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Rats were pretreated for a number of weeks with the liver tumour promoters phenobarbital and 2,3,7,8-tetrachlorodibenzo-p-dioxin, the direct alkylating agents N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea, and the hepatocarcinogens ethionine and diethylnitrosamine. A subsequent challenge with a single, low dose of radioactively labelled dimethylnitrosamine was given to assay the capacity of the liver for O6-methylguanine repair. Pretreatment with 0.05% phenobarbital in the diet for 8 weeks (a promoting regimen) resulted in significantly enhanced O6-methylguanine repair; shorter pretreatment periods (3 days or 2 weeks) had no significant effect. Repeated injection of another liver tumour promoter, 2,3,7,8-tetrachlorodibenzo-p-dioxin, also resulted in enhancement of O6-methylguanine repair. Pretreatment for 2 weeks with N-ethyl-N-nitrosourea resulted in strongly enhanced O6-methylguanine repair, as did a similar pretreatment with diethylnitrosamine, which was included as a positive control. The same pretreatment scheme which was highly effective in the case of N-ethyl-N-nitrosourea, was found to be totally ineffective in the case of N-methyl-N-nitrosourea. When N-methyl-N-nitrosourea was administered for 8 weeks instead of 2, a small but statistically significant increase in O6-methylguanine repair was observed. It is concluded that two factors are responsible for the low effectivity of N-methyl-N-nitrosourea. The first is the relatively low extent of liver DNA methylation by this compound when compared with dimethylnitrosamine. The second is the low efficiency of methylating agents (expressed per extent of DNA alkylation) to induce O6-methylguanine repair in rat liver when compared with ethylating agents. Pretreatment for 2 weeks with a diet containing DL-ethionine also resulted in a substantially increased O6-methylguanine repair capacity. Neither this enhancement, nor that induced by a pretreatment with diethylnitrosamine, could be inhibited by simultaneous feeding of a methionine-enriched diet. Our results indicate that neither increased hepatocellular proliferation nor direct interaction with DNA are necessary for the induction of O6-methylguanine repair enhancement. It is concluded that the capacity of an agent to enhance O6-methylguanine repair in rat liver reflects the hepato(co)carcinogenic capacity of that agent.

Effects of diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) on the sedimentation pattern of [3H]thymidine-labelled Sprague-Dawley female rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. In experiments at 1--56 days after a single injection it was observed that (potential) single strand breaks induced by DEN were repaired at a low rate. At 56 days the sedimentation pattern was still grossly abnormal. Half-life values of 27 and 46 days were observed after 134 mg/kg DEN (approx. 45% of the LD50) and 13.4 mg/kg DEN, respectively. Identical experiments after DMN (10 mg/kg, corresponding to about 35% of the LD50) showed return to (almost) completely control sedimentation patterns within 56 days after injection (t 1/2 = 8 days). Experiments at 6 or 56 days after the last of a series of 5 or 10 weekly injections of DEN (13.4 mg/kg) showed that a major part of DEN-induced damage (measured as single strand breaks) is of a persistent and accumulating character. No accumulation of DMN-induced rat liver lesions was observed. It is concluded that DNA fragmentation and lack of DNA repair is not a consequence of hepatotoxicity. Since at equimolar doses DEN gives appreciably less DNA alkylation (including O6-alkylguanine) but is much more effective both as an inducer of preneoplastic liver lesions and as a hepatocarcinogen when compared with DMN, we believe that the formation of persistent (and accumulating) DNA damage after DEN administration might be relevant in the process of liver tumour formation.

In vivo alkylation of DNA leads to DNA fragmentation in alkaline sucrose gradients. In a previous paper (Chem.-Biol. Interact., 19 (1977) 111) we presented evidence that, depending on the experimental conditions, a major fraction of the single-stranded breaks observed might be derived from alkali-labile alkylphosphotriesters. Using alkaline gradients the present paper shows that injection of ethyl methanesulphonate (EMS) into Sprague-Dawley female rats results in significantly increased liver DNA fragmentation up to at least 56 days after injection. Accumulation of single-strand breaks was indicated by experiments in which at 6 days after the last of a series of 5 weekly EMS injections (5 X 110 mg/kg) 11.4 breaks/10(9) Dalton were found, being 3 times more than the number of breaks observed at 6 days after a single injection of 110 mg/kg EMS (3.8 breaks/10(9) Dalton). In animals treated with methyl methanesulphonate (MMS) single-strand breaks were observed at 4 h, 1 day and 2 days, but not at 6 days after injection (40 mg/kg). Repeated weekly injections of MMS (5 X 40 mg/kg) did not result in increased numbers of breaks when compared with animals receiving a single injection of this agent (1 X 40 mg/kg; animals were killed 1 day after (the last) injection). It is suggested that MMS-induced breaks are derived, either on the gradient or in situ, from apurinic sites, whereas persistent EMS-induced breaks reflect the presence of ethylphosphotriesters. The results are discussed in relation to the lacking capacity of EMS to induce foci of precancerous lesions in rat liver and the non-hepatocarcinogenic properties of both MMS and EMS.

Rat-liver DNA alkylation by diethylnitrosamine (DEN), dimethylnitrosamine (DMN) and ethyl methanesulphonate (EMS) was studied in an attempt to relate chromosome-damaging effects of these agents (the formation of micronuclei in hepatocytes; see preceding paper) to specific alkylation patterns. No correlation was observed between the induction of micronuclei and liver DNA N-alkylation, measured as 3- and 7-alkyl-purines. O6-Alkylguanine is probably not involved in micronucleus induction because it is lost from DNA too rapidly to explain the much more persistent clastogenic effects. In contrast, both the initial amounts of alkylphosphotriesters and the persistencies of these products roughly paralleled the respective effects on micronucleus induction. The possible involvement of alkylphosphotriesters or other O-alkylation products of comparable stabilities is discussed. Results with DMN suggest that part of the primary DNA methylation damage is converted into a secondary (DNA) lesion and that both the primary and secondary lesion(s) contribute to the process of micronucleus formation.