Bacterial and mammalian DNA alkyltransferases sensitize Escherichia coli to the lethal and mutagenic effects of dibromoalkanes

Abstract

Here we confirm and extend our previous studies demonstrating that the mutagenic potency of 1,2-dibromoethane (DBE) and dibromomethane (DBM) is markedly enhanced (not prevented) in bacteria expressing the O6-alkylguanine-DNA alkyltransferase (ATase) encoded by the Escherichia coli ogt gene. We demonstrate that, in close parallel with mutagenesis, the Ogt ATase sensitizes the bacteria to the lethal effects of these carcinogens, suggesting that one or more of the potentially mutagenic lesions induced by DBE and DBM in the presence of Ogt has additional lethal capacity. We further demonstrate that the sensitization to both lethality and mutagenesis by DBE and DBM is a property shared by other DNA alkyltransferases. This objective was accomplished by quantifying the induction of mutations and lethal events in ogt- ada- E. coli expressing an exogenous bacterial or mammalian ATase from a multicopy plasmid. Mammalian recombinant ATases enhanced the lethal and mutagenic actions of DBE and suppressed the lack of sensitivity of the vector-transformed bacteria to DBM. In most cases the order of effectiveness of the ATases ranked: murine > human > Ogt > rat. Further comparisons included the full-length Ada ATase from E. coli and a truncated Ada version (T-ada) that retains the O6-methylguanine binding domain of the protein. The full-length Ada ATase was effective in enhancing the lethality but not the mutagenicity induced by DBE and DBM. The T-ada ATase provided less sensitization than Ada to lethality by DBE, but of the three bacterial ATases T-ada yielded the highest sensitization to mutagenesis by this compound. T-ada and Ada ATases were in general less effective than the mammalian versions, with the exception of the rat recombinant ATase. The effectiveness of the different mammalian and bacterial ATases in promoting the deleterious actions of dibromoalkanes was compared with the effectiveness of these proteins in suppressing the lethal and mutagenic effects induced by N-nitroso-N-methylurea. The ability to sensitize E. coli to the lethal and mutagenic effects of DBE and DBM seems restricted to DNA alkyltransferase, since overexpression of thioredoxin (Trx) or glutaredoxin (Grx1) in ogt- ada- cells showed no effect, in spite of the reported potential of bioactive dihaloethane-derived species to alkylate Trx.

Paradoxical Enhancement of the Toxicity of 1,2-Dibromoethane byO 6-Alkylguanine-DNA Alkyltransferase

“…As shown in Fig. 1, BE is neither cytotoxic nor mutagenic in TRG8 cells irrespective of their AGT status, suggesting that the observed DBE toxicity in E. coli expressing high levels of Ogt, Ada, or hAGT (7,8,10,12) and Chinese hamster lung fibroblasts expressing Ogt (12) results from unmetabolized DBE rather than from BE. The difference of DBE and BE in inducing toxicity in AGT-expressing cells indicates that having two good leaving groups is critical for bringing about this toxicity.…”

Activation of bis-Electrophiles to Mutagenic Conjugates by Human O6-Alkylguanine-DNA Alkyltransferase

“…During the 1990s, a number of papers appeared demonstrating that expression of the DNA repair protein AGT 1 increased the toxicity and mutagenicity of Br(CH 2 ) 2 -Br in bacterial systems (38)(39)(40)(41). These findings were unexpected, but results with hAGT and Br(CH 2 ) 2 Br now demonstrate that the mechanism involves attack of the highly nucleophilic Cys 145 (42) of (h)AGT on Br(CH 2 ) 2 Br to generate AGT-Cys 145 -S(CH 2 ) 2 Br, which appears to undergo similar reactions to GS(CH 2 ) 2 Br (12) and react with DNA, leading to cross-linking (43).…”

Characterization of a Mutagenic DNA Adduct Formed from 1,2-Dibromoethane by O6-Alkylguanine-DNA Alkyltransferase

“…AGT is inactivated by the alkyl group transfer, and therefore additional repair requires new protein synthesis. Although an early study reported that the mutagenicity of 1,2-dibromoethane (DBE) was not affected by enzymes that repair alkylation lesions (4), there is now convincing data that AGT paradoxically promotes the toxicity of DBE (5,6). Overexpression of human AGT (hAGT) or AGTs from other species enhances the mutagenicity and lethality of DBE in Escherichia coli (5)(6)(7)(8)(9) and induces growth retardation in mammalian cells (10).…”