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

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.

Comparison of dose-response relationships for ethyl methanesulfonate and 1-ethyl-1-nitrosourea in Drosophila melanogaster spermatozoa.

The relative importance of different sites of alkylation on DNA was determined by comparing two ethylating agents. 1-Ethyl-1-nitrosourea (ENU) ethylates DNA with a higher proportion of total adducts on ring oxygens than ethyl methanesulfonate, which ethylates with a higher proportion of total adducts on the N-7 of guanine. Research with somatic cells in culture and prokaryotes strongly suggests that O6-guanine (O6-G) is the principal genotoxic site.

Dosage-response relationships for methyl methanesulfonate in Drosophila melanogaster spermatozoa: DNA methylation per nucleotide vs. sex-linked recessive lethal frequency.

Two different mechanisms for mutagenesis following treatment with methyl methanesulfonate (MMS) are suggested from the dose-response curve that is best fit by the linear quadratic model where m = 0.130D + 0.038D2 (D = dose measured as alkylations per nucleotide X 10(3), APdN; m = percent sex-linked recessive lethals, SLRL).

Different mutational profiles induced by N-nitroso-N-ethylurea: effects of dose and error-prone DNA repair and correlations with DNA adducts.

The DNA adducts and mutational profile produced by N-nitroso-N-ethylurea (ENU) in Salmonella are examined. The adduct profile produced by ENU in isolated DNA and at two doses in Salmonella were similar, with one exception: O6-ethylguanine (O6-EtG) was not detected at the low dose in Salmonella. This adduct was presumably repaired by a constitutive repair system.

Relationships between the DNA adducts and the mutations and sister-chromatid exchanges produced in Chinese hamster ovary cells by N-hydroxy-2-aminofluorene, N-hydroxy-N'-acetylbenzidine and 1-nitrosopyrene.

Chinese hamster ovary cells were exposed to N-hydroxy-2-aminofluorene, N-hydroxy-N'-acetylbenzidine and 1-nitrosopyrene, and the resulting DNA adducts, sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine-guanine phosphoribosyl transferase locus were quantified. Each agent produced a major DNA adduct substituted through the C8 of deoxyguanosine. When the data from all three agents were combined, both mutation and SCE induction correlated strongly with the concentration of DNA adducts.

Human alphoid family of tandemly repeated DNA. Sequence of cloned tetrameric fragments and analysis of familial divergence.

Three tetramers of the 170 base-pair monomer repeat unit of human centromeric DNA (alphoid DNA) have been cloned and sequenced. Adjacent subunits differed in sequence by 30 to 45%, while dimers varied by 13 to 20% whether adjacent or not. Divergence was distributed unevenly across the monomeric sequence, such that two highly conserved segments adjoined clusters of insertions/deletions.

Characterization of the purine ring-opened 7-methylguanine and its persistence in rat bladder epithelial DNA after treatment with the carcinogen N-methylnitrosourea.

Purine ring-opened 7-methylguanine, prepared in vitro by alkaline treatment of 7-methylguanosine or of methylated calf thymus DNA, was extensively characterized by chromatographic and spectral techniques as N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine. This modified base chromatographed as an early-eluting peak on an ion-exchange column but separated into two interconvertible components after reversed-phase or porous-resin h.p.

Relationships between specific DNA adducts, mutation, cell survival, and SCE formation.

The induction of biological responses and the formation of specific DNA adducts by 2 classes of carcinogens have been compared in CHO cells. The simple alkylating agents reacted with a variety of nucleophilic centers within DNA while the N-oxidized arylamines reacted only at C8 of deoxyguanosine. With the alkylating agents, mutation induction, which correlated strongly with 0(6)-alkylguanine levels, did not correlate well with either SCE frequency or cell survival.

Correlation between specific DNA-methylation products and mutation induction at the HGPRT locus in Chinese hamster ovary cells.

Suspension cultures of Chinese hamster ovary (CHO) cells were exposed to methyl methanesulfonate (MMS) or methylnitrosourea (MNU) and assayed for mutation induction (6-thioguanine resistance) and for specific DNA adducts. DNA methylation at the 1-, 3- and 7-positions of adenine, the 3-, O6- and 7-positions of guanine, and phosphate was detected in cultures exposed to MMS, while MNU produced 3- and 7-methyladenine, 3-methylcytosine, 3-, O6- and 7-methylguanine, O4-methylthymidine and methylated phosphodiesters. When mutations induced by MMS and MNU were compared by linear correlation analysis with levels of each of these adducts, only O6-methylguanine displayed a strong correlation with mutations (r = 0.

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 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 mumole/kg), and the bladder epithelium DNA adducts were quantified at various times after treatment.

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.

A major and previously undetected carcinogen-DNA adduct was found in the livers of rats given N,N-dimethylnitrosamine or 1,2-dimethylhydrazine. This adduct, which accounted for 55% of the total methyl residues in DNA at 72 hours after carcinogen treatment, was chromatographically identical to a synthetic purine ring-opened derivative of 7-methylguanine and could be released from the isolated hepatic DNA by a specific E. coli glycosylase.

Effect of bacterial concentration on reversions induced in Salmonella typhimurium TA1538 by N-hydroxy-2-acetylaminofluorene.

Reversions induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF) were measured in the Salmonella/microsome quantitative plate assay using various concentrations of Salmonella typhimurium strain TA1538. The number of induced revertants increased with an increasing number of bacteria/plate, but the variation in reversion frequency was not as great as the variation in bacterial concentration. The effects of bacterial concentration on reversion fixation, phenotypic expression, selection of his+ revertants, and the interaction of the mutagen with bacterial DNA were examined.

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

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.

Aminofluorene-DNA adduct formation in Salmonella typhimurium exposed to the carcinogen N-hydroxy-2-acetylaminofluorene.

The DNA adducts formed during incubation of the hepatocarcinogen N-hydroxy-2-acetylaminofluorene with Salmonella typhimurium tester strain TA1538 were investigated to determine if the covalently bound products were identical to those adducts found in rat liver DNA and to establish the biological significance of the adducts in a mutational assay. When bacteria were exposed to N-hydroxy-2-acetylaminofluorene in the presence of a 9,000 x g supernatant from a rat liver homogenate (S9), only one adduct was detected. This adduct had chromatographic, pH-dependent partitioning, and UV spectral characteristics identical to those of N-(deoxyguanosin-8-yl)-2-aminofluorene.

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

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.