Effect of O⁶‐alkylguanine‐DNA alkyltransferase on genotoxicity of epihalohydrins

Abstract

The effect of O 6 -alkylguanine-DNA alkyltransferase (AGT) on the toxicity and mutagenicity of epihalohydrins was studied. AGT is a DNA repair protein that protects cells from agents that produce genotoxic O 6 -alkylguanine lesions by transferring the alkyl group to an internal cysteine residue (Cys 145 in human AGT) in a single-step. This cysteine acceptor site is highly reactive and epihalohydrins reacted readily with AGT at this site with a halide order of reactivity of Br > Cl > F. AGT expression in bacterial cells caused a very large increase in the mutagenicity and cytotoxicity of epibromohydrin. The mutations were almost all G:C to A:T transitions. Epichlorohydrin also augmented AGTmediated mutagenesis but to a lesser extent than epibromohydrin. In vitro experiments showed that AGT was covalently cross-linked to DNA in the presence of epibromohydrin and that this conjugation occurred predominantly at Cys 145 , and to a smaller extent at Cys 150 , a less reactive residue also located within the active site pocket. Two pathways yielding the AGT-DNA adduct were found to occur. The predominant mechanism results in an AGT-epihalohydrin intermediate, which, facilitated by the DNA binding properties of AGT, then reacts covalently with DNA. The second pathway involves an initial reactive DNAepihalohydrin intermediate that subsequently reacts with AGT. Our results show that the paradoxical AGT-mediated increase in genotoxicity which has previously been shown to occur with dihaloalkanes, butadiene diepoxide and nitrogen mustards, also occurs with epihalohydrins and is likely to contribute to their toxicity and mutagenicity.

Alkyltransferase-mediated toxicity of bis-electrophiles in mammalian cells

“…Activation mediated via an hAGT-linked pathway is therefore an additional way in which the genotoxicity of these compounds may occur. Although we did not test this directly, it is very likely that the underlying mechanism of this enhanced toxicity is the formation of DNA-hAGT cross-links as indicated in studies with bacterial cells and in vitro experiments [3,23,24,28]. Attempts to isolate the hAGT-DNA crosslink directly from the mammalian cells were not successful but this experiment is technically very challenging since the number of adducts is likely to be very low and the presence of other proteins interferes with immunoprecipitation techniques.…”

“…TRG8 E. coli cells expressing hAGT demonstrated a very large increase (2–3 orders of magnitude) in the mutation rate with a lesser decrease in cell survival in response to these bifunctional agents [3,23,24,28]. In contrast, the sensitization of the CHO-hAGT cells to the cytotoxic effects of DBE, DBM and EBH was much more drastic than the increase in mutagenic potential in these cells.…”

“…The intermediate formed is highly unstable in an aqueous environment but can react with cellular DNA. EBH may act predominantly in the same way but can also form adducts at Cys150 of hAGT and can react first directly with DNA with the resulting adduct then forming a covalent bond with hAGT [28]. The improved stability of the intermediates formed in the these reactions may account for the huge increase (about 1400-fold) in mutations when EBH was used in bacterial test systems [28].…”

“…Exposure to other bis -electrophiles including butadiene diepoxide (BDO) [25–27], epibromohydrin (EBH) [28] and nitrogen mustards [29] was subsequently shown to lead to hAGT-mediated toxicity and mutagenesis due to the formation of hAGT-DNA cross-links in a similar bacterial test system. Epoxides such as EBH are widely used in the chemical industry [30].…”

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].…”