Toby G. Rossman

Although the epidemiological evidence linking arsenic exposure with increased cancer risk is strong, attempts to induce carcinomas with arsenite in animals have generally failed. This has hindered mechanistic studies of arsenic carcinogenesis.

We previously found that arsenite at low concentrations is not mutagenic but acts as a comutagen, most likely a result of the inhibition of DNA repair (both base and nucleotide excision repair). However, in biochemical assays, no specific repair enzyme has been found to be sensitive to low concentrations of arsenite, leading to the hypothesis that the effects of arsenite on DNA repair may result when DNA damage–induced signaling, which regulates DNA repair—is faulty.

In a test of this hypothesis, we recently showed that in cells treated with 0.1 µM arsenite and ionizing radiation, the p53-dependent increase in p21 expression—which normally blocks cell cycle progression after DNA damage—is deficient. In addition, arsenite treatment increased the abundance of the cell-cycle progression regulator cyclin D1.

To determine if arsenite acts as an enhancing agent (cocarcinogen) with a genotoxic partner, we chose solar ultraviolet radiation (UVR) to induce skin cancer in hairless SKH1 mice. Mice given 10 mg/L sodium arsenite in drinking water for 26 weeks had a 2.4-fold increase in tumor yield after 1.7 KJ/m² UVR three times weekly, as compared with mice exposed to UVR alone.

In a second experiment, we found a dose-related cocarcinogenic effect with a peak enhancement (almost fivefold) at 5 mg/L arsenite plus 1.0 KJ/m² UVR. The tumors were mostly squamous cell carcinomas, and those occurring in mice given UVR plus arsenite appeared earlier and were much larger and more invasive than in mice given UVR alone.

Normal skin obtained at the end of the experiment showed an increased epidermal thickness and an increased fraction of epidermal cells expressing proliferating cell nuclear antigen in mice exposed to arsenite, as compared with control mice. In mice exposed to arsenite plus UVR there was a synergistic effect on proliferation. However, the increase was already apparent at the lowest arsenite dose used (1.25 mg/L) and did not increase further at higher doses.

These results are consistent with the hypothesis that arsenite acts as a cocarcinogen with a second (genotoxic) agent by inhibiting DNA repair and increasing cell proliferation. In addition, our data suggest that arsenite-induced increases in epithelial-cell proliferation might be a necessary, though insufficient, cause of cocarcinogenesis with UVR, given that increased proliferation alone does not lead to skin cancer and does not correlate with cocarcinogenesis.

Another area of study involves the identification of possible biomarkers of genetic susceptibility to arsenic toxicity and carcinogenicity. We have demonstrated for the first time that expression levels of GGT1 might be useful as a biomarker of genetic susceptibility to arsenite.

Human lymphoblast cell lines from normal unexposed donors showed variable sensitivities to the toxic effects of arsenite. We used microarray analysis to compare the basal gene expression profiles between two arsenite-resistant lymphoblast cell lines (GMO02707, GMO00893A) and two arsenite-sensitive lymphoblast cell lines (GMO00546B, GMO00607C). A number of genes were differentially expressed between arsenite-sensitive and arsenite-resistant cells. Among these, γ-glutamyltranspeptidase 1 (GGT1) showed higher expression levels in arsenite-resistant cells. Gene expression analysis using reverse transcriptase polymerase chain reaction (RT-PCR) using gene-specific primers confirmed these results. Reducing GGT1 expression levels with GGT1-specific small interfering RNA in arsenite-resistant lymphoblasts resulted in increased cell sensitivity to arsenite.