methylazoxymethanol and dimethyl-sulfate

Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag(-/-)) or O(6)-methylguanine methyltransferase (Mgmt(-/-)), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt(-/-) neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag(-/-) neurons were for the most part significantly less sensitive than wild type or Mgmt(-/-) neurons to MAM and HN2. Aag(-/-) neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt(-/-) mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM-treated Aag(-/-) or MGMT-overexpressing (Mgmt(Tg+)) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in Mgmt(Tg+) mice treated with HN2. Collectively, these in vitro and in vivo studies demonstrate that the type of DNA lesion and the efficiency of DNA repair are two important factors that determine the vulnerability of the developing brain to long-term injury by a genotoxicant.

Topics: Alkylating Agents; Animals; Cattle; Cell Survival; Cerebellum; Chickens; DNA; DNA Fragmentation; DNA Glycosylases; DNA Modification Methylases; DNA Repair; DNA Repair Enzymes; Ethylamines; Humans; Mechlorethamine; Methylazoxymethanol Acetate; Mice; Motor Activity; Neurons; Sulfuric Acid Esters; Tumor Suppressor Proteins

Methylazoxymethanol (MAM) and dimethyl sulfate (DMS) are mutagens whose genetic effects can be ascribed to the methylation of DNA. While both methylate the N7 position of guanine heavily, only MAM strongly methylates the O(6) position of guanine. We evaluated the relative effectiveness and specificity of MAM and DMS in bacterial assays for the induction of point mutations and the formation of chromosomal duplications by genetic recombination. Salmonella typhimurium strain TS1121 was used to measure the formation of genetic duplications on the basis of the aroC321 allele and mutations by reversion of the hisG46 allele. Specific base pair substitutions and frameshift mutations were measured in a reversion assay based on lacZ alleles of Escherichia coli. The results show MAM to be the more potent mutagen and DMS the stronger recombinagen in the Salmonella assay. In the lacZ assay DMS induced several classes of base pair substitutions (GC-->AT transitions, GC-->TA transversions and AT-->TA transversions), as well as lower frequencies of +1, -1 and -2 frameshift mutations. The activity of MAM as a base pair substitution mutagen was more specific than that of DMS, inducing only GC-->AT transitions. It also induced +G, -G, -A and -CG frameshift mutations, though more weakly than it induced GC-->AT transitions. Long known as a base pair substitution mutagen, the induction of frameshifts by MAM was unexpected. The results show that both DMS and MAM are effective inducers of base pair substitutions and modest inducers of frameshifts and that DMS exhibits a broader spectrum of mutagenic activity than does MAM.

Topics: Alleles; Base Pairing; Carcinogens; DNA Methylation; DNA Repair; DNA, Bacterial; Gene Duplication; Histidine; Lac Operon; Methylazoxymethanol Acetate; Mutagenicity Tests; Mutagens; Phosphorus-Oxygen Lyases; Point Mutation; Recombination, Genetic; Salmonella typhimurium; Sulfuric Acid Esters; Tryptophan