Anatoly Zhitkovich

Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens.

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.

Chromium in Drinking Water: Sources, Metabolism, and Cancer Risks

Drinking water supplies in many geographic areas contain chromium in the +3 and +6 oxidation states. Public health concerns are centered on the presence of hexavalent Cr that is classified as a known human carcinogen via inhalation. Cr(VI) has high environmental mobility and can originate from anthropogenic and natural sources. Acidic environments with high organic content promote the reduction of Cr(VI) to nontoxic Cr(III). The opposite process of Cr(VI) formation from Cr(III) also occurs, particularly in the presence of common minerals containing Mn(IV) oxides. Limited epidemiological evidence for Cr(VI) ingestion is suggestive of elevated risks for stomach cancers. Exposure of animals to Cr(VI) in drinking water induced tumors in the alimentary tract, with linear and supralinear responses in the mouse small intestine. Chromate, the predominant form of Cr(VI) at neutral pH, is taken up by all cells through sulfate channels and is activated nonenzymatically by ubiquitously present ascorbate and small thiols. The most abundant form of DNA damage induced by Cr(VI) is Cr-DNA adducts, which cause mutations and chromosomal breaks. Emerging evidence points to two-way interactions between DNA damage and epigenetic changes that collectively determine the spectrum of genomic rearrangements and profiles of gene expression in tumors. Extensive formation of DNA adducts, clear positivity in genotoxicity assays with high predictive values for carcinogenicity, the shape of tumor–dose responses in mice, and a biological signature of mutagenic carcinogens (multispecies, multisite, and trans-sex tumorigenic potency) strongly support the importance of the DNA-reactive mutagenic mechanisms in carcinogenic effects of Cr(VI). Bioavailability results and kinetic considerations suggest that 10–20% of ingested low-dose Cr(VI) escapes human gastric inactivation. The directly mutagenic mode of action and the incompleteness of gastric detoxification argue against a threshold in low-dose extrapolation of cancer risk for ingested Cr(VI).

Mismatch Repair Proteins Are Activators of Toxic Responses to Chromium-DNA Damage

ABSTRACT Chromium(VI) is a toxic and carcinogenic metal that causes the formation of DNA phosphate-based adducts. Cr-DNA adducts are genotoxic in human cells, although they do not block replication in vitro. Here, we report that induction of cytotoxicity in Cr(VI)-treated human colon cells and mouse embryonic fibroblasts requires the presence of all major mismatch repair (MMR) proteins. Cr-DNA adducts lost their ability to block replication of Cr-modified plasmids in human colon cells lacking MLH1 protein. The presence of functional mismatch repair caused induction of p53-independent apoptosis associated with activation of caspases 2 and 7. Processing of Cr-DNA damage by mismatch repair resulted in the extensive formation of γ-H2AX foci in G2 phase, indicating generation of double-stranded breaks as secondary toxic lesions. Induction of γ-H2AX foci was observed at 6 to 12 h postexposure, which was followed by activation of apoptosis in the absence of significant G2 arrest. Our results demonstrate that mismatch repair system triggers toxic responses to Cr-DNA backbone modifications through stress mechanisms that are significantly different from those for other forms of DNA damage. Selection for Cr(VI) resistant, MMR-deficient cells may explain the very high frequency of lung cancers with microsatellite instability among chromate workers.

Ascorbate acts as a highly potent inducer of chromate mutagenesis and clastogenesis: linkage to DNA breaks in G2 phase by mismatch repair

Here we examined the role of cellular vitamin C in genotoxicity of carcinogenic chromium(VI) that requires reduction to induce DNA damage. In the presence of ascorbate (Asc), low 0.2–2 μM doses of Cr(VI) caused 10–15 times more chromosomal breakage in primary human bronchial epithelial cells or lung fibroblasts. DNA double-strand breaks (DSB) were preferentially generated in G2 phase as detected by colocalization of γH2AX and 53BP1 foci in cyclin B1-expressing cells. Asc dramatically increased the formation of centromere-negative micronuclei, demonstrating that induced DSB were inefficiently repaired. DSB in G2 cells were caused by aberrant mismatch repair of Cr damage in replicated DNA, as DNA polymerase inhibitor aphidicolin and silencing of MSH2 or MLH1 by shRNA suppressed induction of γH2AX and micronuclei. Cr(VI) was also up to 10 times more mutagenic in cells containing Asc. Increasing Asc concentrations generated progressively more mutations and DSB, revealing the genotoxic potential of otherwise nontoxic Cr(VI) doses. Asc amplified genotoxicity of Cr(VI) by altering the spectrum of DNA damage, as total Cr-DNA binding was unchanged and post-Cr loading of Asc exhibited no effects. Collectively, these studies demonstrated that Asc-dependent metabolism is the main source of genotoxic and mutagenic damage in Cr(VI)-exposed cells.

Bioavailability, intracellular mobilization of nickel, and HIF-1α activation in human lung epithelial cells exposed to metallic nickel and nickel oxide nanoparticles

Micron-sized particles of poorly soluble nickel compounds, but not metallic nickel, are established human and rodent carcinogens. In contrast, little is known about the toxic effects of a growing number of Ni-containing materials in the nano-sized range. Here, we performed physicochemical characterization of NiO and metallic Ni nanoparticles and examined their metal ion bioavailability and toxicological properties in human lung epithelial cells. Cellular uptake of metallic Ni and NiO nanoparticles, but not metallic Ni microparticles, was associated with the release of Ni(II) ions after 24-48 h as determined by Newport Green fluorescence. Similar to soluble NiCl₂, NiO nanoparticles induced stabilization and nuclear translocation of hypoxia-inducible factor 1α (HIF-1α) transcription factor followed by upregulation of its target NRDG1 (Cap43). In contrast to no response to metallic Ni microparticles, nickel nanoparticles caused a rapid and prolonged activation of the HIF-1α pathway that was stronger than that induced by soluble Ni(II). Soluble NiCl₂ and NiO nanoparticles were equally toxic to H460 human lung epithelial cells and primary human bronchial epithelial cells; metallic Ni nanoparticles showed lower toxicity and Ni microparticles were nontoxic. Cytotoxicity induced by all forms of Ni occurred concomitant with activation of an apoptotic response, as determined by dose- and time-dependent cleavage of caspases and poly (ADP-ribose) polymerase. Our results show that metallic Ni nanoparticles, in contrast to micron-sized Ni particles, activate a toxicity pathway characteristic of carcinogenic Ni compounds. Moderate cytotoxicity and sustained activation of the HIF-1α pathway by metallic Ni nanoparticles could promote cell transformation and tumor progression.

DNA-PROTEIN CROSS-LINKS PRODUCED BY VARIOUS CHEMICALS IN CULTURED HUMAN LYMPHOMA CELLS

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 Burkitts 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.

Interlaboratory validation of a new assay for DNA-protein crosslinks

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 Burkitts 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.

The Chromate-Inducible chrBACF Operon from the Transposable Element TnOtChr Confers Resistance to Chromium(VI) and Superoxide

Large-scale industrial use of chromium(VI) has resulted in widespread contamination with carcinogenic chromium(VI). The abilities of microorganisms to survive in these environments and to detoxify chromate require the presence of specific resistance systems. Here we report identification of the transposon-located (TnOtChr) chromate resistance genes from the highly tolerant strain Ochrobactrum tritici 5bvl1 surviving chromate concentrations of >50 mM. The 7,189-bp-long TnOtChr of the mixed Tn21/Tn3 transposon subfamily contains a group of chrB, chrA, chrC, and chrF genes situated between divergently transcribed resolvase and transposase genes. The chrB and chrA genes, but not chrF or chrC, were essential for establishment of high resistance in chromium-sensitive O. tritici. The chr promoter was strongly induced by chromate or dichromate, but it was completely unresponsive to Cr(III), oxidants, sulfate, or other oxyanions. Plasmid reporter experiments identified ChrB as a chromate-sensing regulator of chr expression. Induction of the chr operon suppressed accumulation of cellular Cr through the activity of a chromate efflux pump encoded by chrA. Expression of chrB, chrC, or chrF in an Escherichia coli sodA sodB double mutant restored its aerobic growth in minimal medium and conferred resistance to superoxide-generating agents menadione and paraquat. Nitroblue tetrazolium staining on native gels showed that ChrC protein had superoxide dismutase activity. TnOtChr appears to represent a mobile genetic system for the distribution of the chromate-regulated resistance operon. The presence of three genes protecting against superoxide toxicity should provide an additional survival advantage to TnOtChr-containing cells in the environments with multiple redox-active contaminants.

Rapid DNA Double-Strand Breaks Resulting from Processing of Cr-DNA Cross-Links by Both MutS Dimers

Mismatch repair (MMR) strongly enhances cyto- and genotoxicity of several chemotherapeutic agents and environmental carcinogens. DNA double-strand breaks (DSB) formed after two replication cycles play a major role in MMR-dependent cell death by DNA alkylating drugs. Here, we examined DNA damage detection and the mechanisms of the unusually rapid induction of DSB by MMR proteins in response to carcinogenic chromium(VI). We found that MSH2-MSH6 (MutSalpha) dimer effectively bound DNA probes containing ascorbate-Cr-DNA and cysteine-Cr-DNA cross-links. Binary Cr-DNA adducts, the most abundant form of Cr-DNA damage, were poor substrates for MSH2-MSH6, and their toxicity in cells was weak and MMR independent. Although not involved in the initial recognition of Cr-DNA damage, MSH2-MSH3 (MutSbeta) complex was essential for the induction of DSB, micronuclei, and apoptosis in human cells by chromate. In situ fractionation of Cr-treated cells revealed MSH6 and MSH3 chromatin foci that originated in late S phase and did not require replication of damaged DNA. Formation of MSH3 foci was MSH6 and MLH1 dependent, whereas MSH6 foci were unaffected by MSH3 status. DSB production was associated with progression of cells from S into G(2) phase and was completely blocked by the DNA synthesis inhibitor aphidicolin. Interestingly, chromosome 3 transfer into MSH3-null HCT116 cells activated an alternative, MSH3-like activity that restored dinucleotide repeat stability and sensitivity to chromate. Thus, sequential recruitment and unprecedented cooperation of MutSalpha and MutSbeta branches of MMR in processing of Cr-DNA cross-links is the main cause of DSB and chromosomal breakage at low and moderate Cr(VI) doses.

Analysis of DNA–protein crosslinking activity of malondialdehyde in vitro

In an attempt to identify endogenous chemicals producing DNA-protein crosslinks, we have studied in vitro crosslinking potential of malondialdehyde, a bifunctional chemical that is ubiquitously formed as a product of lipid peroxidation of polyunsaturated fatty acids. We have found that malondialdehyde readily forms crosslinks between DNA and histones under physiological ionic and pH conditions. Formation of DNA-protein crosslinks was limited to proteins that were able to bind to DNA. Malondialdehyde failed to form DNA-protein crosslinks when histone binding to DNA was prevented by elevated ionic strength or when bovine serum albumin was used in the reaction mixture. Malondialdehyde-produced DNA-histone crosslinks were relatively stable at 37 degrees C with t1/2=13.4 days. Crosslinking of histones to DNA proceeds through the initial formation of protein adduct followed by reaction with DNA. Modification of DNA by malondialdehyde does not lead to a subsequent crosslinking of proteins. Significant formation of DNA-protein crosslinks was also registered in isolated kidney and liver nuclei treated with malondialdehyde. Based on its reactivity and stability of the resulting crosslinks, it is suggested that malondialdehyde could be one of the significant sources of endogenous DNA-protein crosslinks.