Epigenetic changes are intimately linked to cancer and other human diseases. This science has been developed on the basis of chemical analysis of DNA methylation. In most of these studies, the data are obtained by use of bisulfite genomic sequencing. Bisulfite deaminates specifically cytosine residues in single-stranded DNA molecules. 5-Methylcytosine resists the action of bisulfite. This difference in bisulfite reaction constitutes the principle for the detection of methylated sites. This review examines the details of chemistry involved in the bisulfite modification of DNA; deamination steps, single strand specificity, and DNA degradation in relation to chain cleavage. Points of uncertainty in these processes are mentioned. Recent advances by use of a 10 M bisulfite solution are explained, and a convenient laboratory protocol for quantifying bisulfite is described.
In this article, we described the spontaneously occurring mutation specificities of defects that are involved in translesion polymerase, mutS mismatch correction and polA mismatch correction in Escherichia coli. We argue that 1) there is no contribution of translesion polymerase to E. coli chromosomal mutation, 2) mutS system recognizes and corrects transition and frameshift mismatches and 3) polA system recognizes and corrects deletion, frameshift and transition mismatches. We also characterized the genetic alterations that inactivate either the CAN1 gene of Saccharomyces cerevisiae haploid cells or heterozygously situated in diploid cells. The characteristics of mutation in haploid yeast are essentially consistent with those in E. coli, suggesting that similar mechanisms are operating to form spontaneous mutation. CAN1+/can1- (CanS) to can1-/can1- (CanR) mutations in diploid cells could occur through recombination, mainly allelic crossover and gene conversion.
In order to understand how mutagens behave and act in vivo, it is important to understand how the integrity of the genome is maintained and protected in each specific tissue. Although several lines of evidence suggest that the systems used to protect and maintain the genome can change or are modified during cellular differentiation processes, and when cells alter their status from a proliferating to a non-proliferating state, data on individual tissues is very limited. Recent studies on the age-dependent accumulation of spontaneous mutations in transgenic mice clearly indicate that there is tissue-specificity when examining genome maintenance and protection. The genome is most unstable in epithelial tissues of the small and large intestine when compared to 13 other organs and tissues. The genome is highly protected in brain, skin and testis. Studies of the molecular nature of specific mutations suggest the presence of unique tissue-specific mechanisms leading to the formation of mutations in specific tissues. Studies of mutations in DNA repair gene deficient mice have shown that some of the genes involved in mismatch repair are indispensable in many tissues for the maintenance of the genome. The Xpa and Xpc genes involved in nucleotide excision repair have also been shown to be important in some tissues. However, the studies reported to date are only a beginning, and a complete comprehensive picture of the maintenance and repair of the genomic integrity in individual tissues remains to be developed.
Benzo[a]pyrene (B[a]P), one of polycyclic aromatic hydrocarbons (PAHs), is a ubiquitous environmental pollutant and a potent mutagen and carcinogen. To sensitively detect the genotoxicity of PAHs in complex mixtures extracted from environmental pollutants, Salmonella enterica serovar Typhimurium (S. typhimurium) strain YG5161 is engineered by introduction of plasmid pYG768 carrying the dinB gene encoding Escherichia coli DNA polymerase IV into standard Ames tester strain S. typhimurium TA1538 (Matsui et al., DNA Repair in press). Strain YG5161 exhibits higher sensitivity to the genotoxicity of B[a]P and other PAHs than do strain TA1538 and TA98. As the conventional Ames tester strains do, however, strain YG5161 also detects the mutagenicity of aromatic amines and nitroaromatics with high sensitivity, which may veil the genotoxicity of PAHs in complex mixtures. S. typhimurium possesses strong enzyme activities of nitroreductase and O-acetyltransferase, which mediate the metabolic activation of aromatic amines and nitroaromatics and enhance the potent genotoxicity. In this study, we disrupted the nfsB and oat genes encoding the activation enzymes in strain TA1538 to reduce the cross sensitivity, and introduced plasmid pYG768 into the ΔnfsBΔoat strain. The resulting strain YG5185 retained similar high mutability to various chemicals including PAHs as did strain YG5161 and substantially decreased the sensitivity to 1-nitropyrene, 1,8-dnitropyrene and 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-1). We propose that the novel tester strain YG5185 is useful to specifically and sensitively detect the genotoxic PAHs in complex mixtures from various polluted environmental sources.
Three lots of kojic acid (KA) which were produced for use as a reagent, food additive and in cosmetics were shown to be mutagenic in S. typhimurium TA100 with or without S9 mix, with a specific activity of around 100 revertants per mg of KA. Since there are contradictory reports on genotoxicity of KA, we examined, using HPLC, whether the mutagenicity to S. typhimurium is due to KA itself, or due to contaminants present in the KA samples. Although two UV absorbing fractions were separated by HPLC, mutagenicity was detected only in the major fraction and the specific mutagenic activity of KA did not change before and after HPLC separation. The material in the major peak fractions on HPLC was confirmed to be KA by NMR. Thus it was demonstrated that KA itself is mutagenic and no mutagenic contaminants were detected in the three lots of samples. Since KA is known to produce liver tumors in mice, we further examined the genotoxicity of KA in the liver of rodents. KA induced micronuclei (MN) in the regenerating liver of adult mice by its gastric intubation at 1 g per kg body weight. However, no MN were induced in young mice (3 weeks old) without partial hepatectomy. Since it was recently found that KA had no tumor-initiating activity in the liver of mice in a two-step carcinogenicity study, there is no evidence that the genotoxicity detected in the mouse liver is involved in liver carcinogenesis.
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