https://mmbr.asm.org/content/mmbr/48/1/60.full.pdf

Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli.

INTRODUCTION . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 201 S OURCES OF AP SITES . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 202 Spontaneous DepurinationlDepyrimidination . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 Chemically Induced DepurinationlDepyrimidination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 203 DepurinationlDepyrimidination by DNA Glycosylases . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 207 REPAIR OF AP SITES . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 209 MUTAGENESIS BY AP SITES . . . .... . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 Misincorporation by DNA Polymerases in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 210 Mutagenesis in E. coli... . . . . . . . . . ..... . . . . . . . . . 2 1 2 Mutagenesis in Animal Cells . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 3 A P Sites a s Intermediates in Spontaneous Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 AP Sites as Intermediates in Chemical Mutagenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 MOLECULAR MECHANISMS FOR MUTAGENESIS BY AP S ITES . . . . . . . . .. . . . . . . . . . 219

Mutagenesis by Apurinic/Apyrimidinic Sites

Alkylating agents, because of their ability to react directly with DNA either in vitro or in vivo, or following metabolic activation as in the case of the dialkylnitrosamines, have been used extensively in studying the mechanisms of mutagenicity and carcinogenicity. Their occurrence is widespread in the environment and human exposure from natural and pollutant sources is universal. Since most of these chemicals show varying degrees of both carcinogenicity and mutagenicity, and exhibit compound-specific binding patterns, they provide an excellent model for studying molecular dosimetry. Molecular dosimetry defines dose as the number of adducts bound per macromolecule and relates the binding of these adducts to the human mutagenic or carcinogenic response. This review complies DNA alkylation data for both methylating and ethylating agents in a variety of systems and discusses the role these alkylation products plays in molecular mutagenesis.

Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents.

DNA damage-induced cell death: From specific DNA lesions to the DNA damage response and apoptosis

Results from the testing of 108 coded chemicals in Chinese hamster ovary (CHO) cells for the induction of chromosome aberrations and sister chromatid exchanges (SCEs) are presented. All chemicals were tested with and without exogenous metabolic activation, using protocols designed to allow testing up to toxic doses. Cell harvest times could also be extended if chemical-induced cell cycle delay was seen. Chromosome aberrations were induced by 43 of the chemicals, and 66 induced SCEs; 37 of the chemicals were positive for both endpoints.

Chromosome aberrations and sister chromatid exchanges in chinese hamster ovary cells: Evaluations of 108 chemicals

Methods were developed for the efficient routine degradation and fractionation of ethylated and methylated DNA. Alkylated DNA was hydrolyzed by a neutral thermal method to yield 3- and 7- alkylpurines and O2-alkylcytosines. The partially apurinic DNA was separated from the bases by precipitation in 0.1 N HCl. Portions of the DNA precipitate were further hydrolyzed either by 0.1 N HCl to yield purine bases, or by enzymes to yield nucleosides and phosphotriesters. The chemical and enzymic digests were fractionated by a combination of high pressure liquid chromatography systems to yield quantitative estimates of the following products from methylated or ethylated DNA: 1-, 3-, and 7-alkyladenines, O2-alkylcytosines, 3-, O6-, and 7- alkylguanines and O2-, 3-, and O4-alkylthymines. N6-Alkyladenines, 1-alkylguanines and N2-alkylguanines were not detected and the 3- alkylcytosines were detected but not quantified. Phosphotriesters were estimated from the amounts of recovered alkyl phosphotriesters of thymidylyl (3'-5') thymidine. Using these methods, it was possible to account for 98, 81, 98, and 92% of the DNA bound alkyl groups obtained from DNA reacted with [14C]methyl methanesulfonate, [3H]ethyl methanesulfonate, N-[3H]-methyl-N-nitrosourea, and N-[14C]ethyl-N-nitrosourea, respectively. The methods described provide reproducible and quantitative methods of analysis for all the known methylated or ethylated products in a single DNA sample.

A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography

Hepatic N-oxidation, followed by N-glucuronidation, has been proposed as a route of metabolic activation for arylamine bladder carcinogens. It is postulated that the N-glucuronides are transported to the bladder lumen where they are hydrolyzed under slightly acidic conditions to release direct-acting carcinogenic and mutagenic N-hydroxyarylamines. In this study, 4-aminobiphenyl (ABP), 1-naphthylamine (1-NA), 2-naphthylamine (2-NA), 2-acetylaminofluorene (AAF), 4-nitrobiphenyl (NBP), benzidine (BZ), and N-acetylbenzidine (ABZ) were administered to male beagle dogs (60 μmole/kg), and the bladder epithelium DNA adducts were quantified at various times after treatment. At 24-48 hr after administration, the order of binding to bladder epithelium DNA was: ABP >> AAF > NBP 2-NABZ ABZ >> 1-NA. The level of DNA modification by ABP remained constant for 7 days, whereas 2-NA and AAF residues decreased by 35% and 80%, respectively. The extent and relative persistence of total DNA binding correlated with the compounds' ability to induce bladder tumors in dogs. ABP, AAF, NBP, 2-NA and ABZ administration resulted in DNA binding sufficient for adduct analysis. Enzymatic hydrolysis of the DNA and examination of the adducts by high pressure liquid chromatography indicated that arylamine substitution at C8 of deoxyguanosine was the dominant product. Additional adducts were detected in animals treated with ABP, NBP, and 2-NA. Furthermore, the profiles of adducts obtained in vivo were remarkably similar to the profiles obtained when the N-hydroxy arylamine metabolites of these carcinogens were reacted with DNA in vitro at pH 5.0. To evaluate the mutagenic potential of these arylamine-DNA adducts, Salmonella typhimurium strains TA 1535 and TA 1538 were incubated with N-hydroxy-2-NA, N-hydroxy-2-aminofluorene (AF), N-hydroxy-ABP, and N-hydroxy-ABZ and the resulting DNA adducts and reversions were quantified. Arylamine-C8-deoxyguanosine substitution was correlated with frameshift reversions induced by these agents, with the lesions showing a relative order of mutagenic efficiency of ABZ>AF2-NA>ABP. These data suggest that mutagenic N-hydroxyarylamines may be ultimate carcinogens for the bladder epithelium. Furthermore, if one assumes that a mutagenic lesion is important for tumor initiation, then C8-deoxyguanosine substitution by these compounds may be significant for urinary bladder carcinogenesis.

Arylamine-DNA adducts in vitro and in vivo: their role in bacterial mutagenesis and urinary bladder carcinogenesis.

Hepatic N-oxidation, followed by N-glucuronidation, has been proposed as a route of metabolic activation for arylamine bladder carcinogens. It is postulated that the N-glucuronides are transported to the bladder lumen where they are hydrolyzed under slightly acidic conditions to release directacting carcinogenic and mutagenic N-hydroxyarylamines. In this study, 4-aminobiphenyl (ABP), 1naphthylamine (1-NA), 2-naphthylamine (2-NA), 2-acetylaminofluorene (AAF), 4-nitrobiphenyl (NBP), benzidine (BZ), and N-acetylbenzidine (ABZ) were administered to male beagle dogs (60 pmole/kg), and the bladder epithelium DNA adducts were quantified at various times after treatment. At 24-48 hr after administration, the order of binding to bladder epithelium DNA was: ABP >> AAF > NBP a 2-NA-BZ - ABZ >> 1-NA. The level of DNA modification by ABP remained constant for 7 days, whereas 2-NA and AAF residues decreased by 35% and 80%, respectively. The extent and relative persistence of total DNA binding correlated with the compounds' ability to induce bladder tumors in dogs. ABP, AAF, NBP, 2-NA and ABZ administration resulted in DNA binding sufficient for adduct analysis. Enzymatic hydrolysis of the DNA and examination of the adducts by high pressure liquid chromatography indicated that arylamine substitution at C8 of deoxyguanosine was the dominant product. Additional adducts were detected in animals treated with ABP, NBP, and 2NA. Furthermore, the profiles of adducts obtained in vivo were remarkably similar to the profiles obtained when the N-hydroxy arylamine metabolites of these carcinogens were reacted with DNA in vitro at pH 5.0. To evaluate the mutagenic potential of these arylamine-DNA adducts, Salmonella typhimurium strains TA 1535 and TA 1538 were incubated with N-hydroxy-2-NA, N-hydroxy-2aminofluorene (AF), N-hydroxy-ABP, and N-hydroxy-ABZ and the resulting DNA adducts and reversions were quantified. Arylamine-C8-deoxyguanosine substitution was correlated with frameshift reversions induced by these agents, with the lesions showing a relative order of mutagenic efficiency of ABZ>AF-2-NA>ABP. These data suggest that mutagenic N-hydroxyarylamines may be ultimate carcinogens for the bladder epithelium. Furthermore, if one assumes that a mutagenic lesion is important for tumor initiation, then C8-deoxyguanosine substitution by these compounds may be significant for urinary bladder carcinogenesis.

Arylamine-DNA Adducts In Vitro and In Vivo: Their Role in Bacterial Mutagenesis and Urinary Bladder

Chinese hamster ovary (CHO) cells were exposed to [3H]ethyl nitrosourea (ENU) or [3H]ethyl methanesulfonate (EMS) and the following DNA ethylation products were quantitated: 3- and 7-ethyladenine, O2-ethylcytosine, 3-, 7- and O6-ethylguanine, O2- and O4-ethyldeoxythymidine and the representative ethylated phosphodiester, deoxythymidylyl (3'-5')ethyl-deoxythymidine. When mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus induced by these same treatments were compared with the observed ethylation products, mutations were found to correlate best with 3- and O6-ethylguanine. EMS induced approximately twice as many sister-chromatid exchanges (SCEs) as ENU at doses yielding equal mutation frequencies. When SCEs were indirectly compared with DNA ethylation products, 3-ethyladenine and ethylated phosphodiesters related best to SCE formation. Because mutation and SCE induction appear, at least in part, to be related to different DNA adducts, SCE induction by simple ethylating agents may not be a quantitative indicator of potentially mutagenic DNA damage.

Induction of mutations and sister-chromatid exchanges in Chinese hamster ovary cells by ethylating agents.

Identification of n5-methyl-n5-formyl-2,5,6-triamino-4-hydroxypyrimidine as a major adduct in rat liver dna after treatment with the carcinogens, n,n-dimethylnitrosamine or 1,2-dimethylhydrazine

David T. Beranek

Papers

A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography

Arylamine-DNA adducts in vitro and in vivo: their role in bacterial mutagenesis and urinary bladder carcinogenesis.

Arylamine-DNA Adducts In Vitro and In Vivo: Their Role in Bacterial Mutagenesis and Urinary Bladder

Induction of mutations and sister-chromatid exchanges in Chinese hamster ovary cells by ethylating agents.

Identification of n5-methyl-n5-formyl-2,5,6-triamino-4-hydroxypyrimidine as a major adduct in rat liver dna after treatment with the carcinogens, n,n-dimethylnitrosamine or 1,2-dimethylhydrazine