Alkyltransferase-mediated toxicity of bis-electrophiles in mammalian cells

Abstract

The primary function of O(6)-alkylguanine-DNA alkyltransferase (AGT) is to maintain genomic integrity in the face of damage by both endogenous and exogenous alkylating agents. However, paradoxically, bacterial and mammalian AGTs have been shown to increase cytotoxicity and mutagenicity of dihaloalkanes and other bis-electrophiles when expressed in bacterial cells. We have extended these studies to mammalian cells using CHO cells that lack AGT expression and CHO cells stably transfected with a plasmid that expresses human AGT. The cytotoxicity of 1,2-dibromoethane, dibromomethane and epibromohydrin was significantly increased by the presence of AGT but cytotoxicity of butadiene diepoxide was not affected. Mutations caused by these agents were assessed using hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a reporter gene. There was a small (c. 2-3-fold) but statistically significant AGT-mediated increase in mutations caused by 1,2-dibromoethane, dibromomethane and epibromohydrin. Analysis of the mutation spectrum induced by 1,2-dibromoethane showed that the presence of AGT also altered the types of mutations with an increase in total base substitution mutants due to a rise in transversions at both G:C and A:T sites. AGT expression also led to mutations arising from the transcribed strand, which were not seen in cells lacking AGT. Although the frequency of deletion mutations was decreased by AGT expression, the formation of large deletions (> or = 3 exons) was increased. This work demonstrates that interaction of AGT with some bis-electrophiles can cause mutagenicity and diminished cell survival in mammalian cells. It is consistent with the hypothesis that DNA-AGT cross-links, which have been characterized in experiments with purified AGT protein and such bis-electrophiles, can be formed in mammalian cells.

Figure 1. Effect of hAGT expression on toxicity of DBE, DBM, EBH and BDO in CHO cells

CHO cells stably transfected with a pCMV expression vector or with hAGT were treated with the compounds shown for 2 h. Cell survival rates were determined as described in Materials and Methods and expressed as the percentage of survival rates of DMSO treated cells. Results are shown in Panel (A) for DBE, Panel (B) for DBM, Panel (C) for EBH and Panel (D) for BDO. Two-way ANOVA was used to analyze the effect of hAGT expression: Panel (A), pCMV versus hAGT: P <0.01; Panel (B), pCMV versus hAGT: P < 0.01; Panel (C), pCMV versus hAGT: P < 0.01; Panel (D) pCMV versus hAGT: P = 0.6.

Figure 2. Effect of hAGT expression on the productions of mutations in the HPRT gene by DBE, DBM, EBH and BDO in CHO cells

CHO cells stably transfected with a pCMV expression vector or with hAGT were treated with the compounds shown for 2 h. Mutations were determined as described in Materials and Methods and the mutation rate expressed per 10⁶ surviving cells grown in the absence of 6-TG. Results are shown in Panel (A) for DBE, Panel (B) for DBM, Panel (C) for EBH and Panel (D) for BDO. Two-way ANOVA was used to analyze the effect of hAGT expression: Panel (A), pCMV versus hAGT, P < 0.01; Panel (B), pCMV versus hAGT: P = 0.02; Panel (C), pCMV versus hAGT: P < 0.01; Panel (D), pCMV versus hAGT: P = 0.1. Comparison of the individual points plus and minus hAGT for each dose showed that the number of mutations for DBE doses 0.5 and 1 mM, DBM doses 0.1 and 0.25 mM and all of the doses of EDB were significantly different (P < 0.05).