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 (≥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.

DNA–protein crosslinks processed by nucleotide excision repair and homologous recombination with base and strand preference in E. coli model system

“…1.A-B). This result is reasonable, for previous reports have shown that EBH can react with hAGT more efficiently and formed intermediates are more stable 39 . The reason will be explained in detail in next section.…”

“…Previous reports show that DBE can form DPCs only at the C145 site and DPCs are formed through a highly unstable intermediate produced by reacting hAGT with DBE firstly. However, EBH can form DPCs at both C150 and C145 sites and DPCs can also be formed through a stable intermediate produced by reacting with DNA directly 22; 39 . EBH can react with hAGT more efficiently and form intermediates that are more stable 39 .…”

Multifaceted Roles of Alkyltransferase and Related Proteins in DNA Repair, DNA Damage, Resistance to Chemotherapy, and Research Tools

“…Chinese hamster lung fibroblasts, which contain little or no endogenous AGT, were sensitized to the growth inhibitory effects of dihaloalkanes by expression of E. coli AGTs (145). More extensive studies carried out with CHO cells stably transfected with a plasmid that expresses human AGT showed that the cytotoxicity of 1,2-dibromoethane, dibromomethane and epibromohydrin was significantly increased by the presence of AGT (146). Mutations caused by these agents in the hypoxanthine-guanine phosphoribosyltransferase gene were increased by 2-3-fold in the presence of AGT.…”

DNA repair systems

“…The ac vity of the enzyme may be modulated in vitro and in vivo, increasing or decreasing the poten al of some DNA damaging agents for causing damage (e.g. halogenated hydrocarbons, epihalohydrins, and others) [206,207]. The toxicity of some an cancer agents(for example, pla num deriva ves) may be enhanced by modula on of the ac vity of O6-methylguanine DNA transferase [208,209].…”