Choua C. Vu

Translesion Synthesis across O6-Alkylguanine DNA Adducts by Recombinant Human DNA Polymerases

Previous studies have shown that replicative bacterial and viral DNA polymerases are able to bypass the mutagenic lesions O6-methyl and -benzyl (Bz) G. Recombinant human polymerase (pol) δ also copied past these two lesions but was totally blocked by O6-[4-oxo-4-(3-pyridyl)butyl] (Pob)G, an important mutagenic lesion formed following metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. The human translesion pols ι and κ produced mainly only 1-base incorporation opposite O6-MeG and O6-BzG and had very low activity in copying O6-PobG. Human pol η copied past all three adducts. Steady-state kinetic analysis showed similar efficiencies of insertion opposite the O6-alkylG adducts for dCTP and dTTP with pol η and κ; pol ι showed a strong preference for dTTP. pol η, ι, and κ showed pre-steady-state kinetic bursts for dCTP incorporation opposite G and O6-MeG but little, if any, for O6-BzG or O6-PobG. Analysis of the pol η O6-PobG products indicated that the insertion of G was opposite the base (C) 5′ of the adduct, but this product was not extended. Mass spectrometry analysis of all of the pol η primer extension products indicated multiple components, mainly with C or T inserted opposite O6-alkylG but with no deletions in the cases of O6-MeG and O6-PobG. With pol η and O6-BzG, products were also obtained with –1 and –2 deletions and also with A inserted (opposite O6-BzG). The results with pol η may be relevant to some mutations previously reported with O6-alkylG adducts in mammalian cells.

GLUTATHIONE TRAPPING TO MEASURE MICROSOMAL OXIDATION OF FURAN TO CIS-2-BUTENE-1,4-DIAL

Furan is a liver carcinogen and toxicant. Furan is oxidized to the reactive dialdehyde, cis-2-butene-1,4-dial, by microsomal enzymes. This reactive metabolite readily reacts with glutathione nonenzymatically to form conjugates. A high-performance liquid chromatography-electrochemical method for the detection of cis-2-butene-1,4-dial-glutathione (GSH) conjugates in microsomal preparations was developed to measure the extent of furan metabolism to cis-2-butene-1,4-dial in vitro. Previously unobserved mono-GSH reaction products of cis-2-butene-1,4-dial were detected in addition to the already characterized bis-GSH conjugates. Chemical characterization of these compounds indicated that the α-amino group of glutathione had reacted with cis-2-butene-1,4-dial to form a thiol-substituted pyrrole adduct. The analytical method was used to estimate the extent of furan oxidation in rat liver microsomes from untreated or acetone-pretreated F344 rats as well as in human P450 2E1 Supersomes. Our results confirm that cytochrome P450 2E1 can catalyze the oxidation of furan to cis-2-butene-1,4-dial. However, the data are also consistent with the involvement of other P450 enzymes in the oxidation of furan in untreated animals. This assay will be a valuable tool to explore tissue and species differences in rates of furan oxidation.

DNA Sequence Context Affects Repair of the Tobacco-Specific Adduct O6-[4-Oxo-4-(3-pyridyl)butyl]guanine by Human O6-Alkylguanine-DNA Alkyltransferases

The repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) protects cells from the mutagenic and carcinogenic effects of alkylating agents by removing O(6)-alkylguanine adducts from DNA. Recently, we established that AGT protects against the mutagenic effects of pyridyloxobutylation resulting from the metabolic activation of the tobacco-specific nitrosamines (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosonornicotine by repairing O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine (O(6)-pobG). There have been several epidemiologic studies examining the association between the I143V/K178R AGT genotype and lung cancer risk. Two studies have found positive associations, suggesting that AGT proteins differ in their repair of DNA damage caused by TSNA. However, it is not known how this genotype alters the biochemical activity of AGT. We proposed that AGT proteins may differ in their ability to remove large O(6)-alkylguanine adducts, such as O(6)-pobG, from DNA. Therefore, we examined the repair of O(6)-pobG by wild-type (WT) human, I143V/K178R, and L84F AGT proteins when contained in multiple sequence contexts, including the twelfth codon of H-ras, a mutational hotspot within this oncogene. The AGT-mediated repair of O(6)-pobG was more profoundly influenced by sequence context than that of O(6)-methylguanine. These differences are not the result of secondary structure (hairpin) formation in DNA. In addition, the I143V/K178R variant seems less sensitive to the effects of sequence context than the WT or L84F proteins. These studies indicate that the sequence dependence of O(6)-pobG repair by human AGT (hAGT) varies with subtle changes in protein structure. These data establish a novel functional difference between the I143V/K178R protein and other hAGTs in the repair of a toxicologically relevant substrate, O(6)-pobG.

Role of aldehydes in the toxic and mutagenic effects of nitrosamines.

α-Hydroxynitrosamine metabolites of nitrosamines decompose to a reactive diazohydroxide and an aldehyde. To test the hypothesis that the aldehydes contribute to the harmful effects of nitrosamines, the toxic and mutagenic activities of three model methylating agents were compared in Chinese hamster ovary cells expressing or not expressing human O⁶-alkylguanine DNA alkyltransferase (AGT). N-Nitrosomethylurethane (NMUr), acetoxymethylmethylnitrosamine (AMMN), and 4-(methylnitrosamino)-4-acetoxy-1-(3-pyridyl)-1-butanone (NNK-4-OAc) are all activated by ester hydrolysis to methanediazohydroxide. NMUr does not form an aldehyde, whereas AMMN generates formaldehyde, and NNK-4-OAc produces 4-oxo-1-(3-pyridyl)-1-butanone (OPB). Since these compounds were likely to alkylate DNA to different extents, the toxic and mutagenic activities of these compounds were normalized to the levels of the most cytotoxic and mutagenic DNA adduct, O⁶-mG, to assess if the aldehydes contributed to the toxicological properties of these methylating agents. Levels of 7-mG indicated that the differences in cytotoxic and mutagenic effects of these compounds resulted from differences in their ability to methylate DNA. When normalized against the levels of O⁶-mG, there was no difference between these three compounds in cells that lacked AGT. However, AMMN and NNK-4-OAc were more toxic than NMUr in cells expressing AGT when normalized against O⁶-mG levels. In addition, AMMN was more mutagenic than NNK-4-OAc and MNUr in these cells. These findings demonstrate that the aldehyde decomposition products of nitrosamines can contribute to the cytotoxic and/or mutagenic activity of methylating nitrosamines.