Effect of O6-alkylguanine-DNA alkyltransferase on genotoxicity of epihalohydrins.
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 AGT-mediated 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 DNA-epihalohydrin 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.
Kalapila
Aley G
AG
Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Loktionova
Natalia A
NA
Pegg
Anthony E
AE
D018736:DNA Adducts / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases; D004789:Enzyme Activation / Q000187:drug effects; D004811:Epichlorohydrin / Q000737:chemistry / Q000633:toxicity; D004852:Epoxy Compounds / Q000737:chemistry / Q000633:toxicity*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D008954:Models, Biological; D009152:Mutagenicity Tests; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000187:drug effects; D018675:Toxicity Tests
D018736:DNA Adducts; D004852:Epoxy Compounds; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; C540081:rpoB protein, E coli; D004811:Epichlorohydrin; D019853:O(6)-Methylguanine-DNA Methyltransferase; D012321:DNA-Directed RNA Polymerases