Carcinogenic metals and the epigenome: understanding the effect of nickel, arsenic, and chromium

Abstract

Carcinogenic metals, such as nickel, arsenic, and chromium, are widespread environmental and occupational pollutants. Chronic exposure to these metals has been connected with increased risks of numerous cancers and as well as non-carcinogenic health outcomes, including cardiovascular disease, neurologic deficits, neuro-developmental deficits in childhood, and hypertension. However, currently the specific molecular targets for metal toxicity and carcinogenicity are not fully understood. Here, we propose that the iron- and 2-oxoglutarate-dependent dioxygenase family enzymes, as well as, other histone modifying enzymes are important intracellular targets that mediate the toxicity and carcinogenicity of nickel, and maybe potential targets in chromium and arsenic induced carcinogenesis. Our data demonstrate that all three metals are capable of inducing post-translational histone modifications and affecting the enzymes that modulate them (i.e. the iron- and 2-oxoglutarate-dependent dioxygenase family, including HIF-prolyl hydroxylase PHD2, histone demethylase JHDM2A/JMJD1A, and DNA repair enzymes ABH3 and ABH2, and histone methyltransferases, G9a). Given the effects that these metals can exert on the epigenome, future studies of their involvement in histone modifying enzymes dynamics would deepen our understanding on their respective toxicities and carcinogenicities.

Figure 1

Model for iron- and 2-oxoglutarate-dependent dioxygenase dimethylation of lysines.

Figure 2

Model of particular nickel compound uptake and intracellular dissolution.

Figure 3

Newport Green fluorescence showing the intracellular distribution of nickel ions following treatment of human lung cells with NiCl₂ and Ni₃S₂. From “Fluorescent tracking of nickel ions in human cultured cells” vol. 219(1) by Ke Q. et al. Copyright 2007 by Toxicology and Applied Pharmacology. Reproduced with permission of Toxicology and Applied Pharmacology via Copyright Clearance Center.

Figure 4

The changes of global histone H3K9 methylation following nickel ion exposure. (a) A549 cells were exposed to 0.5 mM or 0.75 mM NiCl₂ for 24 h. (b) A dose-dependent increase of global H3K9 dimethylation by nickel ions. A549 cells were exposed to various concentrations of NiCl₂ for 24 h. (c) A time course study on global H3K9 dimethylation following nickel ion exposure. A549 cells were exposed to 1 mM NiCl² for selected time intervals as indicated. (d) Exposure to 1 mM NiCl₂ for 24 h increased H3K9 dimethylation in different cell types. HOS, human osteosarcoma; MES, murine embryonic stem. Histones were extracted and separated in a 15% SDS-polyacrylamide gel and immunoblotted with various antibodies as indicated. Loading of the histones in all gels was assessed using Coomassie blue staining. From “Ions Increase Histone H3 Lysine 9 Dimethylation and Induce Transgene Silencing” vol. 26(10) by Haobin Chen et al. Copyright 2006 by Molecular and Cellular Biology. Reproduced with permission of Molecular and Cellular Biology via Copyright Clearance Center

Figure 5

A kinetic study on Ni inhibition of JHDM2A and ABH3 demethylase activity. (a) Purified Flag- JHDM2A was assayed for its demethylase activity in the presence of different concentrations of Ni(II) ions as indicated. The assay with addition of EDTA, a chelator of divalent metals, was performed in parallel as a negative control. (a) Data quantification of (a). (c) Purified 69His-tagged ABH3 was assayed for its demethylase activity in the presence of different concentrations of Ni(II) ions as indicated. (d) Data quantification of (c). (e) Purified aconitase, an Fe–S cluster-containing enzyme, was incubated with different concentrations of Ni(II) ions for 4 h. After incubation, the aconitase activity was measured immediately as previously described. Aconitase activity is presented as that relative to levels in the control samples. Each bar represents the mean (±SD) from three samples per treatment. *Statistically significant change (P<0.05) compared to control samples. From “Iron- and 2-oxoglutarate-dependent Dioxygenases:an emerging group of molecular targets for nickel toxicity and carcinogenicity” vol. 22 by Chen H. and Costa M. Copyright 2009 by Biometals. Reproduced with permission of Biometals via Copyright Clearance Center

Figure 6

Nickel ions bind to the iron-binding site ofABH2in cells. (a) measurement of FLAG-ABH2 and ABH2(D173A) expression levels in the nickel-treated 293T cells. 293T cells were transiently transfected with FLAG-ABH2 and FLAG-ABH2(D173A) expression vectors and then treated with 1mMNiCl2 that contained 0.22 mCi of 63NiCl2. Expression of FLAG-ABH2 or ABH2(D173A) in cell lysates was measured by Western blot using anti-FLAG antibody. The intensity of bands was quantified using ImageJ software and marked below the graph. The quantification results were graphed on the right. (b), cell lysates collected in (a) were subject to IP with anti-FLAG resin. The FLAG-tagged recombinant proteins were eluted with FLAG peptide, and their associated radioactivity was measured. (c) 63Ni-specific radioactivity associated with FLAG-ABH2 or ABH2(D173A) was calculated. The experiment was conducted in triplicate, and values are means_S.D. for triplicates. The difference in 63Ni-specific radioactivity between FLAG-ABH2 and FLAG-ABH2(D173A) samples is statistically significant because a two-tailed Student t test analysis gives a p value of 0.044. From “Nickel Ions Inhibit Histone Demethylase JMJD1A and DNA Repair Enzyme ABH2 by Replacing the Ferrous Iron in the Catalytic Centers” vol. 285(10) by Chen et al. Published, JBC Papers in Press 2009.

Figure 7

Gene expression profiles of PBMCs of subjects with occupational exposure compared to subjects with environmental exposure to nickel. (a) The number of entities with more than 1.25-, 1.5-, and 2-fold change in expression level with pairwise comparison. (b) Principal component analysis revealed distinct separation between subjects with environmental exposure to low levels of nickel (red circle0 and subjects with occupational exposure to high levels of nickel (blue circle). (c) Hierarchical cluster analysis of genes with more than 1.5-fold changes expression in PBMCs of all 5 subjects occupationally exposed to nickel compared to subjects with environmental exposure. The color bar related color code to the expression value determined after quantile normalization and baseline transformation to the median levels of all samples.

Figure 8

Cr(VI) exposure increased G9a protein levels. A549 cells were treated with 5 or 10 µM of Cr(VI) for 24 hours. Total protein lysates were extracted and analyzed with antibody against G9a. Antibody against tubulin was used to assess the loading of proteins. (b) A549 cells were exposed to 5 or 10 µM Cr(VI) for 24 hours. The global levels of H3K9me2 were measured using specific antibodies. Coomassie blue staining was used to assess the equal loading of the histones. (C and D) Chromate exposure increased G9a mRNA levels after 24 hr exposure. A549 cells were treated with 5 or 10 µM Cr(VI) for 1 (d) or 24 (c) hours. G9a mRNA levels were analyzed by Northern blotting. The ethidium bromide staining of 28S and 18S RNA was performed to assess the loading of RNA samples. The relative intensity of the bands was measured and plotted as the mean ratio of G9a mRNA to 18S RNA ± SE (error bars). * P < 0.05, ** P < 0.01, *** P < 0.001. From “Modulation of histone methylation and MLH1 gene silencing by hexavalent chromium” vol. 237(3) by Sun et al. Copyright 2009 Toxicology and Applied Pharmacology. Reproduced with permission of Toxicology and Applied Pharmacology via Copyright Clearance Center.

Figure 9

Gene expression profiles of control, Cr(VI) transformed and parental BEAS-2B cells. Hierarchical cluster analysis of genes with more than 2-fold changed expression in one out of three groups (control, Cr_small, Cr_large) compared to parental BEAS-2B cells. The color bar related color code to the expression value determined after quantile normalization and baseline transformation to the median levels of all samples. From “Comparison of gene expression profiles in chromate transformed BEAS-2B cells” vol. 6(3) by Sun et al. Published in PloS One in 2011.

Figure 10

Distinct localization of H3K9 di-methylation (H3K9me2) and H3K4 tri-methylation (H3K4me3) in arsenite exposed cells. A549 cells were exposed 5 µM arsenite for 24 h. After exposure, cells were co-stained with di-methylated H3K9 (red) and tri-methylated H3K4 (green) antibodies. The nucleus was counterstained with DAPI (blue). Merge images show a merge of the red, green and blue staining. The pictures were taken using a confocal microscope. From “Effects of nickel, chromate, and arsenite on histone 3 lysine methylation” vol. 236(1) by Zhou X. et al. Copyright 2009 Toxicology and Applied Pharmacology. Reproduced with permission of Toxicology and Applied Pharmacology via Copyright Clearance Center.