David T. Beranek

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. 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.

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 = 0.879, p less than 0.001). The relationship between O6-methylguanine and induced mutations in CHO cells is similar to that previously reported in CHO cells for O6-ethylguanine and mutations (Heflich et al., 1982) and indicates that alkylation-induced mutations at the HGPRT locus in CHO cells are primarily associated with O6-alkylguanine formation.

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. The synthetic ring-opened adduct was characterized by mass and NMR spectroscopy as N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine and appears to exist in two rotameric forms.

The relationship between sister-chromatid exchange induction and the formation of specific methylated DNA adducts in Chinese hamster ovary cells

The ability of simple alkylating carcinogens to induce sister-chromatid exchange (SCE) has been well established (Perry and Evans, 1975; Carrano et al., 1978), although the alkyl lesion(s) leading to SCE formation have not been delineated. Several lines of evidence have led to the suggestion that persistent DNA damage, particularly O6-alkylguanine, is important in inducing SCE (Wolff, 1978, 1982; Goth-Goldstein, 1977). After treatment with simple alkylating agents, human cells which are defective in the removal of O6-alkylguanine have higher SCE frequencies than cells with normal levels of O6-methylguanine removal (Wolff et al., 1977; Day et al., 1980). The results of liquid-holding studies, however, argue that repairable DNA damage is involved in SCE formation. In these studies, the mutation rate (Jostes, 1981) or the level of O6-methylguanine (Connell and Medcarf, 1982) remained constant while SCE frequencies decreased after holding the cells using conditions that prohibited replication. These results suggested that SCE were the result of repairable DNA damage such as 3-alkyladenine or 7-alkylguanine (Jostes, 1981; Connell and Medcalf, 1982), although no single DNA adduct was identified which was repaired at the rate that SCE-causing damage was removed. The results of time-course experiments are consistent with the liquid-holding studies. In these experiments, the initial SCE frequency induced by exposure to methyl methanesulfonate (MMS) decreased in subsequent generations after treatment (Ockey, 1981; Muscarella and Bloom, 1982). SCE frequency returned to near control levels approximately 10 generations after treatment (Muscarella and Bloom, 1982). Studies in which levels of specific DNA adducts induced by different alkylating agents were quantified and compared with SCE frequencies have not yet yielded conclusive results. The results of Swenson et al. (1980) were supportive of

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. To determine the importance in germ-line mutagenesis of the O6-G site relative to the N-7 of guanine, dose-response curves were constructed for both ENU and EMS, where dose was measured as total adducts per deoxynucleotide (APdN) and response as sex-linked recessive lethals (SLRL) induced in Drosophila melanogaster spermatozoa. For both mutagens the dose response curve was linear and extrapolated to the origin. The dose-response curve for ENU was fit to an equation m = 6.2D, and the dose response curve for EMS, from this and previous experiments, was m = 3.2D where m = %SLRL and D = APdN X 10(-3). Therefore, ENU is 1.9 times more efficient per adduct in inducing SLRL mutations than EMS. In vitro studies showed that ENU induced 9.5% of its total adducts on O6-G while EMS induced 2.0% of its adducts on O6-G. If O6-G was the sole genotoxic site, then ENU should be 4.8 times more efficient per adduct than EMS. In contrast, if N-7 G was the sole genotoxic site, ENU would be only 0.19 as effective as EMS. It was concluded that while O6-G was the principal genotoxic site, N-7 G made a significant contribution to germ-line mutagenesis.

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). A predominant component of the dose-response curve at moderate to high dose is the quadratic component which is interpreted as the result of two single-strand breaks. The distribution of methyl adducts in vivo is consistent with the previously determined in vitro distribution of methyl adducts on DNA following treatment with MMS. With the use of HPLC, 82% of the 3H-labeled adducts are found on the N-7 of guanine. It has previously been shown by both in vitro studies and in vivo correlation with mutagenesis that the N-7 alkyl guanine is not itself a predominately genotoxic lesion; however, N-7 alkyl guanine destabilizes guanine resulting in an increased rate of hydrolysis producing apurinic sites. In data presented in this paper, the loss of labeled adducts is shown to be at a rate consistent with hydrolysis of the destabilized alkyl guanine. The apurinic site thus produced should be converted to single-strand breaks by AP endonucleases once sperm has fertilized the egg. Single-strand breaks are repaired by excision repair which is not error-prone; however, multiple breaks producing a proximity effect should lead to double-strand breaks that are repaired by an error-prone process. Mutations that are induced by a proximity effect would account for the quadratic term. It is hypothesized that a proximity effect is produced when two breaks are sufficiently close together to prevent using the complementary strand as a template. The linear component of the dose-response curve is probably due to alkylation of oxygens in the purine or pyrimidine ring leading to mispairing. However, due to the low frequency of ring-oxygen alkylation following treatment with MMS, this important genotoxic site is not the predominant one observed at experimental levels normally used in the laboratory. From the dose-response curve, it is calculated that at mutation frequencies of 10 times the spontaneous frequency or higher, the predominant mechanism is the multi-hit component; however, at mutation induced frequencies of 0.1 of the spontaneous frequency, which are levels more likely to be encountered in mans exposure to environmental mutagens, the dominant mechanism is the linear component.(ABSTRACT TRUNCATED AT 400 WORDS)