Arsenic exposure is associated with decreased DNA repair in vitro and in individuals exposed to drinking water arsenic

Abstract

The mechanism(s) by which arsenic exposure contributes to human cancer risk is unknown ; however, several indirect cocarcinogenesis mechanisms have been proposed. Many studies support the role of As in altering one or more DNA repair processes. In the present study we used individual-level exposure data and biologic samples to investigate the effects of As exposure on nucleotide excision repair in two study populations, focusing on the excision repair cross-complement 1 (ERCC1) component. We measured drinking water, urinary, or toenail As levels and obtained cryopreserved lymphocytes of a subset of individuals enrolled in epidemiologic studies in New Hampshire (USA) and Sonora (Mexico). Additionally, in corroborative laboratory studies, we examined the effects of As on DNA repair in a cultured human cell model. Arsenic exposure was associated with decreased expression of ERCC1 in isolated lymphocytes at the mRNA and protein levels. In addition, lymphocytes from As-exposed individuals showed higher levels of DNA damage, as measured by a comet assay, both at baseline and after a 2-acetoxyacetylaminofluorene (2-AAAF) challenge. In support of the in vivo data, As exposure decreased ERCC1 mRNA expression and enhanced levels of DNA damage after a 2-AAAF challenge in cell culture. These data provide further evidence to support the ability of As to inhibit the DNA repair machinery, which is likely to enhance the genotoxicity and mutagenicity of other directly genotoxic compounds, as part of a cocarcinogenic mechanism of action.

Figure 1

ERCC1 gene expression (mean ± SE) by drinking water As level for individuals from the New Hampshire (NH) and Mexican populations and for both populations combined (n = 53). ERCC1 levels were assessed by RT-PCR and normalized to 18s or GAPDH as described in “Materials and Methods.” *Statistically significant compared to the ≤ 5 μg/L group (p < 0.05).

Figure 2

Association of drinking water As exposure > 5 μg/L with decreased ERCC1 protein levels in human lymphocytes from the New Hampshire population. (A) Immunoblot of protein extracts from human lymphocytes obtained from a subset of eight individuals assessed using antibody to ERCC1 or β -actin. (B) The ratio of band densities of ERCC1 to β-actin from the immunoblot shown in (A) graphed by drinking water As concentration; values shown are mean ± SD. *Statistically significant compared to the control group (p < 0.05).

Figure 3

Comet assay on human lymphocytes obtained from New Hampshire subjects exposed in vivo to low or high drinking water As levels. Cells were analyzed at baseline, after in vitro challenge with 2-AAAF, and after a 4-hr repair period.

Figure 4

Comet assay results associated with in vivo As exposure in human lymphocytes from the New Hampshire population. Cells from 12 individual subjects were each divided into three subsets and analyzed immediately after harvest (time = 0 hr; p < 0.001), after a 2-hr in vitro challenge with 4 μM 2-AAAF (time = 2 hr; p = 0.248), and after a 4-hr repair period (time = 6 hr; p < 0.001). See “Materials and Methods” for details.

Figure 5

Effect of in vitro As exposure on ERCC1 expression in a cultured Jurkat lymphoblast cell line. Cells were harvested after exposure to As (0.01–10 μM) for 24 hr. ERCC1 mRNA expression level was assessed by RT-PCR, quantitated using a standard curve using known amounts of cDNA, and normalized to 18s rRNA, as described in “Materials and Methods.” Values shown are mean ± SD. *Statistically significant compared to the control group (p < 0.05).

Figure 6

Effect of in vitro As exposure on DNA damage and repair function in a cultured Jurkat lymphoblast cell line. Cultured cells were exposed in vitro to 0 or 1 μM As for 24 hr (n = 6 independent cultures). Cells from each culture were analyzed by single-cell gel electrophoresis immediately after harvest (time = 0 hr), after a 2-hr in vitro challenge with 4 μM 2-AAAF (time = 2 hr), and after a 4-hr repair period (time = 6 hr). See “Materials and Methods” for details. Values represent mean ± 95% confidence interval. *Statistically significant compared to the control group (p < 0.05).