjacent base on the mutation frequency at G:C pairs
— id: 6030, year: 1997, vol: 38, page: A271, stat: Journal Article,

Carcinogenicity and genotoxicity of lead, beryllium, and other metals
Cohen MD; Bowser DH; Costa M
Toxicology of metals Boca Raton FL : Lewis Publishers, 1996,
— id: 4422, year: 1996, vol: , page: 253, stat: Chapter,

Mechanisms of nickel genotoxicity and carcinogenicity
Costa M
Toxicology of metals Boca Raton FL : Lewis Publishers, 1996,
— id: 4421, year: 1996, vol: , page: 245, stat: Chapter,

Overview : an introduction to metal toxicity and carcinogenicity of metals
Costa M
Toxicology of metals Boca Raton FL : Lewis Publishers, 1996,
— id: 4420, year: 1996, vol: , page: 203, stat: Chapter,

Interlaboratory validation of a new assay for DNA-protein crosslinks
Costa M; Zhitkovich A; Gargas M; Paustenbach D; Finley B; Kuykendall J; Billings R; Carlson TJ; Wetterhahn K; Xu J; Patierno S; Bogdanffy M
1996 Jul 10;369(1-2):13-21, Mutation research
In 1992, a simple and sensitive assay for detecting DNA-protein crosslinks was developed [1]. In an effort to facilitate the greater use of the assay, a number of studies were conducted to evaluate its reliability and reproducibility. During this work, the assay was used to assess whether various metals and other compounds could induce crosslinks in cultured human lymphocytes (Epstein-Barr virus-transformed Burkitt's Lymphoma cell line). Potassium permanganate, mercury chloride, lead nitrate, magnesium perchlorate, aluminum chloride, and cadmium chloride did not induce DNA-protein crosslinks at either cytotoxic or non-cytotoxic levels. Copper sulfate, arsenic trioxide, and potassium chromate induced DNA-protein crosslinks only at cytotoxic concentrations. Acute lethality of the cells was assessed immediately after exposure to metals by trypan blue exclusion while long-term lethality was assessed by cell proliferation and trypan blue exclusion following an incubation period of 5 days after exposure to the metal compound. All metals exhibited more toxicity in the long-term lethality assay compared to the short-term assay. The cultured human lymphocytes treated with various doses of lead acetate, cadmium chloride, arsenic trioxide and copper sulfate, as well as cis-platinum and chromate, were sent to four different laboratories to compare the reliability and reproducibility of the DNA-protein crosslink assay. Depending on the chemical studied, there were quantitative differences in the results observed among the various laboratories using the assay. However, all laboratories generally showed that cis-platinum, chromate, arsenic trioxide and copper sulfate induced DNA-protein crosslinks at levels that produced acute cytotoxicity, whereas cadmium chloride and lead acetate did not
— id: 10371, year: 1996, vol: 369, page: 13, stat: Journal Article,

Monitoring human lymphocytic DNA-protein cross-links as biomarkers of biologically active doses of chromate
Costa M; Zhitkovich A; Toniolo P; Taioli E; Popov T; Lukanova A
1996 Oct;104 Suppl 5:917-919, Environmental health perspectives
A simple and sensitive assay for DNA-protein cross-links has been used as a biomarker of chromate exposure and early carcinogenic effects. Pilot studies of DNA-protein cross-links in peripheral blood lymphocytes have been conducted with individuals who had higher exposure to chromate, including welders, and with individuals who had lower levels of exposure such as residents living in a chromium-contaminated area in Jersey City, New Jersey. Studies were also conducted in two Bulgarian cities (Jambol and Burgas) with different levels of air pollution and Cr(VI) exposure and in chrome platers in Bulgaria who had high exposure to chromate. DNA-protein cross-links in U.S. welders and in individuals living in Hudson County, New Jersey around chromium-contaminated areas were significantly higher compared to matched controls. Although blood and urinary levels of chromium were not extensively studied in these populations, we were able to obtain these measurements in the Bulgarian population. Chromium levels in red blood cells of controls living in Burgas were in the order of 1 to 2 ppb chromium, and these individuals had the lowest levels of DNA-protein cross-links. However, the chromium levels in Jambol ranged from about 2 to 7 ppb in red blood cells of city residents to about 22 ppb in chrome platers. DNA-protein cross-links were saturated at about 7 to 8 ppb chromium in the red blood cells, and cross-links correlated well only with chromium levels in red blood cells. Urinary chromium levels did not correlate well with either DNA-protein cross-links or chromium levels in with red blood cells
— id: 12521, year: 1996, vol: 104 Suppl 5, page: 917, stat: Journal Article,

Peroxidase deficiency of nickel-transformed hamster cells correlates with their increased resistance to cytotoxicity of peroxides
Dowjat WK; Huang X; Cosentino S; Costa M
1996 Apr;9(2):151-156, Biometals
Using a procedure aimed at isolation of genes that are inactivated during nickel-induced carcinogenesis in Chinese hamster cells, a homolog of genes encoding human and mouse heme containing peroxidases has been cloned. Northern blot analysis of normal cultured fibroblasts and two nickel-transformed cell lines confirmed that this gene was expressed in normal but not in transformed cells. Nickel-transformed cells also tested negative for peroxidase activity using a sensitive fluorescence assay. Cultured embryo cells or fibroblasts that express peroxidase activity and their nickel-transformed peroxidase-deficient counterparts were employed to investigate the role of peroxidase-catalyzed processes in cytotoxicity induced by tert-butyl hydroperoxide or cumene hydroperoxide. It has been found that peroxidase-deficient cells were significantly more resistant to cytotoxic effect of these compounds suggesting that cytotoxic effect of hydroperoxides may be mediated in part by free radicals generated in the course of peroxidase-catalyzed reactions
— id: 10372, year: 1996, vol: 9, page: 151, stat: Journal Article,

Mutagenicity of cobalt and reactive oxygen producers
Kitahara J; Yamanaka K; Kato K; Lee YW; Klein CB; Costa M
1996 Oct 1;370(3-4):133-140, Mutation research
Oxidative stress has been implicated in carcinogenesis yet there are chemicals that produce oxidative stress that are not carcinogenic. Mutations are the inherited results of DNA damage and are critical events in carcinogenesis. The mutagenicity of oxidative stress induced by peroxide, paraquat and cobalt compounds was examined in transgenic gpt+ Chinese hamster cell lines (G12 and G10). These two cell lines are known to be more sensitive to mutagens such as X-rays and UV than their parental V-79 cells. In these studies, the mutagenic activity of cobalt chloride, a metal that induces oxidative stress but is not carcinogenic, was measured to be 7.7 times higher than the spontaneous mutant frequency in G12, but was only 1.5 to 2.5 times higher than spontaneous mutant frequency in G10 cells. The mutant frequency of cobalt sulfide was somewhat lower. Hydrogen peroxide was found to be only weakly mutagenic in G12 cells, and treatment of cells with a combination of hydrogen peroxide and cobalt did not alter the mutation frequency induced by cobalt sulfide alone. Paraquat did not elicit mutagenesis in either cell line. These results indicate that agents producing oxidative stress are not necessarily mutagenic and these results are discussed in the context of the oxidative stress produced by other carcinogens such as nickel compounds
— id: 10369, year: 1996, vol: 370, page: 133, stat: Journal Article,

Occupational exposure to Cr(VI): comparison between chromium levels in lymphocytes, erythrocytes, and urine
Lukanova A; Toniolo P; Zhitkovich A; Nikolova V; Panev T; Popov T; Taioli E; Costa M
1996 ;69(1):39-44, International archives of occupational & environmental health
The relationships between chromium (Cr) levels in lymphocytes, erythrocytes, urine, and ambient air were compared among 14 chrome-platers from a metallurgic plant in Bulgaria and two groups of local controls, one from the same heavily polluted industrial town as the chrome-platers (n = 11) and one from a seaside resort town 100 km away (n = 6). Among the chrome-platers, the Cr concentration in peripheral lymphocytes was positively correlated with total Cr and Cr(VI) levels in ambient air and with Cr excretion in urine. As compared to the controls, the chrome-platers had mean Cr levels in lymphocytes twice as high, in erythrocytes ninefold higher, and in urine fourfold to eightfold higher. Although Cr levels in urine and lymphocytes were similar between the two control groups, levels in erythrocytes were 3 times higher among subjects from the industrial area than among those from the seaside town. The study suggests that lymphocyte Cr could be a good indicator of the Cr body burden caused by high exposures to Cr(VI), such as in electroplating operations. In these conditions, erythrocyte Cr may be less useful, possibly owing to increased toxicity due to the high affinity of erythrocytes for Cr. However, when exposure is lower, such as in most environmental situations, erythrocyte Cr should provide a better and more sensitive index than lymphocyte Cr. By contrast, urinary Cr, which provides information on total Cr exposure, including Cr(III) from dietary and environmental sources, does not seem to be of value for studying occupational exposure to Cr(VI)
— id: 10374, year: 1996, vol: 69, page: 39, stat: Journal Article,

DNA-protein crosslinks as a biomarker of cis-platinum activity in cancer patients
Taioli, E; Zhitkovich, A; Toniolo, P; Bernstein, J; Blum, R; Costa, M
1996 MAY-JUN ;3(3):439-441, Oncology reports
We have developed a new method to assess the amount of DNA-protein crosslinks (DNA-PC) in peripheral lymphocytes, based on the selective precipitation of the DNA crosslinked to proteins. We assessed the amount of DNA-PC in peripheral lymphocytes of 18 cancer patients who underwent chemotherapy with cis-platinum for the first time. Since the chemotherapy was administered over a two-day period, blood samples were drawn at baseline (before starting the therapy), 4 h after the infusion of the first dose of cis-platinum, the next day (24 h after the first dose, and immediately before the infusion of the second dose), and 2 days later (48 h after the first dose). The mean change of DNA-PC 4 h after therapy was 0.8+/-0.8% (p=0.006), 0.5+/-0.6% after 1 day (p=0.007), and 0.1+/-0.5% after two days (ns). The correlation between DNA-PC changes and cumulative dose of cis-platinum was -0.22 at 4 h, -0.19 after 1 day, and -0.68 at 2 days (p=0.005). The crosslinking effect of cis-platinum seems to vary among individuals and with dose; the DNA-PC may be used to define sub-populations of patients with various degree of sensitivity to the pharmacologic action of this chemotherapeutic agent, and thus to adjust the dosage on an individual basis
— id: 52979, year: 1996, vol: 3, page: 439, stat: Journal Article,

Formation of the amino acid-DNA complexes by hexavalent and trivalent chromium in vitro: importance of trivalent chromium and the phosphate group
Zhitkovich A; Voitkun V; Costa M
1996 Jun 4;35(22):7275-7282, Biochemistry
We have recently shown that a substantial fraction of all Cr-DNA adducts in chromate-exposed cells are represented by ternary complexes involving amino acids or glutathione bridged by Cr-(III) to DNA. The tridentate amino acids such as cysteine, glutamic acid, and histidine were predominantly found cross-linked to DNA. The mechanism by which Cr can cross-link these amino acids to DNA has been modeled by reacting DNA and trivalent and hexavalent chromium with cysteine and histidine. The formation of a Cr(III)-amino acid binary complex was required before Cr(III) reacted with DNA to yield a ternary complex. Cr(III)-pretreated DNA did not bind cysteine or histidine even after prolonged incubations. Reduction of Cr(VI) in the presence of DNA gave rise to an extensive cross-linking of cysteine and histidine. Addition of DNA to Cr(VI) mixtures at the start of reduction or after the reduction was complete had little effect on the level of ternary complexes indicating that Cr(III)-amino acid binary complexes were DNA-attacking species. In order to identify DNA groups involved in the ternary complex formation, pre-formed Cr(III)-histidine complexes were reacted with nucleosides and nucleotide monophosphates followed by separation and analysis of the products. The incubation of the Cr(III)-histidine complexes with nucleotide monophosphates but not with nucleosides gave rise to ternary complexes that contained both histidine and Cr, showing the primary importance of the phosphate group in this reaction. All four DNA nucleotides were capable of the ternary complex formation with Cr(III) and histidine. No apparent base preference in the amino acid cross-linking was also found in the reaction of Cr(III)/cysteine and Cr(VI)/cysteine mixtures with oligonucleotides of base-specific composition
— id: 7063, year: 1996, vol: 35, page: 7275, stat: Journal Article,

DNA-protein crosslinks in peripheral lymphocytes of individuals exposed to hexavalent chromium compounds
Zhitkovich, A; Lukanova, A; Popov, T; Taioli, E; Cohen, H; Costa, M; Toniolo, P
1996 APR-JUN ;1(2):86-93, Biomarkers
DNA-protein crosslinks were measured in peripheral blood lymphocytes of chrome-platers and controls from Bulgaria in order to evaluate a genotoxic effect of human exposure to carcinogenic Cr(VI) compounds, Chrome-platers and most of the unexposed controls were from the industrial city of Jambol; some additional controls were recruited from the seaside town of Burgas, The chrome-platers had significantly elevated levels of chromium in pre- and post-shift urine, erythrocytes and lymphocytes compared with the control subjects, The largest differences between the two groups were found in erythrocyte chromium concentrations which are considered to be indicative of Cr(VI) exposure, Despite the significant differences in internal chromium doses, levels of DNA-protein crosslinks were not significantly different between the combined controls and exposed workers, Individual DNA-protein crosslinks, however, correlated strongly with chromium in erythrocytes at low and moderate doses but at high exposures, such as among the majority of chrome-platers, these DMA adducts were saturated at maximum levels, The saturation of DNA-protein crosslinks seems to occur at 7-8 pg I-1 chromium in erythrocytes whereas a mean erythrocyte chromium among the chrome platers was as high as 22.8 mu g I-1. Occupationally unexposed subjects exhibited a significant variability with respect to the erythrocyte chromium concentration, however erythrocyte chromium levels correlated closely with DNA-protein crosslinks in lymphocytes, The controls from Jambol had higher chromium concentrations in erythrocytes and elevated levels of DNA-protein crosslinks compared with Burgas controls, Occupational exposure to formaldehyde among furniture factory workers did not change levels of DNA-protein crosslinks in peripheral lymphocytes. DNA-protein crosslink measurements showed a low intraindividual variability and their levels among both controls and exposed indivduals were not affected by smoking, age or weight
— id: 52703, year: 1996, vol: 1, page: 86, stat: Journal Article,

The receptor tyrosine kinase ARK mediates cell aggregation by homophilic binding
Bellosta P; Costa M; Lin DA; Basilico C
1995 Feb;15(2):614-625, Molecular & cellular biology
The ARK (AXL, UFO) receptor is a member of a new family of receptor tyrosine kinases whose extracellular domain contains a combination of fibronectin type III and immunoglobulin motifs similar to those found in many cell adhesion molecules. ARK mRNA is expressed at high levels in the mouse brain, prevalently in the hippocampus and cerebellum, and this pattern of expression resembles that of adhesion molecules that are capable of promoting cell aggregation through homophilic or heterophilic binding. We report here the ability of the murine ARK receptor to mediate homophilic binding. Expression of the ARK protein in Drosophila S2 cells induces formation of cell aggregates consisting of ARK-expressing cells, and aggregation leads to receptor activation, with an increase in receptor phosphorylation. Homophilic binding does not require ARK tyrosine kinase activity, since S2 cells expressing a receptor in which the intracellular domain was deleted were able to undergo aggregation as well as cells expressing the wild-type ARK receptor. Similar results were obtained with NIH 3T3 and CHO cells expressing high levels of ARK, although in this case ARK expression appeared to be accompanied by constitutive activation. The purified recombinant extracellular domain of ARK can induce homotypic aggregation of coated fluorescent beads (Covaspheres), and this protein can also function as a substrate for adhesion by S2 and NIH 3T3 cells expressing ARK. These results suggest that ARK represents a new cell adhesion molecule that through its homophilic interaction may regulate cellular functions during cell recognition
— id: 8207, year: 1995, vol: 15, page: 614, stat: Journal Article,

Chemical mechanisms of metal carcinogenesis
Costa M
Handbook of metal-ligand interactions in biological fluids : bioinorganic chemistry New York : Dekker, 1995,
— id: 4418, year: 1995, vol: , page: 838, stat: Chapter,

Diverse aspects of metal carcinogenesis
Costa M
Handbook of metal-ligand interactions in biological fluids : bioinorganic chemistry New York : Dekker, 1995,
— id: 4419, year: 1995, vol: , page: 1003, stat: Chapter,

Inactivation of critical cancer-related genes by nickel-induced DNA hypermethylation and increased chromatin condensation
Costa M
Genetic response to metals New York : Dekker, 1995,
— id: 4417, year: 1995, vol: , page: 53, stat: Chapter,

Model for the epigenetic mechanism of action of nongenotoxic carcinogens
Costa M
1995 Mar;61(3 Suppl):666S-669S, American journal of clinical nutrition
On the basis of studies with carcinogenic nickel compounds, we propose a new model of how epigenetic carcinogens might act. This model is based on the fact that nickel compounds induce an increase in chromatin condensation, causing neighboring genes that are actively expressed in euchromatin to be condensed into heterochromatin. Such redistribution in condensation of chromatin would probably only be transient were it not for the DNA cytosine methyl transferase enzyme, which through de novo methylation can cause genes to be inherited in an active state. Actively expressed genes have less cytosine methylation in their promoter whereas hypermethylation of cytosine in promoters is characteristic of inactive genes. Therefore, nickel, through induction of an enhanced condensation state of chromatin that results in the incorporation of critical genes such as the senescence and tumor suppressor genes into heterochromatin (ie, thread on a spool) and the subsequent methylation of this DNA, silences the genetic activity that might be essential for maintenance of a normal cell. This model is consistent with the literature on cytosine methylation and is also consistent with studies of nickel carcinogenesis showing that it increases cytosine methylation. It is also consistent with nickel carcinogenesis being synergistic with many other mutagenic carcinogens (ie, x rays, benzopyrene, or ultraviolet light), which has always suggested that it has a unique component that is not part of the mechanism of these mutagenic carcinogens
— id: 6596, year: 1995, vol: 61, page: 666S, stat: Journal Article,

MOLECULAR RESPONSE TO NICKEL - INHERITED EFFECTS ON TRANSCRIPTION
COSTA, M
1995 MAR 10 ;14(1-6):242-242, Journal of cellular biochemistry
— id: 86744, year: 1995, vol: 14, page: 242, stat: Journal Article,

NICKEL CARCINOGENESIS - INACTIVATION OF SENESCENCE AND OTHER CANCER-RELATED GENES BY ENHANCED HETEROCHROMATIN CONDENSATION AND DNA HYPERMETHYLATION
COSTA, M
1995 JAN 5 ;336(3):185-185, Journal of cellular biochemistry
— id: 87350, year: 1995, vol: 336, page: 185, stat: Journal Article,

INACTIVATION OF RETINOBLASTOMA PROTEIN ASSOCIATED WITH NICKEL-INDUCED TRANSFORMATION OF HUMAN-CELLS
DOWJAT, WK; LIN, X; COSTA, M
1995 JAN 5 ;336(3):195-195, Journal of cellular biochemistry
— id: 87352, year: 1995, vol: 336, page: 195, stat: Journal Article,

Unlike chromium(III) alone, low concentrations of amino acid-chromium(III)-DNA-crosslinks act as strong blocks to DNA replication
Gao M; Snow ET; Singh J; Zhitkovich A; Costa M
1995 ;25 (Suppl. 25):17-17, Environmental & molecular mutagenesis
When hexavalent metal salts of the carcinogen chromate, CrO4, enter mammalian cells they are rapidly reduced via reactive Cr(V) and other intermediates to stable Cr(III) species. This process induces oxidative DNA damage as well as the formation of long-lasting DNA-protein and DNA-amino acid crosslinks. DNA-amino acid crosslinks are among the most numerous of all types of Cr-mediated DNA damage, however, the biological consequence of this type of DNA adduct is unknown. Using M13 DNA as a model system, we have previously shown that low concentrations of Cr(III), when complexed to the DNA template, can facilitate DNA replication and enhance DNA polymerase processivity (Snow and Xu, Biochemistry 30:11238, 1991). We have now employed a similar system to investigate the effect of DNA-Cr-histidine complexes on DNA replication in vitro using the Klenow Fragment of E coli DNA polymerase I. Although histidine competes with DNA to reduce the rate of formation of DNA-Cr(III) complexes, as many as 50% of the Cr(III)-complexes that are formed contain histidine. Furthermore, the histidine-Cr(III)-DNA complexes are more stable than complexes formed with Cr(III) alone. Moreover, unlike Cr(III) alone, we find that the Cr(III)-histidine-DNA complexes act as strong, dose-dependent, blocks to replication. We also find that these complexes are mutagenic when transfected into repair proficient E coli (greater than 3-fold increase in mutant frequency at a concentration Cr(III) and histidine that results in an 85% decrease in survival of the phage DNA). These results indicate that the amino acid-Cr-DNA complexes may be an important class of chromium-mediated genotoxic lesions
— id: 6033, year: 1995, vol: 25 (Suppl. 25), page: 17, stat: Journal Article,

Heterochromatic proteins specifically enhance nickel-induced 8-oxo-dG formation
Huang X; Kitahara J; Zhitkovich A; Dowjat K; Costa M
1995 Aug;16(8):1753-1759, Carcinogenesis
7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) was measured as an indicator of nickel-induced oxidative base damage in the presence of H2O2. Heterochromatic proteins isolated from Chinese hamster liver cells enhanced the formation of 8-oxo-dG induced by NiCl2 and H2O2 in vitro, whereas euchromatic proteins inhibited this reaction. The inhibitory effect of euchromatic proteins on dG oxidation may be due to the oxygen radical scavenging effects of low molecular weight protein-rich fractions. Gel electrophoresis confirmed that histone H1 was present at a higher concentration in heterochromatin than in euchromatin. It is believed that the presence of nickel-protein complexes in cells is crucial for the formation of reactive oxygen species (ROS). We found that Ni2+ binds to histone H1 and core histones as determined by 63Ni autoradiography of proteins on nitrocellulose membranes. In vitro studies showed that commercially purified histone H1, and to a considerably lesser extent core histones, enhanced the NiCl2 and H2O2 catalyzed formation of 8-oxo-dG in a reaction containing free dG base. Since histone H1 is a lysine- and alanine-rich protein, the levels of 8-oxo-dG induced by NiCl2 and H2O2 were studied in the presence of these amino acids and found to be enhanced by them. These results suggest that nickel may specifically produce oxidative DNA damage in heterochromatin because of the nature of its binding to histone H1 and core histones. This selective oxidation of genetically inactive heterochromatin may explain why nickel compounds which generate oxygen radicals and oxidize DNA bases are inactive in most gene mutation assays
— id: 6833, year: 1995, vol: 16, page: 1753, stat: Journal Article,

Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens
Lee YW; Klein CB; Kargacin B; Salnikow K; Kitahara J; Dowjat K; Zhitkovich A; Christie NT; Costa M
1995 May;15(5):2547-2557, Molecular & cellular biology
A transgenic gpt+ Chinese hamster cell line (G12) was found to be susceptible to carcinogenic nickel-induced inactivation of gpt expression without mutagenesis or deletion of the transgene. Many nickel-induced 6-thioguanine-resistant variants spontaneously reverted to actively express gpt, as indicated by both reversion assays and direct enzyme measurements. Since reversion was enhanced in many of the nickel-induced variant cell lines following 24-h treatment with the demethylating agent 5-azacytidine, the involvement of DNA methylation in silencing gpt expression was suspected. This was confirmed by demonstrations of increased DNA methylation, as well as by evidence indicating condensed chromatin and heterochromatinization of the gpt integration site in 6-thioguanine-resistant cells. Upon reversion to active gpt expression, DNA methylation and condensation are lost. We propose that DNA condensation and methylation result in heterochromatinization of the gpt sequence with subsequent inheritance of the now silenced gene. This mechanism is supported by direct evidence showing that acute nickel treatment of cultured cells, and of isolated nuclei in vitro, can indeed facilitate gpt sequence-specific chromatin condensation. Epigenetic mechanisms have been implicated in the actions of some nonmutagenic carcinogens, and DNA methylation changes are now known to be important in carcinogenesis. This paper further supports the emerging theory that nickel is a human carcinogen that can alter gene expression by enhanced DNA methylation and compaction, rather than by mutagenic mechanisms
— id: 6675, year: 1995, vol: 15, page: 2547, stat: Journal Article,

THE LOW-LEVELS OF SOME TRANSCRIPTION FACTORS BINDING IN NICKEL RESISTANT CELLS CORRELATES WITH SILENCING OF THROMBOSPONDIN EXPRESSION
SALNIKOW, K; GAO, M; WANG, S; COSTA, M
1995 JAN 5 ;336(3):190-190, Journal of cellular biochemistry
— id: 87351, year: 1995, vol: 336, page: 190, stat: Journal Article,

Increased DNA-protein crosslinks in lymphocytes of residents living in chromium-contaminated areas
Taioli E; Zhitkovich A; Kinney P; Udasin I; Toniolo P; Costa M
1995 Dec;50(3):175-180, Biological trace element research
It has been known for a number of years that chromium-containing mine slags were used as landfill in residential areas of Hudson County, New Jersey. Since one of the major lesions induced in intact cells by chromate is the DNA-Protein crosslink, we have used this lesion as a biomarker of biological effect of chromium (Cr) exposure. We have previously developed a sensitive and easy-to-perform assay to detect DNA-Protein crosslinks, based on the selective K SDS precipitation of DNA associated with protein. We examined the levels of DNA-Protein crosslinks in peripheral blood mononuclear cells of 33 individuals determined to be at risk for chromium exposure by virtue of their residence in Hudson County and their urinary Cr levels. These data were compared to the levels of DNA-Protein crosslinks among 49 controls who resided in noncontaminated areas. A complete clinical examination and urine analysis did not show any Cr-related abnormalities among the exposed population. The mean DNA-Protein crosslink level in the lymphocytes of the exposed group was 1.3 +/- 0.5% (SD), whereas the unexposed group had 0.8 +/- 0.4% (p < 0.001), after adjustment for age, gender, race, smoking, and weight. Further studies in this population are needed to confirm the possible association between the high levels of DNA-Protein crosslink and Cr exposure
— id: 10377, year: 1995, vol: 50, page: 175, stat: Journal Article,

NORMAL VALUES OF DNA-PROTEIN CROSS-LINKS IN MONONUCLEAR BLOOD-CELLS OF A POPULATION OF HEALTHY CONTROLS
TAIOLI, E; ZHITKOVICH, A; TONIOLO, P; COSTA, M
1995 MAR-APR ;8(2):76-79, CANCER JOURNAL
Background - We have previously used a sensitive and simple assay to detect DNA-protein crosslinks, based upon the selective potassium SDS precipitation of DNA associated with protein, DNA-protein crosslinks are a promising biomarker of environmental exposure to carcinogens such as chromate. Methods - We have examined the levels of DNA-protein crosslinks in peripheral blood mononuclear cells of 68 healthy subjects, to determine the normal values of this biomarker in the general population, and to analyze the effect of possible confounding factors, such as age, gender, ethnicity, smoking habits, weight. Results - The mean value of DNA-protein crosslinks was 0.78 +/- 0.04%, No statistically significant differences in DNA-protein crosslinks were observed with gender, ethnicity or weight, No statistically significant correlations were found with age and humber of cigarettes smoked per day. Conclusions - These results set a reference value for DNA-protein crosslinks in healthy subjects,
— id: 87362, year: 1995, vol: 8, page: 76, stat: Journal Article,

Glutathione and free amino acids form stable complexes with DNA following exposure of intact mammalian cells to chromate
Zhitkovich A; Voitkun V; Costa M
1995 Apr;16(4):907-913, Carcinogenesis
Exposure of cells to carcinogenic Cr(VI) compounds results in the formation of several types of DNA lesions such as strand breaks, DNA-protein crosslinks and uncharacterized DNA-Cr adducts. Hexavalent chromium compounds are positive in most bacterial and eukaryotic mutagenic systems, although the nature of DNA modifications underlying the chromium-induced mutagenesis is not known. Hexavalent chromate(VI) is very active in cellular systems because it is actively transported into cells, but intracellularly it is ultimately reduced to Cr(III). Here we show that exposure of Chinese hamster ovary (CHO) cells to potassium chromate(VI) leads to the formation of stable complexes between DNA and amino acids or glutathione. Cysteine, glutamic acid and histidine were the major amino acids crosslinked to DNA in chromate-treated cells. Incubation of purified DNA in the presence of EDTA dissociated SDS stable amino acid-DNA complexes, which indicates that these DNA adducts are most likely to represent ternary coordination complexes mediated by Cr(III) rather than covalent linkage between amino acids/glutathione and DNA. The amino acids that were found complexed with DNA purified from chromate-exposed cells did not orginate from previously crosslinked proteins during DNA isolation, but represented authentic reactions of free amino acids and glutathione with chromium and DNA in cells. Ternary complexes of glutathione or amino acids with Cr(III) and DNA were estimated to account for as much as 50% of DNA-bound chromium following exposure to < or = 25 microM chromate
— id: 6795, year: 1995, vol: 16, page: 907, stat: Journal Article,

GLUTATHIONE AND FREE AMINO-ACIDS FORM STABLE ADDUCTS WITH DNA FOLLOWING EXPOSURE OF CHO CELLS TO CHROMATE
ZHITKOVICH, A; VOITKUN, V; COSTA, M
1995 JAN 5 ;336(3):198-198, Journal of cellular biochemistry
— id: 87354, year: 1995, vol: 336, page: 198, stat: Journal Article,

The role of oxygen radicals in the nickel-induced toxicity and carcinogenicity
Costa M
Frontiers of reactive oxygen species in biology and medicine Amsterdam : Excerpta Medica, 1994,
— id: 4416, year: 1994, vol: , page: 539, stat: Chapter,

Molecular mechanisms of nickel carcinogenesis
Costa M; Salnikow K; Cosentino S; Klein CB; Huang X; Zhuang Z
1994 Sep;102 Suppl 3:127-130, Environmental health perspectives
Carcinogenic, water-insoluble Ni compounds are phagocytized by cells; and the particles undergo dissolution inside the cell, releasing Ni ions that interact with chromatin. Ni produces highly selective damage to heterochromatin. The longest contiguous region of heterochromatin in the Chinese hamster genome is found on the q arm of the X chromosome, and this region is selectively damaged by Ni. More than half of the male mice in which there were Ni-induced transformations of Chinese hamster cells exhibited complete deletion of the long arm of the X chromosome. The introduction of a normal X chromosome into these cells resulted in cellular senescence, suggesting that the Ni interacted with Chinese hamster genome to inactivate a senescence gene. Investigations were conducted into the mechanisms by which Ni produced damage to chromatin. Ni ions have a much higher affinity for proteins and amino acids than for DNA (by five to seven orders of magnitude). Therefore, Ni interacted with chromatin because of the protein present, not because of its reactivity for DNA. Studies have shown that Ni produced an increase in oxidative products in cells as indicated by oxidation of the fluorescent dye dichlorofluorescein; Ni has also been shown to produce oxidation of proteins in cells, as measured by carbonyl formation. Ni cross-linked certain amino acids and proteins to DNA. These covalent cross-links were not dissociated by EDTA and are inconsistent with direct Ni involvement, but they are consistent with Ni acting catalytically. Using subtractive hybridization, we have isolated a number of clones that are expressed in normal but not in Ni-transformed cells.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 8073, year: 1994, vol: 102 Suppl 3, page: 127, stat: Journal Article,

Molecular mechanisms of nickel carcinogenesis
Costa M; Zhuang Z; Huang X; Cosentino S; Klein CB; Salnikow K
1994 Jun 6;148(2-3):191-199, Science of the total environment
Nickel treatment of intact cultured cells oxidized dichlorofluorescin to a fluorescent product indicating that nickel elevated the level of oxidants in cells. Nickel also caused an increase in crosslinking of amino acids to DNA and these complexes did not appear to involve the direct participation of Ni2+. Histidine, cysteine and tyrosine were prominent among the amino acids crosslinked to DNA. Nickel selectively damaged heterochromatin and this resulted in deletions of heterochromatic regions during nickel carcinogenesis. Thrombospondin was one of the genes expressed in normal cells that was not expressed in nickel-transformed cells. Other aspects of the molecular mechanism of nickel carcinogenesis are discussed
— id: 10380, year: 1994, vol: 148, page: 191, stat: Journal Article,

MOLECULAR MECHANISMS OF NICKEL CARCINOGENESIS - INHERITED EFFECTS ON TRANSCRIPTION
COSTA, M
1994 MAR 13 ;207(11):421-INOR, Abstracts of papers (American Chemical Society)
— id: 52570, year: 1994, vol: 207, page: 421, stat: Journal Article,

2ND INTERNATIONAL MEETING ON MOLECULAR MECHANISMS OF METAL TOXICITY AND CARCINOGENICITY - JANUARY 10-17, 1993 MADONNA-DI-CAMPIGLIO, ITALY - PREFACE
COSTA, M; CANTONI, O; KLEIN, C; ROSSMAN, T; SNOW, E; SUK, W
1994 SEP ;102(1):2-2, Environmental health perspectives
— id: 52335, year: 1994, vol: 102, page: 2, stat: Journal Article,

ALTERATION IN THE PHOSPHORYLATION OF THE RB PROTEIN IN NICKEL-TRANSFORMED HUMAN-CELLS
COSTA, M; DOWJAT, WK; LIN, XL
1994 FEB 13 ;15(3):194-194, Journal of cellular biochemistry
— id: 52509, year: 1994, vol: 15, page: 194, stat: Journal Article,

Second International Meeting on Molecular Mechanisms of Metal Toxicity and Carcinogenicity : January 10-17, 1993, Madonna di Campiglio, Italy
Costa, Max
Research Triangle Park, NC : National Institute of Environmental Health Sciences, 1994,
— id: 1219, year: 1994, vol: , page: , stat: ,

Crystalline Ni3S2 specifically enhances the formation of oxidants in the nuclei of CHO cells as detected by dichlorofluorescein
Huang X; Klein CB; Costa M
1994 Mar;15(3):545-548, Carcinogenesis
Dichlorofluorescein (DCF) was used as a fluorescent probe to detect oxidants formed in cultured CHO cells during nickel treatment. Crystalline Ni3S2 specifically enhanced the formation of oxidants in the nuclei of these living cells, but Ni3S2 particles did not enhance DCF fluorescence as much when added in vitro to isolated nuclei. Our results add to the emerging concept that oxidants mediated by nickel compounds may play an important role in nickel-induced genotoxicity
— id: 6409, year: 1994, vol: 15, page: 545, stat: Journal Article,

The role of nickel and nickel-mediated reactive oxygen species in the mechanism of nickel carcinogenesis
Huang X; Zhuang Z; Frenkel K; Klein CB; Costa M
1994 Sep;102 Suppl 3:281-284, Environmental health perspectives
Increasing evidence demonstrates the reactive oxygen species (ROS) are implicated in metal carcinogenesis. Exposure of cultured Chinese hamster ovary (CHO) cells to several nickel compounds, i.e. NiS, Ni3S2, NiO (black and green), and NiCl2 has been shown to increase oxidation of 2',7-dichlorofluorescein to the fluorescent 2',7-dichlorofluorescein (DCF), suggesting that nickel compounds increased the concentration of oxidants in CHO cells. This fluorescence can be attenuated by addition of exogenous catalase to the extracellular media, indicating that H2O2 is one of the formed oxidants in this system. Fluorimetric measurements of chromogens following thiobarbituric acid reaction showed that nickel compounds also induce lipid peroxidation with a decreasing potency NiS, Ni3S2 > black NiO > green NiO > NiCl2. These results suggest that lipid hydroperoxides may also be produced through the action of nickel in intact cells. MgCl2, an antagonist of Ni-induced DNA strand breaks and cell transformation, has no effect on the formation of DCF fluorescence induced in CHO cells by nickel. The results suggest that nickel is an active inducer of ROS in intact mammalian cells and that the molecular mechanism of nickel carcinogenesis may involve multiple steps of nickel-mediated ROS
— id: 6647, year: 1994, vol: 102 Suppl 3, page: 281, stat: Journal Article,

Metal mutagenesis in transgenic Chinese hamster cell lines
Klein CB; Kargacin B; Su L; Cosentino S; Snow ET; Costa M
1994 Sep;102 Suppl 3:63-67, Environmental health perspectives
Metals are toxic agents for which genotoxic effects are often difficult to demonstrate. To study metal mutagenesis, we have used two stable hprt/gpt+ transgenic cell lines that were derived from Chinese hamster V79 cells. Both the G12 and G10 cell lines are known to be very sensitive to clastogens such as X-rays and bleomycin, with the mutagenic response of the integrated xanthine guanine phosphoribosyl transferase (gpt) gene in G10 usually exceeding that of the same gene in the transgenic G12 cells. In studies with carcinogenic insoluble nickel compounds, a high level of mutagenesis was found at the gpt locus of G12 cells but not at the endogenous hypoxanthine phosphoribosyl transferase (hprt) locus of V79 cells. We have since demonstrated the similar recovery of a high frequency of viable G12 mutants with other insoluble nickel salts including nickel oxides (black and green). The relative mutant yield for the insoluble nickel compounds (G12 > G10) is the opposite of that obtained with nonmetal clastogens (G10 > G12). In the G12 cells, nickel mutagenesis may be related to the integration of the gpt sequence into a heterochromatic region of the genome. For some of the insoluble nickel compounds, significant inhibition of both cytotoxicity and mutant yield resulted when the G12 cells were pretreated with vitamin E. In comparison with the nickel studies, the mutagenic responses to chromium compounds in these cell lines were not as dramatic. Mutagenesis of the gpt target could not be demonstrated with other metals such as mercury or vanadium
— id: 6661, year: 1994, vol: 102 Suppl 3, page: 63, stat: Journal Article,

Transformation of human osteoblasts to anchorage-independent growth by insoluble nickel particles
Lin X; Costa M
1994 Sep;102 Suppl 3:289-292, Environmental health perspectives
Nickel compounds are well established by epidemiologic studies as human carcinogens. Although the carcinogenicity of nickel compounds has been studied in experimental animals and in a variety of cultured mammalian cells, there are only sporadic reports of nickel-induced transformation of human cells. In attempts to study the mechanisms of nickel-induced carcinogenesis in human cells, an immortalized human osteoblastic cell line (HOS) that could not grow in soft agar or form tumors in athymic nude mouse was repeatedly treated with a water-soluble nickel compound (NiCl2) or a less water-soluble nickel compound crystalline (NiS). After three rounds of NiS treatment, there was an increase in anchorage-independent (AI) colony formation. This was not found in untreated or NiCl2-treated cells. Ten AI colonies obtained from NiS-treated cells were isolated. All of these clones showed changes in cell morphology, including the appearance of uniform polygon shape, growth in multilayers, and heavy staining with Giemsa. Most of these clones were retested for their ability to grow in soft agar and showed growth efficiencies of 5 to 50%. It has been shown by other investigators that aggregate growth is well correlated with tumorigenic potential in viral or chemical transformants of HOS cells. Four of seven tested NiS-transformed clones were able to form large aggregates compared to their untransformed counterparts, and continued to proliferate in aggregate form when they were plated on 0.9% agar. Current investigations focus on the molecular and genetic changes induced by nickel compounds in these human cells
— id: 6682, year: 1994, vol: 102 Suppl 3, page: 289, stat: Journal Article,

Nickel-induced transformation of human cells causes loss of the phosphorylation of the retinoblastoma protein
Lin X; Dowjat WK; Costa M
1994 May 15;54(10):2751-2754, Cancer research
The retinoblastoma (Rb) protein (pRb) has been studied in various crystalline NiS-transformed cell clones derived from the human osteoblast cell line, HOS TE-85. The parental HOS cells were not able to proliferate in soft agar medium, but they acquired this property following treatment with crystalline NiS. The pRb was found only in the hypophosphorylated form in 8 of 9 nickel-transformed clones examined, whereas in the parental cells the pRb appeared in both phosphorylated and unphosphorylated forms. Neither Rb gene expression nor its phosphorylation was affected by acute nickel treatments of HOS cells. The nickel-transformed HOS clones expressed the major regulators of Rb phosphorylation, cyclin E and cdk-2, at levels similar to those of the parental cells. In coimmunoprecipitation assays with cell lysates from the transformed clones that exhibited the hypophosphorylated form of pRb, the Rb protein failed to form a complex with simian virus 40 large T-antigen, indicating a lack of functional activity. When a plasmid containing the normal Rb gene was transfected into these nickel-transformed cells, it restored the Rb phosphorylation pattern observed in parental cells and the cells acquired a normal phenotype (i.e., they were no longer able to grow in soft agar). This suggested that a mutation was induced in nickel-transformed cells that affected the ability of the Rb protein to be phosphorylated and function normally, and this mutation allowed the human nickel-transformed cells to acquire anchorage-independent growth
— id: 6440, year: 1994, vol: 54, page: 2751, stat: Journal Article,

Loss of thrombospondin transcriptional activity in nickel-transformed cells
Salnikow K; Cosentino S; Klein C; Costa M
1994 Jan;14(1):851-858, Molecular & cellular biology
mRNA from normal Chinese hamster embryo (CHE) cells was transcribed to cDNA and subtracted with an excess of mRNA from Chinese hamster embryo cells transformed by nickel compounds. Here we report the recovery of a sequence found to be highly homologous to the mouse thrombospondin 1 gene that was obtained by this subtraction procedure. Since thrombospondin is antiangiogenic, cancer cells expressing high levels of thrombospondin cannot grow in vivo because capillaries will not proliferate to cells secreting thrombospondin. To examine expression of thrombospondin, normal CHE cells were stained with monoclonal antibodies to human thrombospondin. The protein was present abundantly in the cytoplasm of normal cells but at greatly reduced levels in Ni-transformed cells. Analysis of mRNA by Northern (RNA) blot revealed transcripts in normal cells but little thrombospondin mRNA in Ni-transformed cells. Loss of thrombospondin mRNA expression was related to Ni treatment rather than transformation, since Ni-resistant cells also exhibited fewer thrombospondin transcripts than did wild-type cells. Digestion of genomic DNA with various combinations of restriction enzymes revealed thrombospondin gene patterns that were identical in both cell types, suggesting that there were no major deletions or rearrangements of the gene in the nickel-transformed cells. The inactivation of the thrombospondin gene was further investigated by analyzing the promoter activity of this gene linked to a chloramphenicol acetyltransferase (CAT) reporter plasmid that was transfected into normal and Ni-transformed cells.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 6507, year: 1994, vol: 14, page: 851, stat: Journal Article,

Altered oxidative stress responses in nickel-resistant mammalian cells
Salnikow K; Gao M; Voitkun V; Huang X; Costa M
1994 Dec 15;54(24):6407-6412, Cancer research
BALB 3T3 cells exposed to NiCl2 acquired resistance to concentrations as high as 200 microM and retain resistance for many generations in the absence of nickel. This resistance was not due to alterations in uptake or to metallothionein overexpression. The nickel-resistant B200 cell line was found to also exhibit cross-resistance to hydrogen peroxide and menadione. These nickel-resistant cells had 1.8 times higher basal levels of glutathione compared to wild-type cells. Studies with the glutathione synthesis inhibitor buthionine sulfoximine showed that while glutathione turnover was more rapid in the nickel-resistant cells, its depletion following NiCl2 treatment of the parental BALB 3T3 cell line was greater than in the nickel-resistant B200 cells. The reduced level of binding of NFkB and AP-1 transcription factors to their DNA consensus sequences in B200 cells compared to wild-type cells, and their more reactive response following treatment of resistant cells with H2O2 or buthionine sulfoximine, strengthens the hypothesis that nickel resistance is closely allied to oxidative stress responses
— id: 6738, year: 1994, vol: 54, page: 6407, stat: Journal Article,

The loss of thrombospondin expression in Ni-resistant and Ni-transformed cells
Salnikow K; Wang S; Gao M; Klein C; Costa M
1994 ;35:A1122-A1122, Proceedings of the annual meeting of the American Association for Cancer Research
Using subtractive hybridization, we have cloned and identified several genes which were not expressed in Ni-transformed Chinese hamster cells. It appeared that one of these genes, thrombospondin-1, was also downregulated in Ni-resistant mouse cells suggesting that this gene can be specifically affected by Ni. Using different fragments of the thrombospondin promoter linked to the CAT reporter gene, we investigated mechanisms of decreased expression of this gene in Ni-resistant and Ni-transformed cells. Cotransfection of these reporter plasmids with an Rb expression vector significantly stimulated activity of the thrombospondin promoter, showing that the absence of some other transcription factor(s) that interacts with the Rb product resulted in the decrease of thrombospondin expression. Band-shift analysis confirmed that nuclear extracts of Ni-resistant cells less efficiently bind to an Sp-1 consensus sequence oligonucleotide. Identification of the transcriptional regulators involved in thrombospondin expression and its downregulation in Ni-resistant and Ni-transformed cells is ongoing
— id: 6019, year: 1994, vol: 35, page: A1122, stat: Journal Article,

EFFECT OF THE RB ON THROMBOSPONDIN TRANSCRIPTION IN NORMAL AND NICKEL-TRANSFORMED CELLS
SALNIKOW, K; COSTA, M
1994 FEB 13 ;15(3):192-192, Journal of cellular biochemistry
— id: 52508, year: 1994, vol: 15, page: 192, stat: Journal Article,

Application of reliability models to studies of biomarker validation
Taioli E; Kinney P; Zhitkovich A; Fulton H; Voitkun V; Cosma G; Frenkel K; Toniolo P; Garte S; Costa M
1994 Mar;102(3):306-309, Environmental health perspectives
We present a model of biomarker validation developed in our laboratory, the results of the validation study, and the impact of the estimation of the variance components on the design of future molecular epidemiologic studies. Four different biomarkers of exposure are illustrated: DNA-protein cross-link (DNA-PC), DNA-amino acid cross link (DNA-AA), metallothionein gene expression (MT), and autoantibodies to oxidized DNA bases (DNAox). The general scheme for the validation experiments involves n subjects measured on k occasions, with j replicate samples analyzed on each occasion. Multiple subjects, occasions, and replicates provide information on intersubject, intrasubject, and analytical measurement variability, respectively. The analysis of variance showed a significant effect of batch variability for DNA-PC and MT gene expression, whereas DNAox showed a significant between-subject variability. Among the amino acids tested, cysteine and methionine showed a significant contribution of both batch and between-subject variability, threonine showed between-subject variability only, and tyrosine showed between-batch and between-subject variability. The total variance estimated through the experiment was used to calculate the minimum sample size required for a future epidemiologic study including the same biomarkers used for the reliability study. Such validation studies can detect the various components of variability of a biomarker and indicate needed improvements of the assay, along with possible use in field studies
— id: 10381, year: 1994, vol: 102, page: 306, stat: Journal Article,

Complexing of amino acids to DNA by chromate in intact cells
Voitkun V; Zhitkovich A; Costa M
1994 Sep;102 Suppl 3:251-255, Environmental health perspectives
Using o-pthaldialdehyde (OPT) fluorescence, the amino acids associated with DNA were studied following exposure of intact Chinese hamster ovary cells to chromate. Rigorous extraction with EDTA, acid, or base was required to release the amino acids cross-linked to the DNA isolated from control or chromate-treated cells by standard procedures (i.e., proteinase K, phenol, etc.). Amino acids resisting extraction from DNA were not studied since analysis was limited to those that could be released by these procedures. There was a chromate dose-dependent increase in amino acids complexed with the DNA that could be released by EDTA, acid, and base, and these amino acids were separated by HPLC and identified. Substantial increases in cysteine, glutamine, glutamic acid, histidine, threonine, and tyrosine were found as a function of increasing concentrations of chromate. There was also a time-dependent increase in complexing of these amino acids to the DNA by chromate. The amino acids found complexed to DNA in intact cells by chromate were thought to originate from reactions of free amino acids or small peptides with the DNA rather than being proteolytic products derived from larger proteins that were cross-linked to the DNA. This was supported by a number of experiments: a) free amino acids or bovine serum albumin (BSA) were cross-linked by chromium to DNA in vitro and the DNA was isolated by standard procedures.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 6768, year: 1994, vol: 102 Suppl 3, page: 251, stat: Journal Article,

Development of an 125I-postlabeling assay as a simple, rapid, and sensitive index of DNA-protein cross-links
Zhuang Z; Costa M
1994 Sep;102 Suppl 3:301-304, Environmental health perspectives
A rapid, simple, and sensitive 125I-postlabeling technique has been developed to allow detection of DNA-protein cross-links induced by environmental contaminants and carcinogens. This method is based on specific incorporation of 125I into tyrosine residues associated with DNA. Cultured Chinese hamster ovary cells were exposed to various crosslinking agents, e.g., UV light, K2CrO4, or NiCl2. DNA was isolated by proteinase K/phenol/chloroform. The residual peptides cross-linked to DNA were radioiodinated with Na125I and chloramine T. After repeated precipitation with ethanol, the radioactivity was determined. The 125I method was compared with a 3[H]-tyrosine prelabeling method and found to be of similar sensitivity
— id: 6796, year: 1994, vol: 102 Suppl 3, page: 301, stat: Journal Article,

Protein oxidation and amino acid-DNA crosslinking by nickel compounds in intact cultured cells
Zhuang Z; Huang X; Costa M
1994 Jun;126(2):319-325, Toxicology & applied pharmacology
Oxidative damage induced by NiCl2 to protein has been investigated. We found that nickel induced a dose-dependent increase in the oxidation of bovine serum albumin (BSA) as detected by carbonyl formation in the presence of H2O2 in vitro, as well as producing carbonyl of proteins in intact cultured Chinese hamster ovary (CHO) cells. Other metals capable of producing oxidative damage to BSA in the presence of H2O2 included Cu, Co, Fe, and chromate. However, Cd2+, Hg2+, and Pb2+ were not active even in the presence of H2O2. As an indicator of nickel-induced genotoxic damage, the crosslinking of amino acids to DNA was also examined. Cysteine, histidine, and tyrosine were increased in their association with DNA based upon their persistent binding to DNA following washes with EDTA or SDS. The results suggest that DNA-protein or DNA-amino acids crosslinks induced by nickel may result from interaction of the nickel-oxidized carbonyl group of protein, peptides, and free amino acids
— id: 6563, year: 1994, vol: 126, page: 319, stat: Journal Article,

Mechanisms of chromium carcinogenicity and toxicity
Cohen MD; Kargacin B; Klein CB; Costa M
1993 ;23(3):255-281, Critical reviews in toxicology
Chromium, like many transition metal elements, is essential to life at low concentrations yet toxic to many systems at higher concentrations. In addition to the overt symptoms of acute chromium toxicity, delayed manifestations of chromium exposure become apparent by subsequent increases in the incidence of various human cancers. Chromium is widely used in numerous industrial processes, and as a result is a contaminant of many environmental systems. Chromium, in its myriad chemical forms and oxidation states, has been well studied in terms of its general chemistry and its interactions with biological molecules. However, the precise mechanisms by which chromium is both an essential metal and a carcinogen are not yet fully clear. The following review does not seek to embellish upon the proposed mechanisms of the toxic and carcinogenic actions of chromium, but rather provides a comprehensive review of these theories. The chemical nature of chromium compounds and how these properties impact upon the interactions of chromium with cellular and genetic targets, including animal and human hosts, are discussed
— id: 6337, year: 1993, vol: 23, page: 255, stat: Journal Article,

Molecular targets of nickel and chromium in human and experimental systems
Costa M
1993 ;19 Suppl 1:71-74, Scandinavian journal of work environment & health
Water-insoluble nickel compounds tend to be carcinogenic because they enter cells through phagocytosis. Highly water-soluble nickel salts, such as nickel chloride, do not enter cells well and thus are not carcinogenic in vivo. Carcinogenic nickel compounds produce selective damage to heterochromatic regions in Chinese hamster chromosomes, probably because of the high concentration of proteins and amino acids in this highly condensed genetically inactive chromatin region. Bivalent nickel ions are highly affinitive to amino acids as compared with deoxyribonucleic acid (DNA). The interaction with proteins is believed to cause this effect. In addition, when nickel binds to proteins, it can be oxidized, and DNA protein damage ensues. A new method detects DNA-protein cross-links induced by various agents, such as nickel, chromium and cis-platinum. It has demonstrated an increase in DNA-protein cross-links in cultured cells from animals treated with chromate and welders exposed to welding fumes
— id: 6342, year: 1993, vol: 19 Suppl 1, page: 71, stat: Journal Article,

Preliminary report on a simple new assay for DNA-protein cross-links as a biomarker of exposures experienced by welders
Costa M; Zhitkovich A; Taioli E; Toniolo P
1993 Oct-Nov;40(2-3):217-222, Journal of toxicology & environmental health
A method originally developed to detect topoisomerase DNA complexes has been adapted to determine DNA-protein cross-links formed in cells following their exposure in vitro and in vivo to cross-linking agents. A preliminary study on welders and controls has been carried out to assess the feasibility of this assay to detect human exposure to chromium (Cr) and nickel (Ni) that are associated with increased DNA-protein cross-links. The percentage of cross-link was significantly higher among welders (1.85% +/- 1.14) than among controls (1.17% +/- 0.46; p = .01) Cross-links were not affected by age, weight, or smoking status. The assay developed has substantial advantages over previous methods, being considerably more simple, inexpensive, and sensitive
— id: 6343, year: 1993, vol: 40, page: 217, stat: Journal Article,

DNA-protein cross-links in welders: molecular implications
Costa M; Zhitkovich A; Toniolo P
1993 Feb 1;53(3):460-463, Cancer research
A new method for detecting DNA-protein cross-links involving selective precipitation of DNA containing cross-linked proteins by K(+)-sodium dodecyl sulfate was utilized in the peripheral WBC of 21 male metal arc welders and in 26 male controls of similar age and racial characteristics who were not exposed to welding fumes. DNA was quantitated by Hoechst fluorescence. Although the concentration of nickel and chromium in the peripheral blood did not differ between subjects in the two groups, one-fourth of the welders had levels of DNA-protein cross-links that were above the upper limit of the controls. Mean cross-link values were 1.85 +/- 1.14% (SD) among the welders and 1.17 +/- 0.46% among the controls, a 58% statistically significant difference (P = 0.01). Thus, many welders appeared to be burdened with an excess of DNA-protein cross-links, suggesting exposure to cross-linking agents and, possibly, a detectable biological effect of potential genotoxic consequences
— id: 10386, year: 1993, vol: 53, page: 460, stat: Journal Article,

Nickel induces increased oxidants in intact cultured mammalian cells as detected by dichlorofluorescein fluorescence
Huang X; Frenkel K; Klein CB; Costa M
1993 May;120(1):29-36, Toxicology & applied pharmacology
Exposure of intact cultured Chinese hamster ovary (CHO) cells to water-soluble nickel (Ni) salts and to relatively water-insoluble crystalline nickel subsulfide (Ni3S2) resulted in an increased formation of the fluorescent oxidized compound, dichlorofluorescein (DCF) from the parent nonfluorescent compound, 2,7-dichlorofluorescin diacetate. This fluorescent product was also formed in vitro following oxidation with relatively strong oxidants such as hydrogen peroxide in the presence of peroxidase, suggesting that Ni increased the concentration of hydrogen peroxide in intact cells. However, formation of other strong oxidants such as hydroperoxides is possible since they have also been shown to cause the oxidation of the nonfluorescent dichlorofluorescin to the fluorescent product DCF in vitro. Localization of the oxidized fluorescent DCF in intact cells was also examined by fluorescence microscopy. Both Ni3S2 and NiCl2 appeared to increase the degree of fluorescence in intact CHO cells around the nuclear membranes. This increase in fluorescence was greater in the presence of relatively water-insoluble Ni3S2 than water-soluble NiCl2. These results add to the emerging concept that Ni-induced genotoxicity may be mediated by oxygen radical intermediates
— id: 10384, year: 1993, vol: 120, page: 29, stat: Journal Article,

Mutagenic responses of nickel oxides and nickel sulfides in Chinese hamster V79 cell lines at the xanthine-guanine phosphoribosyl transferase locus
Kargacin B; Klein CB; Costa M
1993 Jun;300(1):63-72, Mutation research
Mutagenesis of several insoluble nickel compounds--crystalline nickel sulfide NiS, nickel subsulfide Ni3S2, nickel oxides (black and green) and soluble NiCl2 was studied in three Chinese hamster cell lines--at the hprt gene of the well-defined V79 cell line, and at gpt in two transgenic derivative cell lines G12 and G10. The transgenic cell line G12 responded very strongly to the insoluble Ni compounds, such that the gpt mutagenesis was at least 20 times higher than the spontaneous mutagenesis and in some experiments was even higher. In contrast the response of the G10 cells was much lower--the mutant frequencies only increased 2-3 times over the controls. In V79 cells, NiS and NiO (black) did not induce a mutagenic response at hprt. Soluble NiCl2 also exhibited no mutagenic activity in V79 cells and induced considerably lower activity than the insoluble compounds in the transgenic G12 cells. Following vitamin E pretreatment of G12 cells for 24 h prior to nickel exposure, increased cell survival was observed for several insoluble Ni compounds whereas vitamin E had no effect on NiCl2 cytotoxicity. With vitamin E pretreatment, significantly lower mutagenic responses in G12 cells were also noted for some insoluble Ni compounds, while no such effect was observed for NiCl2
— id: 10383, year: 1993, vol: 300, page: 63, stat: Journal Article,

COMPARISON OF THE UPTAKE AND DISTRIBUTION OF CHROMATE IN RATS AND MICE
KARGACIN, B; SQUIBB, KS; COSENTINO, S; ZHITKOVICH, A; COSTA, M
1993 MAR ;36(3):307-318, Biological trace element research
The purpose of this study was to evaluate species differences in tissue accumulation of chromium. Rats and mice were orally exposed to Cr(VI) (potassium chromate) via drinking water (8 mg/d/kg body wt for 4 or 8 wk), or by ip injection (0.3 and 0.8 mg/d/kg for 4 or 14 d). Chromium concentrations were measured by atomic absorption spectrophotometry, and tissues were compared for exposure route and species differences. After oral exposure, irrespective of treatment duration, liver concentrations of chromium were three to four times higher in mice than rats, whereas kidney concentrations were about 50% lower. However, after ip injection, kidney and blood concentrations in rats were two- and four-fold higher, respectively. Both rats and mice showed high values of Cr concentration in the bone. After single ip injection of (Na2CrO4)-Cr-51, Cr concentrations were higher in the blood of rats than mice both after 24 and 72 h. Red blood cell concentrations of Cr were also greater in rats than mice by approximately threefold, whereas white blood cell Cr concentrations were higher in mice than rats. There was also a twofold greater binding of Cr/mumol of hemoglobin in rats compared to mice. These data indicate that species differences exist for Cr metabolism and that they differ with respect to the route of exposure. These results may be owing to species differences in the reduction of Cr and different binding of Cr to hemoglobin
— id: 54334, year: 1993, vol: 36, page: 307, stat: Journal Article,

Development and utilization of a new simple assay for DNA-protein crosslinks as a biomarker of exposure to welding fumes
Toniolo P; Zhitkovich A; Costa M
1993 ;65(1 Suppl):S87-S89, International archives of occupational & environmental health
A new method for DNA-protein crosslinks involving selective precipitation of DNA containing crosslinked proteins by K+ sodium dodecyl sulfate was utilized in the peripheral white blood cells of 21 male metal arc welders and in 26 male controls of similar age and racial characteristics who were not exposed to welding fumes. DNA was quantitated by Hoescht fluorescence. Although the concentration of nickel and chromium in the peripheral blood was low and did not differ between subjects in the two groups, one-fourth of the welders had levels of DNA-protein crosslinks that were above the upper limit of the controls. Mean crosslink values were 1.85% (+/- 1.14) among the welders and 1.17% (+/- 0.46) among the controls, a 58%, statistically significant difference (P = 0.01). Thus, many welders appeared to be burdened with an excess of DNA-protein crosslinks suggesting exposure to crosslinking agents and, possibly, a detectable biologic effect of potential genotoxic consequences
— id: 6540, year: 1993, vol: 65, page: S87, stat: Journal Article,

Trace elements : alumninum, arsenic, cadmium, mercury and nickel
Bhamra RK; Costa M
Environmental toxicants : human exposures and their health effects New York : Van Nostrand Reinhold, 1992,
— id: 4412, year: 1992, vol: , page: 575, stat: Chapter,

Chromium compounds
Cohen MD; Costa M
Environmental and occupational medicine Boston : Little Brown, 1992,
— id: 4413, year: 1992, vol: , page: 799, stat: Chapter,

Forward mutations and DNA-protein crosslinks induced by ammonium metavanadate in cultured mammalian cells
Cohen MD; Klein CB; Costa M
1992 Sep;269(1):141-148, Mutation research
Ammonium metavanadate yielded a dose-dependent increase in mutation frequency at the V79 hprt locus following a 24-h exposure period in serum-free F12 medium. Vanadate also increased the mutation frequency of V79 cells by exposure of cells in salts-glucose medium, but these effects were not as striking, or as dose-dependent as they were in serum-free F12 medium. Ammonium metavanadate enhanced the mutation frequency in a V79 variant containing a transfected bacterial gpt gene. These cells are known to be more responsive to oxidative type mutations, and to mutations involving deletions. Although the absolute level of mutations was greater in these cells with ammonium metavanadate, so was the background, and these cells did not exhibit an enhanced mutagenic response to vanadate when compared to the wild-type V79 cells. The vanadate results were compared to a positive control potassium chromate, which exhibited a dose-dependent increase in mutation frequency. Ammonium metavanadate induced DNA-protein crosslinks formation in both Chinese hamster ovary and human MOLT4 cells, and the role of these relatively unrepaired genetic lesions in the mutations produced by vanadate and chromate are discussed
— id: 13468, year: 1992, vol: 269, page: 141, stat: Journal Article,

Deletion of a senescence gene as a mechanism of nickel carcinogenesis
Costa M
1992 ;4(Special Supplement):481-485, Chemical speciation & bioavailability
Issue titled "Metal compounds in environment and life"
— id: 72790, year: 1992, vol: 4, page: 481, stat: Journal Article,

Involvement of heterochromatin damage in nickel-induced transformation and resistance
Costa M; Conway K; Imbra R; Wang XW
Nickel and human health : current perspectives New York : Wiley, 1992,
— id: 4415, year: 1992, vol: , page: 295, stat: Chapter,

Analysis of residual amino acid--DNA crosslinks induced in intact cells by nickel and chromium compounds
Lin X; Zhuang Z; Costa M
1992 Oct;13(10):1763-1768, Carcinogenesis
Chinese hamster ovary cells were incubated with radioactive amino acids, the DNA was isolated by standard proteinase K/phenol/chloroform extraction and residual amino acids complexed to the DNA were examined as an index of metal induced DNA-protein crosslinks. Using this method, both chromate and nickel caused residual histidine and cysteine to be complexed with the DNA isolated from metal-treated cells. In the case of chromate, a number of amino acids were studied and Tyr, Thr and Cys were found to be complexed to DNA at a level (above the untreated control) that was statistically significant. Stability studies indicated that some of the chromate-induced DNA-protein complexes were mediated by direct participation of chromium(III), whereas others that were resistant to dissociation by EDTA and mercaptoethanol did not seem to involve direct chromium(III) participation. A significant portion of the cysteine complexed to DNA by chromate was believed to involve glutathione since treatment of cells with cycloheximide did not decrease chromate-induced cysteine-DNA crosslinks. In the case of nickel, most of the stable DNA-protein crosslinks did not involve direct metal participation and were probably oxidatively mediated by Ni(II)/Ni(III) redox cycling. These findings present new methodology for analysis of DNA-protein crosslinks by examination of residual amino acids associated with the DNA. This method should be highly sensitive and will yield important information about the mechanism of metal-induced DNA-protein crosslinks
— id: 10393, year: 1992, vol: 13, page: 1763, stat: Journal Article,

Analysis of the binding sites of chromium to DNA and protein in vitro and in intact cells
Salnikow K; Zhitkovich A; Costa M
1992 Dec;13(12):2341-2346, Carcinogenesis
Previous studies have examined Cr(III), or CrO4 reduced to Cr(III), binding in vitro to DNA. However, there have been few studies examining chromate binding to DNA in intact cells. Treatment of intact cells with chromate (Na2(51)CrO4) resulted in chromium (Cr) binding to DNA. The binding of Cr to DNA was much more stable when more residual peptide/amino acids were associated with DNA. A substantial portion of the Cr bound to DNA was released by treatment with EDTA, suggesting that trivalent Cr was the major oxidation state of Cr bound to DNA. Cr(III) stimulated the formation of amino acid-DNA and protein-DNA complexes in vitro. Tyrosine and cysteine exhibited the highest activity in being complexed to DNA by Cr(III) in vitro, while histidine, methionine and threonine also exhibited more activity than any other amino acid. Similar results were found in intact cells. The activity of proteins complexed to DNA by trivalent Cr depended upon the content of these reactive amino acids. Thus, bovine serum albumin was more active than actin, which in turn was more active than histones. These and other studies presented suggested that Cr(III) was involved directly in the formation of DNA-protein complexes in intact cells, unlike other metals such as Ni(II), which are thought to form DNA-protein cross-links catalytically and not participate directly in the complex. The majority of trivalent Cr associated with DNA was bound to the phosphate backbone without exhibiting any base specificity. Collectively, these results indicate that trivalent Cr creates DNA protein crosslinks by binding with reactive amino acids (i.e. cysteine, tyrosine or histidine) and linking these to the phosphate backbone of DNA
— id: 10392, year: 1992, vol: 13, page: 2341, stat: Journal Article,

Nickel and nickel carcinogenesis
Snow ET; Costa M
Environmental and occupational medicine Boston : Little Brown, 1992,
— id: 4414, year: 1992, vol: , page: 807, stat: Chapter,

POTENTIATION OF SODIUM CHROMATE(VI)-INDUCED CHROMOSOMAL-ABERRATIONS AND MUTATION BY VITAMIN-B2 IN CHINESE-HAMSTER V79-CELLS
SUGIYAMA, M; TSUZUKI, K; LIN, XH; COSTA, M
1992 NOV ;283(3):211-214, Mutation research. DNA repair
The effect of vitamin B2, which is capable of reducing chromium(VI) to chromium(V), on chromosomal aberrations and mutation caused by Na2CrO4 was investigated in Chinese hamster V79 cells. Pretreatment with 200 muM vitamin B2 (riboflavin) for 24 h prior to exposure to Na2CrO4 (2.5-5 muM) resulted in an increase of metal-induced chromosomal aberrations and mutation at the HGPRT locus. These and other previous studies suggest that vitamin B2 enhances the clastogenic and mutagenic action of chromate compounds, through its ability to directly reduce chromium(VI) in cells
— id: 51832, year: 1992, vol: 283, page: 211, stat: Journal Article,

A conserved region in human and Chinese hamster X chromosomes can induce cellular senescence of nickel-transformed Chinese hamster cell lines
Wang XW; Lin X; Klein CB; Bhamra RK; Lee YW; Costa M
1992 Apr;13(4):555-561, Carcinogenesis
Cellular senescence is the genetically programmed cessation of cellular proliferation. We have recently mapped a putative senescence gene(s) on the X chromosome of Chinese hamster embryo (CHE) cells. In the present study, we have utilized microcell-mediated chromosome transfer (microcell fusion) to test whether: (i) the human X chromosome exhibits similar genetic potential to induce senescence and (ii) the deletion or inactivation of the X-linked senescence gene(s) in CHE cells is associated with nickel-induced immortalization. A normal CHE or human X chromosome was first introduced into mouse-cell hybrids, then transferred by microcell fusion into a nickel-transformed, immortal male CHE cell line (Ni-2/TGR) with an X deletion (Xq1). Microcell fusion of the normal CHE X chromosome into tumorigenic Ni-2/TGR cells yielded senescence of all X recipient clones. The normal human X chromosome induced dominant senescence of tumorigenic Ni-2/TGR cells in only 17% of the resulting microcell hybrids (14/81). Karyotypic analyses of 13 non-senescing human X chromosome-derived microcell hybrid clones revealed that none of these clones retained the complete X. A normal CHE X chromosome induced senescence of 75% of hybrids obtained with another immortal and tumorigenic nickel-transformed male CHE cell line (Ni-6/TGR), which exhibited no visible deletion of the X chromosome, while the normal human X chromosome, only induced senescence in 19% of these hybrids. Transfer of the normal CHE or human X chromosome into spontaneously transformed and tumorigenic cell lines, CHO/TGR or V79/TGR, had little or no effect on their growth. These data suggest that both human and CHE cells possess similar X-linked genetic activities that regulate the process of cellular senescence, and that in Chinese hamster cells nickel-induced immortalization but not that of CHO or V79 cells is associated with inactivation of an X-linked senescence gene
— id: 13643, year: 1992, vol: 13, page: 555, stat: Journal Article,

A simple, sensitive assay to detect DNA-protein crosslinks in intact cells and in vivo
Zhitkovich A; Costa M
1992 Aug;13(8):1485-1489, Carcinogenesis
Addition of potassium chloride to sodium dodecyl sulfate (SDS) resulted in the formation of an insoluble precipitate that was easily recovered by low-speed centrifugation. Since SDS tightly binds to proteins but not DNA, all proteins and detergent-resistant DNA--protein complexes were also effectively co-precipitated in the presence of potassium--SDS leaving free DNA in the supernatant. The amount of SDS-precipitable DNA represented a measure of DNA--protein crosslinks. We have adapted this method for determining DNA--protein crosslinks formed in cells following their exposure in culture or in vivo to crosslinking agents such as chromate, cis-Pt(II) diammine dichloride and formaldehyde. The critical parameters for application of the K--SDS assay to cells were rigorously reproducible shearing of chromosomal DNA and effective washing steps. We have found that freeze-thawing SDS lysed cells followed by vortexing and repeated resuspensions of the precipitate by pipeting resulted in a low background and high reproducibility of the assay. The method detected in a dose-dependent manner DNA--protein crosslinks induced in CHO cells by chromate, cis-platinum and formaldehyde, with sensitivity similar to the alkaline elution procedure. The K--SDS assay was also successfully utilized to determine DNA--protein crosslinks in rat and mouse white blood cells exposed in vivo to chromate. Its sensitivity and simplicity in sample handling and DNA--protein complex isolation potential allows wide application of the assay to measure formation of DNA--protein crosslinks. The ease of recovery of DNA--protein complexes allows for a more thorough investigation of this lesion
— id: 8420, year: 1992, vol: 13, page: 1485, stat: Journal Article,

Alteration in restriction enzyme digestion patterns detects DNA--protein complexes induced by chromate
Chen Y; Cohen MD; Snow ET; Costa M
1991 Sep;12(9):1575-1580, Carcinogenesis
DNA--protein complexes isolated from CHO cells treated with at least 10-30 microM potassium chromate exhibited an alteration in the degradation of the DNA by restriction enzymes compared to DNA--protein complexes isolated from untreated cells. This alteration in restriction enzyme digestion of DNA--protein complexes induced by chromate was shown to depend upon the binding of trivalent chromium to the DNA and upon the protein associated with the DNA, since both EDTA pretreatment and protease K reversed the inhibition of restriction enzyme degradation of the DNA. The inhibition of restriction enzyme degradation of DNA--protein complexes may be utilized as an indirect way to detect DNA--protein complexing induced by chromate and perhaps other agents
— id: 13934, year: 1991, vol: 12, page: 1575, stat: Journal Article,

Differential DNA-protein crosslinking in lymphocytes and liver following chronic drinking water exposure of rats to potassium chromate
Coogan TP; Motz J; Snyder CA; Squibb KS; Costa M
1991 Jun 1;109(1):60-72, Toxicology & applied pharmacology
Carcinogenic chromium (VI) compounds are persistent environmental contaminants with potential for human exposure through drinking water. One lesion associated with chromium (VI) exposure is the formation of DNA-protein crosslinks (DPC). In an attempt to develop markers of chromium exposure, the formation of DPC in lymphocytes was investigated. Fisher 344 rats were exposed to K2CrO4 in their drinking water for 3 and 6 weeks at concentrations of 100 and 200 ppm chromium. No DPC could be detected in isolated splenic lymphocytes using the alkaline elution technique or by using a polyclonal antibody to chromate-induced DPC. However, increased complexing of proteins with DNA was demonstrated in liver following 3 weeks of exposure at both 100 and 200 ppm chromium. Intraperitoneal administration of potassium chromate did not induce detectable DPC in lymphocytes; however, an increased association of proteins with isolated DNA was detected in the liver. DPC were also induced in isolated splenic lymphocytes following a 2-hr exposure in vitro to 100 microM K2CrO4 in a salts-glucose medium. Although chromium was detected in blood, liver, and kidney, blood levels were comparatively much lower. A comparison of chromium levels required to induce DPC in lymphocytes in vitro and the amount absorbed orally suggests that the white blood cell chromium levels following oral exposure may be too low to induce measurable DNA-protein crosslinks in lymphocytes
— id: 14000, year: 1991, vol: 109, page: 60, stat: Journal Article,

Distribution of chromium within cells of the blood
Coogan TP; Squibb KS; Motz J; Kinney PL; Costa M
1991 Mar 15;108(1):157-166, Toxicology & applied pharmacology
Although a number of investigators have examined the uptake of chromium in red blood cells (RBC) or whole blood, little is known about chromium uptake in white blood cells (WBC). Radiolabeled chromium (51Cr) was used to determine chromium uptake and distribution. Isolated RBC and enriched WBC populations were exposed in vitro to potassium chromate (Cr+6) and uptake was determined over a 2-hr time period. Exposure of either rat or human blood cells to 50 microM K2CrO4 for 2 hr resulted in greater accumulation of chromium within WBC than RBC. Uptake by rat WBC was significantly greater than that of human; whereas, uptake by human RBC was greater than that of the rat. Exposure of human whole blood to 50 microM K2CrO4, prior to isolation of WBC, also resulted in an increased uptake of chromium by WBC. Fisher 344 rats were exposed either orally or intravenously to a single dose of K2CrO4 and the distribution of chromium within blood cells was determined 1 hr, 24 hr, or 7 days following exposure. Regardless of the route or time following exposure, WBC chromium levels were consistently greater than those of RBC. However, the absolute levels of chromium did change with time. A comparison of chromium distribution 24 hr following a single oral exposure (1 ppm Cr+6) to the distribution 7 days following exposure demonstrated a reduction in chromium levels for RBC (10-fold) and for WBC (approximately 2.5-fold). In contrast, intravenous administration of chromate resulted in no significant decrease in RBC chromium levels when compared 1 hr, 24 hr, and 7 days following exposure. Although no difference in WBC chromium content was observed at 1 and 24 hr after exposure, an approximate 1.7-fold decrease in chromium content was detected at Day 7 for WBC. Intravenous administration of chromic chloride (Cr+3) resulted in a low level of chromium associated with RBC following 1 hr, and chromium was undetected in the WBC. These data demonstrate that WBC accumulate hexavalent chromium following both in vitro and in vivo exposure. In addition, white blood cells accumulate chromium to a greater extent than red blood cells. Since WBC accumulate chromium, their use as a target for the development of biomarkers of chromium exposure may be warranted
— id: 14097, year: 1991, vol: 108, page: 157, stat: Journal Article,

DNA-protein complexes induced by chromate and other carcinogens
Costa M
1991 May;92:45-52, Environmental health perspectives
DNA-protein complexes induced in intact Chinese hamster ovary cells by chromate have been isolated, analyzed, and compared with those induced by cis-platinum, ultraviolet light, and formaldehyde. Actin has been identified as one of the major proteins complexed to DNA by chromate based upon its molecular weight, isoelectric point, positive reaction with an actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of similar molecular weight and isoelectric points, and these complexes can be disrupted by chelating agents and sulfhydryl reducing agents, suggesting that the metal itself is participating in binding rather than having a catalytic or indirect role (i.e., oxygen radicals). In contrast, formaldehyde complexed histones to the DNA, and these complexes were not disrupted by chelating or reducing agents. An antiserum raised to chromate-induced DNA-protein complexes reacted primarily with 97,000 kDa protein that did not silver stain. Slot blots, as well as Western blots, were used to detect formation of p97 DNA crosslinks. This protein was complexed to the DNA by all four agents studied
— id: 14041, year: 1991, vol: 92, page: 45, stat: Journal Article,

Molecular mechanisms of nickel carcinogenesis
Costa M
1991 ;31:321-337, Annual review of pharmacology & toxicology
— id: 14156, year: 1991, vol: 31, page: 321, stat: Journal Article,

DNA damage by mercury compounds : an overview
Costa M; Christie NT; Cantoni O; Zelikoff JT; Wang XW; Rossman TG
Advances in mercury toxicology New York : Plenum Press, 1991,
— id: 4397, year: 1991, vol: , page: ?, stat: Chapter,

Senescence of nickel-transformed cells by an X chromosome: possible epigenetic control
Klein CB; Conway K; Wang XW; Bhamra RK; Lin XH; Cohen MD; Annab L; Barrett JC; Costa M
1991 Feb 15;251(4995):796-799, Science
Transfer of a normal Chinese hamster X chromosome (carried in a mouse A9 donor cell line) to a nickel-transformed Chinese hamster cell line with an Xq chromosome deletion resulted in senescense of these previously immortal cells. At early passages of the A9/CX donor cells, the hamster X chromosome was highly active, inducing senescence in 100% of the colonies obtained after its transfer into the nickel-transformed cells. However, senescence was reduced to 50% when Chinese hamster X chromosomes were transferred from later passage A9 cells. Full senescing activity of the intact hamster X chromosome was restored by treatment of the donor mouse cells with 5-azacytidine, which induced demethylation of DNA. These results suggest that a senescence gene or genes, which may be located on the Chinese hamster X chromosome, can be regulated by DNA methylation, and that escape from senescence and possibly loss of tumor suppressor gene activity can occur by epigenetic mechanisms
— id: 8211, year: 1991, vol: 251, page: 796, stat: Journal Article,

The role of oxidative processes in metal carcinogenesis
Klein CB; Frenkel K; Costa M
1991 Nov-Dec;4(6):592-604, Chemical research in toxicology
— id: 13852, year: 1991, vol: 4, page: 592, stat: Journal Article,

DIFFERENCES IN THE EFFECT OF VITAMIN-E ON NICKEL SULFIDE OR NICKEL CHLORIDE-INDUCED CHROMOSOMAL-ABERRATIONS IN MAMMALIAN- CELLS
Lin, XH; Sugiyama, M; Costa, M
1991 Jun;260(2):159-164, Mutation research. DNA repair
Vitamin E (alpha-tocopherol succinate) pretreatment significantly inhibited formation of some but not all of the chromosomal aberrations induced by crystalline NiS particles but it did not affect the chromosome damage induced by NiCl2. Crystalline NiS particles are phagocytized by cells in contrast to water-soluble NiCl2 which enters the cells by a different mechanism. These and other previous studies suggest that the phagocytosis of crystalline NiS particles produces genetic damage as a consequence of oxygen radicals resulting from the irritant particle effect and also from the high intracellular levels of nickel ions dissolving from the endocytized particles
— id: 32170, year: 1991, vol: 260, page: 159, stat: Journal Article,

Complexing of actin and other nuclear proteins to DNA by cis-diamminedichloroplatinum(II) and chromium compounds
Miller CA 3d; Cohen MD; Costa M
1991 Feb;12(2):269-276, Carcinogenesis
Actin was found to be the major protein crosslinked to the DNA of intact Chinese hamster ovary cells that were treated with either potassium chromate (hexavalent) or cis-diamminedichloroplatinum(II) (cis-platinum). This protein was identified as actin by its mol. wt (45 kd), its isoelectric point (pI = 5.4), positive reactivity with an actin antibody, and by protease mapping. Additionally, a purified actin standard migrated to the same location in a two-dimensional gel system as p45. Actin comprised approximately 20% of the protein component in chromate-induced DNA-protein crosslinks. In addition, to actin, several other major proteins (e.g. 53 kd, pI = 5.2, 50 kd, pI = 9) were crosslinked to DNA following exposure to cis-platinum or chromate. These proteins were abundant in the nuclear matrix fraction. Hexavalent chromate is the toxicologically active form because it is readily taken up into cells by an anion transport system. In contrast, trivalent chromium is considerably less toxic because it cannot enter the cell; however, most of the hexavalent form is eventually reduced to the trivalent form inside the cell. Previous studies have suggested that the trivalent form of chromium participates in complexing DNA with proteins. DNA-protein crosslinks were formed in isolated nuclei or in mixtures of purified DNA and protein incubated with trivalent chromium. However, the formation of these complexes required at least 16 h of incubation to exchange the parent compound ligands. Hexavalent chromate did not form these complexes in vitro under similar conditions. Incubation of trivalent chromium with purified actin and DNA resulted in DNA-actin crosslinks as detected by an electrophoretic mobility different from that of either free actin or DNA when the complex was transferred from a gel to nitrocellulose and stained for protein. These studies describe a new technique for detecting DNA-protein complexes and demonstrate that actin-DNA structures in intact cells create sites that selectively react with metal DNA-protein crosslinking agents
— id: 14136, year: 1991, vol: 12, page: 269, stat: Journal Article,

PROTECTIVE EFFECT OF VITAMIN-E AGAINST CHROMOSOMAL-ABERRATIONS AND MUTATION INDUCED BY SODIUM CHROMATE IN CHINESE-HAMSTER V79 CELLS
Sugiyama, M; Lin, XH; Costa, M
1991 May;260(1):19-23, Mutation research. DNA repair
The effect of vitamin E on chromosomal aberrations and mutation caused by Na2CrO4 was investigated in Chinese hamster V79 cells. Pretreatment with 25-mu-M alpha-tocopherol succinate (vitamin E) for 24 h prior to chromate exposure (2.5-5-mu-M) resulted in a decrease of metal-induced chromosomal aberrations. Na2CrO4 (2.5-7.5-mu-M) induced mutations at the HGPRT locus, but only within a very limited concentration range. This mutagenic response could also be suppressed by pretreatment with vitamin E. These results suggest that vitamin E can protect cells from the clastogenic and mutagenic action of chromate compounds, possibly through its ability to scavenge chromium(V) and/or free radicals
— id: 32181, year: 1991, vol: 260, page: 19, stat: Journal Article,

Changes in protein phosphorylation in wild-type and nickel-resistant cells and their involvement in morphological elongation
Wang XW; Costa M
1991 ;4(4):201-206, Biology of metals
Treatment of wild-type Balb/c-3T3 cells with NiCl2 or N6,2-O-dibutyl-adenosine 3',5'-monophosphate (Bt2-cAMP) resulted in a high degree and frequency of cellular elongation. Nickel-resistant Balb/c-3T3 cells (B200) treated with Bt2-cAMP elongated at the same exposure concentration as wild-type cells. In contrast, treatment of the nickel-resistant cells with both non-cytotoxic and cytotoxic doses of NiCl2 failed to induce elongation. Nickel-resistant cells had two-thirds of the total protein-phosphorylation activity of wild-type cells. Both cAMP and NiCl2 enhanced phosphorylation of specific proteins in intact wild-type cells as detected by 32p autoradiography of these proteins separated on two-dimensional gels. A nickel-dependent phosphorylation of specific proteins is seen following NiCl2 treatment of wild-type cells but was not observed in B200 cells. In contrast, the pattern of Bt2-cAMP-stimulated protein phosphorylation was quite similar in both wild-type and nickel-resistant cells. Although it is unclear at present how nickel ions affect the cellular protein-phosphorylation system, these results suggested that targets controlling cellular elongation are sensitive to nickel, are altered in nickel-resistant cells and appear to involve protein phosphorylation. Further characterization of these targets may help in understanding the mechanisms of nickel carcinogenesis
— id: 14236, year: 1991, vol: 4, page: 201, stat: Journal Article,

A blotting method for monitoring the formation of chemically induced DNA-protein complexes
Cohen MD; Miller CA; Xu LS; Snow ET; Costa M
1990 Apr;186(1):1-7, Analytical biochemistry
The formation and identification of DNA-protein crosslinks are usually detected by filter binding assays such as alkaline elution. We describe a modified blotting method to selectively identify DNA-protein complexes (DPCs) formed in vitro by either Cr3+ ion or formaldehyde. This protocol allows DPC formation in vitro to be assayed with various chemical agents, requires minimal usage of radioactivity, and is performed in a shorter time frame than that commonly used to resolve DPCs from free proteins and unbound DNA
— id: 36437, year: 1990, vol: 186, page: 1, stat: Journal Article,

The detection of DNA-protein complexes in vitro by an immunological assay
Cosma, G N; Miller, C A 3rd; Costa, M
1990 ;4(1):17-22, Toxicology in vitro
We have developed an immunological assay to detect DNA-protein complexes (DPCs) in cell cultures treated with environmental toxicants. The assay uses an antiserum developed against K(2)CrO(4)-induced DPCs, which recognizes an acidic protein with a molecular weight of 95 kDaltons. The method uses a filter-binding assay to trap the DPCs from SDS-lysed cell cultures on polycarbonate filters, after which they are immunologically probed with the DPC antiserum. Cultures of Chinese hamster ovary cells were treated with K(2)CrO(4), formaldehyde or UV irradiation. DPCs were detected immunologically in cells treated with K(2)CrO(4) or UV irradiation, but not in those treated with formaldehyde. These results were similar to those obtained when DPCs were detected by filter-binding assay using radiolabelled cell cultures to measure the adherence of protein and DNA to the filters. In addition, both methods gave analogous dose responses of DPC formation in K(2)CrO(4)-treated cells. This novel immunological assay for detecting DNA lesions allows the rapid analysis of samples, which do not need to be radiolabelled, and thus it may have an application as a non-invasive test to screen for DNA-protein complexes
— id: 111599, year: 1990, vol: 4, page: 17, stat: Journal Article,

Analysis of DNA-protein complexes induced by chemical carcinogens
Costa M
1990 Nov;44(3):127-135, Journal of cellular biochemistry
DNA-protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis-platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA-protein complex. An antiserum which was raised to chromate-induced DNA-protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA-protein complexes formed by cis-platinum, UV, formaldehyde, and chromate. Other work in this area, involving studying whether DNA-protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA-protein complexes, the stability and repair of these lesions, and characterization of DNA-protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA-protein complexes and these findings are also reviewed
— id: 14311, year: 1990, vol: 44, page: 127, stat: Journal Article,

DELETION OF A SENESCENCE TUMOR SUPPRESSOR GENE AS A MECHANISM OF NICKEL CARCINOGENESIS
Costa, M
1990 Apr 22;199(3):25CHAS-, Abstracts of papers (American Chemical Society)
— id: 31950, year: 1990, vol: 199, page: 25CHAS, stat: Journal Article,

IMMUNODETECTION OF DNA PROTEIN CROSS-LINKS BY SLOT BLOTTING
Miller, CA; Costa, M
1990 Apr;234(2):97-106, Mutation research. DNA repair
— id: 32089, year: 1990, vol: 234, page: 97, stat: Journal Article,

Nonrandom chromosomal alterations in nickel-transformed Chinese hamster embryo cells
Conway K; Costa M
1989 Nov 1;49(21):6032-6038, Cancer research
The purpose of this study was to determine whether nickel-transformed Chinese hamster cells exhibit nonrandom chromosomal alterations involving heterochromatic regions, particularly the heterochromatic long arm (q) of the X-chromosome, since nickel was previously shown to induce acute chromosomal damage predominantly in heterochromatin. Early passage male or female Chinese hamster embryo cells were transformed to anchorage independence in soft agar following treatment with 10 micrograms/ml NiS, 1 mM NiCl2, or with 10 micrograms/ml 3-methylcholanthrene (MCA). Nickel treatment of Chinese hamster embryo cells resulted in a 2- to 3-fold higher frequency of male (7 of 13) as compared with female (2 of 12) anchorage independent cultures, whereas MCA treatment resulted in equal proportions of male and female anchorage independent cultures. A single soft agar (SA) clone from each of the nickel- or MCA-transformed cultures was karyotyped. Most of the transformed clones had modal chromosome numbers in the diploid range. Four of the 7 male nickel-transformed SA clones exhibited complete or partial deletions of Xq, while one male and one female nickel-transformed SA clone both had an X;5 translocation. In contrast, all of the MCA-transformed SA clones possessed at least one intact X-chromosome. Anchorage-dependent nickel-treated cultures all displayed normal X-chromosomes, suggesting that the Xq deletion was not merely associated with nickel treatment. Structural chromosome alterations were evident in all but one of the anchorage-independent SA clones. None of the anchorage-dependent nickel-treated cultures had rearrangements, although they were generally trisomic for a combination of chromosomes 3 or 3q, 5, and 8. Trisomy of chromosome 3 or 3q occurred in those clones which were tumorigenic in nude mice. Trisomy of chromosome 4 or 4p was a common change which occurred in four nickel-transformed SA clones and a single MCA-transformed SA clone
— id: 10435, year: 1989, vol: 49, page: 6032, stat: Journal Article,

The involvement of heterochromatic damage in nickel-induced transformation
Conway K; Costa M
1989 Jul-Sep;21:437-444, Biological trace element research
Nickel ions produce selective damage in heterochromatic regions of chromosomes. Male Chinese hamster embryo cells, which have heterochromatin along the entire long arm of the X-chromosome, exhibit an unusually high incidence of nickel-induced transformation compared with female cells of the same species. However, 3-methylcholanthrene, a carcinogen that produces a random distribution of chromosome damage, transforms female and male cells equally. Other species that do not have as much heterochromatin on the X-chromosome exhibit similar incidences of nickel-induced tumors in males and females. Four out of five of the male nickel-transformed Chinese hamster cell lines exhibit a deletion of the heterochromatic long arm of the X-chromosome as the only common karyotypic aberration. This result indicates that a deletion of a heterochromatic chromosomal region may be an important feature of the nickel-induced carcinogenic process. All of the male nickel transformed cells lines are able to form tumors in athymic nude mice
— id: 10576, year: 1989, vol: 21, page: 437, stat: Journal Article,

Toxicity and carcinogenicity of nickel compounds [published erratum appears in Crit Rev Toxicol 1989;20(2):135]
Coogan TP; Latta DM; Snow ET; Costa M
1989 ;19(4):341-384, Critical reviews in toxicology
The toxicity and carcinogenicity of nickel compounds are considered in three broad categories: (1) systemic toxicology, (2) molecular toxicology, and (3) carcinogenicity. The systemic toxicity of nickel compounds is examined based upon human and animal studies. The major organs affected are discussed in three categories: (1) kidney, (2) immune system, and (3) other organs. The second area of concentration is molecular toxicology, which will include a discussion of the chemistry of nickel, its binding to small and large molecular weight ligands, and, finally, its cellular effects. The third major area involves a discussion of the carcinogenicity and genotoxicity of nickel compounds. This section focuses on mechanisms, using studies conducted in vivo and in vitro. It also includes a discussion of the assessment of the carcinogenicity of nickel compounds
— id: 10790, year: 1989, vol: 19, page: 341, stat: Journal Article,

Perspectives on the mechanism of nickel carcinogenesis gained from models of in vitro carcinogenesis
Costa M
1989 May;81:73-76, Environmental health perspectives
This article briefly reviews the approach taken to understand the mechanism of nickel-induced neoplastic transformation. The initial phases of the studies were focused on particulate nickel compounds and on the regulation of phagocytosis of nickel compounds by cells undergoing transformation. The particulate nickel compounds most potent in inducing cell transformation were selectively phagocytized by cells, whereas those that were not active were not phagocytized. The intracellular fate of phagocytized nickel sulfide particles is discussed as well as the interaction of nickel with chromatin. Phagocytized nickel sulfide particles were dissolved in the cytoplasm of cells by the acidification of vacuoles containing phagocytized particles. Nickel ions released from the phagocytized particles produced selective damage in heterochromatin. The selective effects of nickel on heterochromatin are also discussed and related to its mechanism of carcinogenesis
— id: 10647, year: 1989, vol: 81, page: 73, stat: Journal Article,

Proceedings of the First International Meeting on Molecular Mechanisms of Metal Toxicity and Carcinogensis, hedl Sept 19-22, 1988 in Urbino, Italy
Costa, Max; Rossman, Toby
Clifton NJ : Humana Press, 1989,
— id: 1218, year: 1989, vol: , page: , stat: ,

Characterization of a nickel resistant mouse cell line
Imbra RJ; Wang XW; Costa M
1989 Jul-Sep;21:97-103, Biological trace element research
Nickel is a potent carcinogen and, at high concentrations, is toxic to mammalian cells. The effects associated with nickel exposure are well-documented but its mechanism of action in the cell has not yet been fully described. In order to understand the metabolic fate of nickel in mammalian cells, a variant cell population has been selected that continues to grow and divide in the presence of nickel chloride concentrations that are toxic to the parental cell line (Balb/c-3T3 mouse fibroblasts). Nickel resistance is not caused by altered uptake of nickel from the medium or increased clearance from the cells and is not associated with changes in metallothionein expression. Compared to the normal cells, the nickel resistant cells have a decreased number of chromosomes and numerous centromeric fusions. The expression of some proteins and the distribution of nickel bound by various proteins are altered in the nickel resistant cells. Preliminary results indicate that the nickel resistant phenotype may be transferred by genomic DNA-mediated transfection into a recipient NIH-3T3 cell line. Current investigations are directed at identifying a gene responsible for nickel resistance
— id: 10574, year: 1989, vol: 21, page: 97, stat: Journal Article,

STUDIES ON THE MECHANISM OF NICKEL-INDUCED HETEROCHROMATIN DAMAGE - EFFECT ON SPECIFIC DNA-PROTEIN INTERACTIONS
Imbra, RJ; Latta, DM; Costa, M
1989 Oct;22(1-4):167-179, Toxicological & environmental chemistry
— id: 31617, year: 1989, vol: 22, page: 167, stat: Journal Article,

Analysis of proteins cross-linked to DNA after treatment of cells with formaldehyde, chromate, and cis-diamminedichloroplatinum(II)
Miller CA 3d; Costa M
1989 Winter;2(1):11-26, Molecular toxicology
The proteins cross-linked to the DNA of cultured Chinese hamster ovary cells after exposure to cis-diamminedichloroplatinum(II) (cis-Pt), chromate, and formaldehyde were compared by two-dimensional (2D) gel electrophoresis, immunoblotting, and centrifugal assays that measured cross-link stability. Chromate and cis-Pt cross-linked seven of the same nonhistone proteins, such as actin, to DNA. In contrast, formaldehyde selectively formed histone-DNA cross-links. Immunoblotting experiments showed that all three chemicals cross-linked a 97-kDa nuclear protein to the DNA despite their different chemical reactivity with DNA and proteins. The chromate- and cis-Pt-induced cross-links were disrupted by thiourea, 2-mercaptoethanol, and EDTA, indicating that the metal could be chemically displaced from the cross-links. The formaldehyde-induced complexes required degradation with DNase 1 for the resolution of histones on 2D gels and were not chemically labile like the metal-induced cross-links. The agents and methodology used here could be applied to the study of additional nuclear proteins that bind or reside near the DNA
— id: 10811, year: 1989, vol: 2, page: 11, stat: Journal Article,

Immunological detection of DNA-protein complexes induced by chromate
Miller CA 3d; Costa M
1989 Apr;10(4):667-672, Carcinogenesis
A select group of non-histone proteins becomes complexed to DNA after Chinese hamster ovary (CHO) cells are treated with potassium chromate. The most abundant complexed protein has a mol. wt of approximately 45 kd and is thought to be actin. An antiserum to the chromate-induced DNA-protein complexes (DPCs) was prepared to facilitate the study of these complexes. Rather than detecting the predominant silver-stained proteins of DPCs described in an earlier study, this antiserum reacts primarily with an acidic 95-kd protein (p95) that does not silver stain. The antiserum can be used routinely to assay for the induction of p95-DNA complexes produced by chromate and perhaps by other carcinogens. Immunofluorescent staining of CHO cells and immunoblotting of cell fractions show the reactive antigens are within the cell nucleus. Blotting experiments with the antiserum indicate the chromate-induced p95-DNA complex dissociates in the presence of 2-mercaptoethanol, suggesting similarity to the DPCs formed by carcinogenic platinum compounds. A reduced species of chromium (probably Cr3+) may form DPCs by binding to the nitrogen, oxygen or sulfur atoms of proteins and DNA. These results illustrate the usefulness of immunological detection methods to study specific DNA-protein interactions induced by carcinogens. The possible relevance of DPCs in the carcinogenic process is discussed
— id: 10687, year: 1989, vol: 10, page: 667, stat: Journal Article,

THE PROCEEDINGS OF THE 1ST INTERNATIONAL MEETING ON MOLECULAR MECHANISMS OF METAL TOXICITY AND CARCINOGENICITY, HELD SE
ROSSMAN, TG; COSTA, M
1989 ;21(1-4):R1-R2, Biological trace element research
— id: 104619, year: 1989, vol: 21, page: R1, stat: Journal Article,

Alteration of nickel-binding proteins in nickel-resistant cells
Wang XW; Costa M
1989 ;1(6):351-358, Cancer communications
Proteins from wild-type and nickel-resistant cells (Balb/c-3T3 and B200, respectively) were studied by one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and detected either by standard protein staining or by fluorographic analysis of [63Ni] binding. Wild-type cells contained three major nickel-binding proteins with molecular masses of 68 kDa (p68), 55 kDa (p55), and 48 kDa (p48). The p55 was present in high concentrations in the microsomal fraction, whereas the p68 nickel-binding protein predominated in the cytosolic fraction. We were unable to demonstrate that p48 was localized in any subcellular fraction. Both the p55 and p48 proteins appeared to be present in similar amounts in wild-type and nickel-resistant cell lines, based upon silver staining of two-dimensional gels, yet in the nickel-resistant B200 cells, these proteins could not be visualized by [63Ni] binding. This suggested that if nickel binding to these proteins were important for nickel toxicity, then nickel resistance in the B200 cells would be associated with the observed loss of metal binding to these proteins. In addition to the changes in [63Ni]-binding proteins, the total concentration of a 44 kDa protein was increased in B200 cells, but its ability to bind nickel could not be established due to its rapid degradation. Among the nickel-binding proteins studied, the p55 contained nickel-binding sites that were the most resistant to exchange by excess nickel ions. The microsomal fraction that contained the highest concentration of p55 also had the highest nickel-binding activity when standardized for protein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 10778, year: 1989, vol: 1, page: 351, stat: Journal Article,

Chromosomal alterations in cell lines derived from mouse rhabdomyosarcomas induced by crystalline nickel sulfide
Christie NT; Sen P; Costa M
1988 ;1(1):43-50, Biology of metals
Prior studies have shown a preferential decondensation (or fragmentation) of the heterochromatic long arm of the X chromosome of Chinese hamster ovary cells when treated with carcinogenic crystalline NiS particles (crNiS). In this report, we show that the heterochromatic regions of mouse chromosomes are also more frequently involved in aberrations than euchromatic regions, although the heterochromatin in mouse cells is restricted to centromeric regions. We also present the karyotypic analyses of four cell lines derived from tumors induced by leg muscle injections of crystalline nickel sulfide which have been analyzed to determine whether heterochromatic chromosomal regions are preferentially altered in the transformed genotypes. Common to all cell lines was the presence of minichromosomes, which are acrocentric chromosomes smaller than chromosome 19, normally the smallest chromosome of the mouse karyotype. The minichromosomes were present in a majority of cells of each line although the morphology of this extra chromosome varied significantly among the cell lines. C-banding revealed the presence of centromeric DNA and thus these minichromosomes may be the result of chromosome breaks at or near the centromere. In three of the four lines a marker chromosome could be identified as a rearrangement between two chromosomes. In the fourth cell line a rearranged chromosome was present in only 15% of the cells and was not studied in detail. One of the three major marker chromosomes resulted from a centromeric fusion of chromosome 4 while another appeared to be an interchange involving the centromere of chromosome 2 and possibly the telomeric region of chromosome 17.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 11256, year: 1988, vol: 1, page: 43, stat: Journal Article,

TRANSFORMATION OF CHINESE-HAMSTER EMBRYO CELLS BY NICKEL YIELDS CELL-LINES WITH ALTERED HETEROCHROMATIC STAINING PATTERNS
Conway, K; Costa, M
1988 Mar;29(5):117-117, Proceedings (American Association for Cancer Research)
— id: 31480, year: 1988, vol: 29, page: 117, stat: Journal Article,

EFFECT OF NICKEL(II) ON DNA-PROTEIN INTERACTIONS
Coogan, TP; Latta, DM; Imbra, RJ; Costa, M
1988 Mar;29(5):87-87, Proceedings (American Association for Cancer Research)
— id: 31478, year: 1988, vol: 29, page: 87, stat: Journal Article,

The role of trace metals in carcinogenesis
Costa, Max; Creasey, William A
[Bethesda MD] : US Dept of Health and Human Services. International Cancer Research Data Bank, 1988,
— id: 1217, year: 1988, vol: , page: , stat: ,

Characterization of DNA-protein complexes induced in intact cells by the carcinogen chromate
Miller CA 3d; Costa M
1988 ;1(2):125-133, Molecular carcinogenesis
Potassium chromate induced the formation of DNA-protein complexes in cultured Chinese hamster ovary cells. The DNA-protein complexes were isolated by ultracentrifugal sedimentation in the presence of 2% sodium dodecyl sulfate (SDS) and 5 M urea. Two-dimensional SDS-polyacrylamide gel electrophoresis analysis of the chromate-induced DNA-protein complexes revealed that two acidic proteins of 53 and 45 kDa and a basic protein of 54 kDa were selectively complexed to the DNA. Numerous other proteins also became associated with the DNA to a lesser degree as the chromate concentration was increased. Nuclease digestion was not a prerequisite for the resolution of the protein component of the DNA-protein complexes using two-dimensional gel electrophoresis. Ultracentrifugal analysis of the DNA-protein complexes in the presence of proteinase K, nucleases, or a chelating agent demonstrated that protein aggregation was not responsible for the increased protein recovery in chromate-treated samples and that the complexes were disrupted by EDTA. These data suggest that the selectively complexed proteins were associated with the DNA through strong interactions that may be mediated by the trivalent form of chromium
— id: 11257, year: 1988, vol: 1, page: 125, stat: Journal Article,

CHANGES IN NUCLEAR PROTEIN-DNA INTERACTION CAUSED BY POTASSIUM CHROMATE
MILLER, CA; COSTA, M
1988 MAR ;29(3-4):112-112, Proceedings (American Association for Cancer Research)
— id: 98523, year: 1988, vol: 29, page: 112, stat: Journal Article,

Stimulation of polyadenosine diphosphoribose synthesis by DNA lesions induced by sodium chromate in Chinese hamster V-79 cells
Sugiyama, M; Costa, M; Nakagawa, T; Hidaka, T; Ogura, R
1988 Mar 1;48(5):1100-1104, Cancer research
Polyadenosine diphosphoribose [poly(ADP-ribose)] synthesis was stimulated by DNA lesions induced with Na2CrO4 and methyl methanesulfonate (MMS) in Chinese hamster V-79 cells. Na2CrO4 and MMS induced DNA single-strand breaks in a concentration-dependent manner; however, the breaks induced by Na2CrO4 were 'protein associated' while those induced by MMS were not. MMS stimulated in a dose-dependent fashion the synthesis of poly(ADP-ribose) up to 6-fold above the control. Na2CrO4 also induced poly(ADP-ribose) synthesis, but the level of synthesis was less than 3-fold. Control experiments demonstrated that Na2CrO4 treatment of cells did not affect their ability to synthesize poly(ADP-ribose) in response to DNA damage. Treatment of cells with Na2CrO4 and MMS induced more poly(ADP-ribose) synthesis than each agent alone; however, whenever Na2CrO4 was utilized, the breaks required proteinase K to be detected. Following removal of extracellular chromate, the DNA strand breaks induced by 0.2 mM Na2CrO4 were repaired quickly during the first hour but more slowly for the next 3 h. In the presence of 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis, the repair of DNA breaks was reduced. These results suggest that DNA protein-associated breaks produced by Na2CrO4 were recognized by poly(ADP-ribose) polymerase and that there are differences in poly(ADP-ribose) synthesis in response to Na2CrO4 and MMS. The results also suggest that the repair of breaks induced by Na2CrO4 are associated with poly(ADP-ribose) synthesis, but perhaps because most of these breaks are protein associated, there is less stimulation of poly(ADP-ribose) synthesis
— id: 141422, year: 1988, vol: 48, page: 1100, stat: Journal Article,

Characterization of mouse cell lines resistant to nickel(II) ions
Wang XW; Imbra RJ; Costa M
1988 Dec 1;48(23):6850-6854, Cancer research
BALB/c-3T3 cells have been isolated that are resistant to NiCl2. The degree of resistance is directly dependent upon the NiCl2 exposure concentration and ranges from 6- to 11-fold. Resistance to NiCl2 does not appear to be due to alterations in cellular uptake, since the entry of Ni(II) into wild-type or resistant cells was similar. Resistance does not appear to be due to alterations in metallothionein expression. Resistant cells have a high incidence of heterochromatic abnormalities involving fusions at the centromeres as determined by C-banding and in situ hybridization utilizing a cloned mouse satellite DNA probe. Cells retain nickel resistance for many generations in the absence or presence of NiCl2 selection; however, with time in the absence of NiCl2, the level of resistance decreases. This loss of resistance is associated with a decreased number of centromeric fusions. These results indicate that nickel resistance is involved with changes in heterochromatin and suggest that this effect of nickel on heterochromatin may be important as an early step in nickel carcinogenesis
— id: 10871, year: 1988, vol: 48, page: 6850, stat: Journal Article,

Genetic toxicology of lead compounds
Zelikoff JT; Li JH; Hartwig A; Wang XW; Costa M; Rossman TG
1988 Oct;9(10):1727-1732, Carcinogenesis
We have investigated the activity of insoluble and soluble lead compounds in inducing mutagenesis, cell transformation and sister chromatid exchange in mammalian cells. Insoluble lead sulfide, readily phagocytized, was more than four times as toxic to V79 cells on a microM basis, than two moderately soluble lead compounds although the exposure time for the soluble salts was five times longer. These findings demonstrate the importance of different cellular mechanism(s) of metal uptake and bioavailability. Both insoluble lead sulfide and more soluble lead nitrate were mutagenic at the HPRT locus in V79 cells. Although less mutagenic at the higher concentrations, lead nitrate at a concentration of 500 microM enhanced the mutation frequency greater than 6-fold above background following a 5-day exposure. Although the mechanism(s) by which lead induces mutations is unknown, failure of both compounds to induce SCE and DNA single-strand breaks, detectable by alkaline elution, suggests that lead-induced mutations may not be a result of direct damage to DNA but may occur via indirect mechanisms including disturbances in enzyme functions important in DNA synthesis and/or repair, or in DNA-helical structure. Lead acetate also transformed SHE cells in a dose-response fashion following a 48-h exposure. Our results indicate that lead compounds may be genotoxic by an indirect mechanism, and lend support to the view that lead is a carcinogen
— id: 10946, year: 1988, vol: 9, page: 1727, stat: Journal Article,

Effect of magnesium on nickel-induced genotoxicity and cell transformation
Conway, K; Wang, X W; Xu, L S; Costa, M
1987 Aug;8(8):1115-1121, Carcinogenesis
Raising the extracellular level of magnesium ions inhibited nickel-induced DNA strand breaks, DNA-protein crosslinks, sister chromatid exchanges, chromosomal aberrations and cell transformation. Carcinogenic nickel ions preferentially damaged centromeres and other heterochromatic regions of Chinese hamster ovary cell chromosomes. Elevation of extracellular magnesium levels prevented the effects of nickel on heterochromatin and inhibited cell transformation, but did not substantially reduce the DNA damage induced by nickel in euchromatic regions. This study suggests that heterochromatic DNA damage may be important to the nickel-induced neoplastic transformation process
— id: 141423, year: 1987, vol: 8, page: 1115, stat: Journal Article,

Principles of industrial toxicology
Costa, M
1987 Jul;4(3):559-570, Clinics in podiatric medicine & surgery
This article offers a brief introduction to the general principles of toxicology, followed by a discussion of the toxicology of heavy metals, solvents, halogenated hydrocarbons, and alcohols. It concludes with an overflow of pesticide toxicology
— id: 111579, year: 1987, vol: 4, page: 559, stat: Journal Article,

EFFECTS OF CHROMATE AND NICKEL ON DNA-PROTEIN INTERACTIONS
Costa, M; Imbra, RJ; Miller, CA; Ashkenazikimmel, T; Latta, DM
1987 Mar;28(3):89-89, Proceedings (American Association for Cancer Research)
— id: 31214, year: 1987, vol: 28, page: 89, stat: Journal Article,

Effects of nickel(II) on nuclear protein binding to DNA in intact mammalian cells
Patierno, S R; Costa, M
1987 May;9(2):113-126, Cancer biochemistry biophysics
An intracellular effect of nickel(II) which may be involved in its carcinogenic action is the alteration of normal DNA-protein binding. This effect of ionic nickel was studied in Chinese hamster ovary cells using several chromatin isolation methods in combination with SDS-polyacrylamide gel electrophoresis. DNA from cells incubated with (35S)-methionine or (35S)-cysteine to radiolabel protein was prepared by three methods: (solation of nuclei or nucleoids followed by chloroform-isoamyl alcohol (24:1 v/v) extraction and in some cases an additional extraction in the absence or presence of 2M NaCl, 40 mM EDTA or SDS; by isopycnic centrifugation through Cs2SO4 gradients containing 0.8% sarkosyl, 2.2 MCs2SO4, 1 mM NaCl and 10 mM EDTA; or by chromatin disaggregation and denaturation using 9 M urea, 2% 2-mercaptoethanol, 4% Nonidet P-40 +/- 2 M NaCl. DNA from nickel-treated cells consistently had more (35S)-methionine radioactivity associated with it than did DNA from untreated cells. This radioactivity was resistant to ribonuclease but sensitive to protease. Differential extraction using denaturing agents and high ionic strength followed by SDS-polyacrylamide gel electrophoresis revealed that most of the tightly bound proteins were nonhistone chromosomal proteins, and possibly histone 1. The enhancement of DNA-protein binding from nickel-treated cells was disrupted by SDS, suggesting that nickel ions do not function as classical bifunctional crosslinking agents. Since regulation of DNA replication and gene expression is dependent upon DNA-protein interactions, the effect of nickel in altering the extent of DNA-protein binding may interfere with this regulation and may contribute to the carcinogenic activity of nickel compounds
— id: 141426, year: 1987, vol: 9, page: 113, stat: Journal Article,

Effect of nickel(II) on DNA-protein binding, thymidine incorporation, and sedimentation pattern of chromatin fractions from intact mammalian cells
Patierno, S R; Sugiyama, M; Costa, M
1987 Spring;2:13-23, Journal of biochemical toxicology
Nuclear uptake and chromatin binding of nickel(II) was investigated in Chinese hamster ovary (CHO) cells. The cytoplasmic:nuclear ratio of nickel immediately following treatment was 5:1, but by 24 and 48 hours this ratio decreased to 4:1 and 2:1, respectively, indicating that nickel is retained longer in the nucleus than cytoplasmic nickel. Chromatin was fractionated by sonication and centrifugation into fast-sedimenting, magnesium-insoluble, or magnesium-soluble components. The magnesium-insoluble portion bound more nickel ions and retained the metal longer than either the magnesium-soluble or the fast-sedimenting fractions. Treatment of cells with nickel chloride (NiCl2) decreased the amount of DNA in the magnesium-insoluble fraction but increased the amount of DNA in the fast-sedimenting chromatin fraction. The magnesium-insoluble fraction isolated from nickel-treated cells contained approximately ten times more [35-S]-methionine-labeled protein per milligram DNA compared with untreated cells. The magnesium-soluble and the fast-sedimenting fractions isolated from the nickel-treated cells did not exhibit a similar increase in [35-S]-methionine-labeled protein per milligram of DNA. Nickel treatment suppressed [14-C]-thymidine incorporation into total DNA by 30% compared with untreated cells. However, the magnesium-insoluble chromatin fraction from nickel-treated cells had a tenfold to 20-fold increase in thymidine incorporation, while the other chromatin fractions did not exhibit an increase in thymidine incorporation. These findings indicate that nickel induced widespread alterations in chromatin conformation and preferentially interacted with an Mg-insoluble component of chromatin
— id: 141427, year: 1987, vol: 2, page: 13, stat: Journal Article,

Comparison of the localization of chromosome damage induced by calcium chromate and nickel compounds
Sen, P; Conway, K; Costa, M
1987 Apr 15;47(8):2142-2147, Cancer research
Chromosomal aberrations were studied in Chinese hamster ovary cells and in C3H10T1/2 cells following treatment with NiCl2, crystalline NiS, and CaCrO4. All three compounds caused an increase in chromosomal aberrations in a concentration- and time-dependent fashion. The chromosomal aberrations induced by NiCl2 and crystalline NiS occurred predominantly in heterochromatic regions of the chromosomes. Additionally, treatment of cells with crystalline NiS and to a smaller extent long-term treatment with NiCl2 caused a preferential effect on the condensation state of the heterochromatic long arm of the X-chromosome in hamster cells. In contrast, treatment of cells with CaCrO4 did not induce aberrations preferentially in heterochromatin. These results are interesting because nickel(II), which is thought to be the ultimate carcinogen of nickel compounds, binds poorly to DNA, is weakly mutagenic, but induces chromosome damage, probably because of its interaction with nuclear proteins in heterochromatin. Chromate binds to DNA, is mutagenic, and interacts with chromatin randomly
— id: 141424, year: 1987, vol: 47, page: 2142, stat: Journal Article,

Physicochemical characteristics and biological effects of nickel oxides
Sunderman, F W Jr; Hopfer, S M; Knight, J A; McCully, K S; Cecutti, A G; Thornhill, P G; Conway, K; Miller, C; Patierno, S R; Costa, M
1987 Feb;8(2):305-313, Carcinogenesis
Ten nickel oxides and nickel-copper oxides, which all contained NiO (bunsenite) as the predominant crystalline phase, were assayed as follows: in vitro dissolution tests in water and body fluids; in vitro phagocytosis tests in Chinese hamster ovary and C3H-10T1/2 cells; morphological transformation and cytotoxicity tests in cultured Syrian hamster embryo (SHE) cells; erythropoiesis stimulation assay by intrarenal administration to Fischer-344 rats; and scoring the renal histopathologic responses in rats killed 3 months post-injection. The test compounds differed substantially in their biological effects when tested in the various experimental systems. Based upon highly significant concordance of ranked results in the assays (P less than 0.001), six colligative biological attributes of the compounds were identified: (i) dissolution half-times in rat serum and renal cytosol; (ii) phagocytosis by C3H-10T1/2 cells; (iii) morphological transformation of SHE cells; (iv) erythropoiesis stimulation in rats; (v) induction of tubular hyperplasia in rat kidneys; and (vi) induction of arteriosclerosis in rat kidneys. Strong rank correlation (P less than 0.01) between results of the cell transformation and erythropoiesis stimulation assays is especially notable, since the compounds were tested by blind protocols in independent laboratories. The presence of high surface area and demonstrable Ni(III) were two physicochemical characteristics that were associated with the greatest biological effects of nickel oxides
— id: 141425, year: 1987, vol: 8, page: 305, stat: Journal Article,

Assessment of the uptake and mutagenicity of nickel chloride in salmonella tester strains
Biggart, N W; Costa, M
1986 Dec;175(4):209-215, Mutation research
NiCl2 was examined for mutagenic activity in a number of Salmonella tester strains. Conditions were established where there was substantial uptake of the metal into the bacterial cells. However, even when the metal ion was apparently taken up, as determined by metal association with cells, there was a lack of mutagenic activity. These results suggest that nickel is unable to induce basepair or frameshift mutations in Salmonella tester strains and are discussed in relationship to the low binding affinity of Ni(II) for DNA
— id: 141434, year: 1986, vol: 175, page: 209, stat: Journal Article,

Differences in the effects of Hg(II) on DNA repair induced in Chinese hamster ovary cells by ultraviolet or X-rays
Christie, N T; Cantoni, O; Sugiyama, M; Cattabeni, F; Costa, M
1986 Feb;29(2):173-178, Molecular pharmacology
The effect of relatively nontoxic levels of HgCl2 on semiconservative DNA synthesis and on DNA repair induced following treatment of intact cells with X-ray or ultraviolet (UV) light has been studied in cultured Chinese hamster ovary cells. In the presence of 1 microM HgCl2 the repair of DNA strand breaks induced by 450 rads of X-rays was reduced by 37%. If a treatment of 2.5 microM HgCl2 was given to cells for only 15 min prior to a 450-rad irradiation, the rate of repair was reduced even further with only 25% of the breaks being repaired in the first hour following irradiation. When comparable treatments of HgCl2 were given to Chinese hamster ovary cells in conjunction with UV irradiation there was no significant effect on either the number of initial strand scission events or the return to high molecular weight DNA following completion of repair. Only after exposure of cells to toxic levels of Hg(II) (higher concentrations or longer treatments) was there measurable inhibition of UV-induced repair as evidenced by a reduced rate of ligation of DNA to a high molecular weight form. Inhibition of the endonuclease step of UV repair was not observed since Hg(II)-treated cells exhibited the same level of strand scission immediately following UV as cells not treated with Hg(II). The observed differences in the effects of Hg(II) on two pathways for DNA repair indicate that the potential for synergistic action between Hg(II) and other DNA damaging agents will be determined in part by the repair pathways induced by each agent. Additionally, it was found that inhibition of semiconservative synthesis also occurs at low concentrations of HgCl2 similar to those affecting X-ray-induced repair. The presence of Hg-DNA adducts in the DNA at these concentrations may cause a reduction in normal replication to facilitate DNA repair
— id: 133270, year: 1986, vol: 29, page: 173, stat: Journal Article,

Antagonistic effect of magnesium chloride on the nickel chloride-induced inhibition of DNA replication in Chinese hamster ovary cells
Conway, K; Sen, P; Costa, M
1986 Jun;1(2):11-25, Journal of biochemical toxicology
The degree of inhibition of semiconservative DNA replication induced by nickel chloride (NiCl2) was analyzed by radiolabeled-thymidine incorporation alone or with cesium chloride (CsCl) density gradient centrifugation. The onset and duration of this Ni2+-induced inhibition was time- and concentration-dependent, but the degree of inhibition was not. A maximal reduction in the rate of DNA synthesis was observed within the first hour of treatment with 2.5 mM NiCl2, which was the highest noncytotoxic concentration utilized. After six hours, 500 microM and 1 mM as well as 2.5 mM NiCl2 all produced the same 50% to 60% reduction in [3H]-thymidine incorporation into DNA. The inhibitory effect of nickel ions on DNA synthesis was reversible. The rate of DNA synthesis following a 500 microM or 1 mM NiCl2 treatment began to increase after washout of nickel, but a six-hour exposure of cells to 2.5 mM NiCl2 produced a sustained 50% to 60% suppression of DNA synthetic activity for at least 36 hours. At all concentrations of NiCl2 used in this study, some inhibition of DNA synthesis persisted for at least 48 hours, but by 72 hours after treatment, the rate of [3H]-thymidine incorporation was actually 10% above the control. Examination of autoradiographic slides of cells treated with 2.5 mM NiCl2 for six hours demonstrated a 60% reduction of silver grains, but there was no preferential reduction in the quantity of grains in the nucleolus or any other region. Cesium chloride density gradient analysis of the replication of nucleolar DNA in cells treated with 2.5 mM nickel supported the autoradiographic findings. The inhibitory effect of NiCl2 on DNA replication was prevented by the addition of magnesium chloride (MgCl2) to cells maintained in a simple salts/glucose medium (SGM). This effect did not appear to be due to an antagonism of the cellular uptake of nickel by Mg2+, since the maximally effective dose of Mg2+ reduced 63Ni2+ uptake by no more than 25% while the inhibition of replication was completely reversed
— id: 133266, year: 1986, vol: 1, page: 11, stat: Journal Article,

Metal ion carcinogenesis: mechanistic aspects
Costa M; Heck JD
1986 ;20:259-278, Metal ions in biological systems
— id: 72788, year: 1986, vol: 20, page: 259, stat: Journal Article,

Methods for the in vitro assessment of metal ion toxicity
Heck JD; Costa M
1986 ;20:279-303, Metal ions in biological systems
— id: 72789, year: 1986, vol: 20, page: 279, stat: Journal Article,

Pathway of nickel uptake influences its interaction with heterochromatic DNA
Sen, P; Costa, M
1986 Jun 30;84(2):278-285, Toxicology & applied pharmacology
Exposure of intact Chinese hamster ovary cells to water-soluble NiCl2 and to particulate crystalline NiS induced a concentration-dependent incidence of chromosomal aberrations which included gaps, breaks, and exchanges. Exposure of cells to crystalline NiS particles caused a high incidence of chromatid exchanges and dicentrics and produced what appears to be an effect on the condensation state of the heterochromatic long arm of the X chromosome. Treatment of cells with NiCl2 did not cause any significant effect on the long arm of the X chromosome, and there was a much lower incidence of the dicentric type of chromosomal aberrations compared to NiS. To examine whether the fragmentation/decondensation of the long arm of the X chromosome produced by crystalline NiS particles was due to a phagocytic pathway of uptake of NiS particles, cells were treated with NiCl2-albumin complexes that had been encapsulated in liposomes. Although treatment of cells with NiCl2-albumin complexes yielded higher intracellular nickel levels than were obtained by treatment of cells with NiCl2, at comparable intracellular levels fragmentation/decondensation of the heterochromatic long arm of the X chromosome was observed when nickel (II) was delivered by way of a liposome but not when cells were treated with unencapsulated NiCl2. Ionic nickel alone irrespective of its delivery mechanism exhibited some preference for heterochromatin, since there was a higher incidence of aberrations observed in the heterochromatic centromeric region of chromosomes. These observations suggest that the pathway of delivery of Ni2+ from NiS particles may be responsible for a preferential interaction of this metal with heterochromatin leading to an effect on the condensation state/fragmentation of the heterochromatic long arm of the X chromosome
— id: 133269, year: 1986, vol: 84, page: 278, stat: Journal Article,

Characterization of DNA lesions induced by CaCrO4 in synchronous and asynchronous cultured mammalian cells
Sugiyama, M; Patierno, S R; Cantoni, O; Costa, M
1986 Jun;29(6):606-613, Molecular pharmacology
Alkaline elution studies demonstrated CaCrO4-induced DNA single strand breaks and DNA-protein crosslinks. DNA single strand breaks increased following treatment with 10-400 microM CaCrO4 in Chinese hamster ovary cells maintained with a minimal salts/glucose medium. DNA single strand breaks were rapidly repaired when extracellular CaCrO4 was removed even following exposure levels of CaCrO4 (200 microM for 2 hr) which reduced survival to 0.6%. Under these exposure conditions the trypan blue exclusion was greater than 80%, whereas cell growth was inhibited by 46% within 24 hr. The DNA-protein crosslinks induced by 10 microM CaCrO4 were repaired in the absence of metal within 24 hr. In contrast, the amount of DNA-protein crosslinks measured 24 hr after a 2-hr treatment with 50, 100, and 200 microM CaCrO4 remained unchanged at the 50 microM level or increased at the two higher concentrations. Thus, at concentrations of 50 microM or greater there was no repair of the DNA protein crosslinks, and this may have been due to cytotoxicity of the metal. CaCrO4 at 10 or 25 microM exposure for 6 hr also induced DNA-protein crosslinking in Chinese hamster ovary cells maintained in normal tissue culture growth media. The lack of repair of DNA-protein crosslinks at the 25 microM level, which did not substantially reduce cell survival, indicated the persistence of these lesions in a noncytotoxic form. Uptake of CaCrO4 was linear with all of the concentrations tested. Analysis of the cell cycle sensitivity to CaCrO4 revealed that cells in early S phase were the most sensitive to the cytotoxic and strand breaking activity of CaCrO4. Compared with other phases of the cell cycle, there was also an elevated level of DNA-protein crosslinks when cells were treated in early S phase and incubated 24 hr without CaCrO4. These results implicate the DNA-protein crosslink as an important lesion that may be responsible for the cytotoxic and carcinogenic properties of chromate
— id: 141428, year: 1986, vol: 29, page: 606, stat: Journal Article,

Comparison of DNA lesions and cytotoxicity induced by calcium chromate in human, mouse, and hamster cell lines
Sugiyama, M; Wang, X W; Costa, M
1986 Sep;46(9):4547-4551, Cancer research
DNA lesions, cytotoxicity, and cellular uptake of CaCrO4 were compared in Chinese hamster ovary, in mouse embryo fibroblast C3H10T1/2, and in human osteosarcoma cells. The concentration of CaCrO4 that reduced colony formation by 50% was 2- or 3-fold less in human osteosarcoma cells than in C3H10T1/2 or Chinese hamster ovary cells. Alkaline elution studies showed that CaCrO4 induced DNA single strand breaks in a concentration dependent manner in all three cell lines. However, the human cells exhibited four times more breaks than the hamster cells and two times more than C3H10T1/2 cells when the CaCrO4 exposure conditions were equivalent. Alkaline elution studies also demonstrated the formation of DNA-protein cross-links by CaCrO4 in all three cell lines. In hamster and mouse cells the induction of these DNA-protein cross-links was dependent on concentrations that ranged from 5 to 50 microM for 6 h; however, the cross-links were saturated at 25 microM in human cells and at 50 microM in mouse and hamster cells. The level of cross-links was four times greater in the human cells compared to the mouse cells and was a factor of 2 greater in the hamster cells compared to the mouse cells. The uptake of CaCrO4 was linear with respect to time and concentration. Uptake of CaCrO4 was equivalent in the human and mouse cells, but was a factor of 4 less in the hamster cells. The single strand breaks were almost entirely repaired after an 18-h incubation in metal free medium in all three cell lines, whereas DNA-protein cross-links persisted in these cell lines in proportion to their initial levels. These results demonstrate differences in the sensitivity of human, hamster, and mouse cells to CaCrO4 and suggest that the repair-resistant DNA-protein cross-link may be important in mediating the long term toxic and carcinogenic effects of CaCrO4
— id: 133267, year: 1986, vol: 46, page: 4547, stat: Journal Article,

DNA-protein cross-links induced by nickel compounds in intact cultured mammalian cells
Patierno, S R; Costa, M
1985 Oct;55(1-2):75-91, Chemico-biological interactions
The carcinogenic activity of crystalline NiS has been attributed to phagocytosis and intracellular dissolution of the particles to yield Ni2+ which is thought to enter the nucleus and damage DNA. In this study the extent and type of DNA damage in Chinese hamster ovary CHO cells treated with various nickel compounds was assessed by alkaline elution. Both insoluble (crystalline NiS) and soluble (NiCl2) nickel compounds induced single strand breaks and DNA protein cross-links. The single strand breaks were repaired relatively quickly but the DNA-protein cross-links were present and still accumulating 24 h after exposure to nickel. Single strand breakage occurred at both non-cytotoxic and cytotoxic concentrations of nickel, however, DNA-protein cross-linking was absent when cells were exposed to toxic nickel levels. The concentration of nickel that induced DNA-protein cross-linking correlated with those metal concentrations that reversibly inhibited cellular replication
— id: 141430, year: 1985, vol: 55, page: 75, stat: Journal Article,

Preferential DNA-protein cross-linking by NiCl2 in magnesium-insoluble regions of fractionated Chinese hamster ovary cell chromatin
Patierno, S R; Sugiyama, M; Basilion, J P; Costa, M
1985 Nov;45(11 Pt 2):5787-5794, Cancer research
Intracellular nickel ions (Ni2+) have been shown to cause single-strand breaks in DNA, that were rapidly repaired, and DNA-protein cross-links, that persisted for at least 24 h following removal of extracellular ionic nickel. In this study, we have used the techniques of alkaline elution, chromatin fractionation, and sodium dodecyl sulfate:polyacrylamide gel electrophoresis to examine the DNA-protein cross-linking induced by NiCl2 in Chinese hamster ovary cells. Continuous treatment of logarithmically growing Chinese hamster ovary cells with 2.5 mM NiCl2 in complete medium resulted in DNA single-strand breaks within 1 h, followed by a time-dependent increase in the induction of DNA-protein cross-links at 2, 3, and 6 h. Since the entry of nickel into cells was maximal within 2 h of exposure, the time delay for the formation of DNA-protein cross-links was not limited by metal uptake. The nickel-induced DNA-protein cross-linking appeared to require active cell cycling, since single-strand breaks but no cross-linking could be detected in confluent cells treated with 1, 2.5, or 5 mM NiCl2 for 3 h. DNA-protein cross-linking induced by nickel occurred in late S phase of the cell cycle. High-molecular-weight nonhistone chromatin proteins and possibly histone H1 migrating at the Mr 30,000 range became cross-linked to DNA after treatment of cells with NiCl2. All nickel-cross-linked proteins were concentrated in the magnesium-insoluble regions of fractionated chromatin and were stable to urea, 2-mercaptoethanol, and Nonidet P-40. Some proteins (Mr 48,000, 52,000, 55,000, 70,000, and 95,000), the association of which with DNA was also stable to Sarkosyl, salt, and EDTA, were detectable in DNA rigorously fractionated from untreated cells. Nickel therefore appeared to cause the cross-linking of proteins that normally reside in close association with DNA. Alterations of the normal association of these proteins with DNA by nickel may be an early event in the nickel transformation process
— id: 141429, year: 1985, vol: 45, page: 5787, stat: Journal Article,

Perspectives on the mechanism of nickel carcinogenesis
Costa M; Heck JD
1984 ;6:285-309, Advances in inorganic biochemistry
— id: 72787, year: 1984, vol: 6, page: 285, stat: Journal Article,

Toxicity and carcinogenicity of essential and non-essential metals
Costa M; Kraker AJ; Patierno SR
1984 ;1:1-49, Progress in clinical biochemistry & medicine
— id: 72786, year: 1984, vol: 1, page: 1, stat: Journal Article,

Growth inhibition and metallothionein induction in cadmium-resistant cells by essential and non-essential metals
Evans, R M; Patierno, S R; Wang, D S; Cantoni, O; Costa, M
1983 Jul;24(1):77-83, Molecular pharmacology
Essential and non-essential metal ions were compared on the basis of their growth-inhibitory potency and their mediation of metallothionein induction in a Chinese hamster ovary cell line resistant to cadmium. Cadmium-resistant cells were found to be 20-fold and 6-fold more resistant than wild-type Chinese hamster ovary cells to the non-essential metals CdCl2 and HgCl2, respectively. In contrast, cadmium-resistant cells showed 2-fold or less resistance to growth inhibition due to the metals with known or possible biological essentiality, ZnCl2, CuSO4, CoCl2, and NiCl2. Resistance to either cadmium or mercury was not due to decreased uptake as measured isotopically or by X-ray fluorescence. At concentrations near the threshold of growth inhibition, CdCl2 and ZnCl2 induced metallothionein 8- to 10-fold above background levels in cadmium-resistant cells within 8-10 hr. A 2- to 3-fold induction of this protein was produced in resistant cells by levels of HgCl2, CuSO4, and CoCl2 near the threshold of growth inhibition whereas NiCl2 produced no measurable elevations of metallothionein at concentrations below, near, and above those that inhibit cell growth. Induction of metallothionein was measured by a modified 203Hg binding assay and by [35S]cysteine incorporation. No measurable induction of metallothionein was evident in wild-type cells with any metal treatment using a reasonable quantity of cells consistent with our assay. These results in cadmium-resistant cells demonstrate selective induction of metallothionein by various metals and suggest that induction of this protein alone is not solely responsible for differences in the growth-inhibitory potential of these elements
— id: 141431, year: 1983, vol: 24, page: 77, stat: Journal Article,

Inhibition of LPS toxicity for macrophages by metallothionein-inducing agents
Patierno, S R; Costa, M; Lewis, V M; Peavy, D L
1983 Apr;130(4):1924-1929, Journal of immunology
Parenteral administration of adrenal corticosteroids or particular transition metal salts are known to protect mice from the lethal effects of bacterial lipopolysaccharides (LPS). To determine if both groups of substances act through similar biologic mechanisms, their capacity to protect macrophages from the direct toxic effects of LPS was examined in vitro. When added simultaneously with LPS at culture initiation, 10 to 100 microM cortisone increased the viability of normal peritoneal macrophages as determined by trypan blue exclusion. Prednisolone and corticosterone protected LPS-treated macrophages at even lower concentrations (0.1 to 1 microM); estradiol and testosterone failed to alter cell viability at any concentration tested. Protection was dependent on de novo synthesis because inclusion of 20 nM actinomycin C1 or 1 microM cycloheximide with 10 microM corticosterone during a 4-hr pretreatment period blocked induction of the protective effect. Murine macrophages were also protected by micromolar concentrations of zinc, cadmium, mercury, and manganese, but not by calcium or lead. As was obtained with corticosteroids, heavy metal-induced protection depended on de novo RNA and protein synthesis. Because all substances that protected against LPS are known inducers of metallothionein in somatic cells, peritoneal macrophages were assayed for the presence of this unique, cytoplasmic protein. Within 2 to 8 hr, 10 microM cadmium caused three to fivefold increases in the incorporation of 35S-cysteine and in the binding of 203Hg into the TCA-soluble fraction of cell lysates that was excluded on centrifugally accelerated Sephadex G-10 columns. These results suggest macrophages may be protected from LPS-mediated cytotoxicity through synthesis of a sulfhydryl-rich, metal-binding protein. Although its mechanism of action remains unknown, it is proposed that metallothionein may function homeostatically by altering intracellular concentrations of zinc or may play a regulatory role by facilitating transfer of heavy metals among metal-requiring apoproteins
— id: 141433, year: 1983, vol: 130, page: 1924, stat: Journal Article,

Application of a modified 203Hg binding assay for metallothionein
Patierno, S R; Pellis, N R; Evans, R M; Costa, M
1983 Apr 4;32(14):1629-1636, Life sciences
A sensitive and rapid method to estimate concentrations of functional metallothionein in small biological samples, based upon the acid stability of 203Hg binding and solubility of this protein in trichloroacetic acid is described. Sephadex G-10 minicolumns supported in centrifuge tubes afforded separation and quantitation of isotope bound metallothionein from unbound metal. Elution of metallothionein bound 203Hg was achieved by short term-low speed centrifugation that segregated chelator-ligand complex into the eluate while unbound ligand remained in the gel. A well characterized standard of pure metallothionein protein was utilized to verify the specificity and sensitivity of the modified assay. Metallothionein levels were estimated by 203Hg binding in extracts of wild type and cadmium resistant Chinese hamster ovary cells treated with maximum tolerable concentrations of CdCl2. Similar separation methods demonstrated [35S]-cysteine incorporation into induced metallothionein. Additionally, induction of metallothionein was observed after treatment with particulate CdS but not crystalline NiS particles. These results demonstrate that the modified assay system is easily applied to serial measurement of metallothionein levels in multiple small biological samples
— id: 141432, year: 1983, vol: 32, page: 1629, stat: Journal Article,

Comparison of protein, RNA, and DNA binding and cell-cycle-specific growth inhibitory effects of nickel compounds in cultured cells
Harnett, P B; Robison, S H; Swartzendruber, D E; Costa, M
1982 Jun 15;64(1):20-30, Toxicology & applied pharmacology
— id: 141437, year: 1982, vol: 64, page: 20, stat: Journal Article,

Factors influencing the phagocytosis, neoplastic transformation, and cytotoxicity of particulate nickel compounds in tissue culture systems
Costa, M; Abbracchio, M P; Simmons-Hansen, J
1981 Sep 15;60(2):313-323, Toxicology & applied pharmacology
— id: 141438, year: 1981, vol: 60, page: 313, stat: Journal Article,

Biochemical and morphological transformation of hamster embryo cells in tissue culture by specific metal compounds
Costa M
Molecular basis of environmental toxicity Ann Arbor MI : Ann Arbor Science, 1980,
— id: 4410, year: 1980, vol: , page: 373, stat: Chapter,

Metal carcinogenesis in tissue culture systems
Costa M; Jones MK; Lindberg O
1980 ;140:45-73, ACS symposium series
— id: 72785, year: 1980, vol: 140, page: 45, stat: Journal Article,

Phagocytosis of particulate nickel compounds is related to their carcinogenic activity
Costa M; Mollenhauer HH
Nickel toxicology London : Academic Press, 1980,
— id: 4411, year: 1980, vol: , page: 43, stat: Chapter,

Metal carcinogenesis testing : principles and in vitro methods
Costa, Max
Clifton NJ : Humana Press, 1980,
— id: 1216, year: 1980, vol: , page: , stat: ,

Preliminary report on nickel-induced transformation in tissue culture
Costa M
Ultratrace metal analysis in biological sciences and environment : a symposium Washington DC : American Chemical Society, 1979,
— id: 4409, year: 1979, vol: , page: 73, stat: Chapter,

Regulation of cAMP-dependent protein kinases by endogenous heat stable inhibitor(s)
Costa M
Proteases and hormones New York : Elsevier, 1979,
— id: 4408, year: 1979, vol: , page: 159, stat: Chapter,

Alteration in morphology of chinese hamster ovary cells by Ni3S2 and dibutyryl cAMP
Costa, M
1978 Jun;44(3):555-566, Toxicology & applied pharmacology
— id: 141439, year: 1978, vol: 44, page: 555, stat: Journal Article,

[Polarographic study of the effect of verapamil on respiratory activity and oxidative phosphorylation of mitochondria]
Rodrigues, L E; Pinto, R S; Costa, M
1978 Feb;31 Suppl 1:15-18, Arquivos brasileiros de cardiologia
— id: 99555, year: 1978, vol: 31 Suppl 1, page: 15, stat: Journal Article,

Cyclic AMP and cyclic AMP-dependent protein kinase in cell growth processes
Costa, Max
[S.l. : s.n.], 1976,
Thesis (Ph.D. - Pharmacology) -- University of Arizona, 1976
— id: 1220, year: 1976, vol: , page: , stat: ,