Adult rats received a single injection of the carcinogens N-ethyl-N-nitrosourea (ENU) or N,N-dimethylnitrosamine (DMN) and were killed after various time intervals (up to 56 days). Liver DNA was analysed by h.p.l.c. for alkylated purines [3-alkyladenine (3-alkylAde), 7-alkylAde, 3-alkylguanine (3-alkylGua), O6-alkylGua, 7-alkylGua, imidazole ring-opened (ro) 7-alkylGua] and alkylated pyrimidines [O2-alkylcytosine (O2-alkylCyt), 3-alkylCyt, O2-alkylthymine (O2-alkylThy), 3-alkylThy, O4-alkylThy]. Alkylphosphotriesters were assayed by both h.p.l.c. [alkylphosphotriester of thymidyl(3'-5')thymidine, (dTp(alkyl)dT)] and alkaline sucrose sedimentation (total phosphotriesters). No significant amounts of 3-alkylCyt and ro 7-alkylGua were observed, but the presence of all other products could be established 2 h after DMN or ENU. At that moment the relative amounts of some products (O2-alkylCyt, O2-alkylThy, O4-alkylThy, O6-alkylGua), when compared with 7-alkylGua, were smaller than those observed after in vitro alkylation of isolated DNA. This suggests either a lower accessibility of some sites in DNA in situ, or (as known already for O6-alkylGua) the presence of rapidly exhausted, fast repair modes for these adducts. The in vivo stability of ethylated products was (much) higher than that of the homologous methylated products. In the case of O-alkylated thymidines this difference was impressive: apparent half-life values of 17 days for O2-ethylthymine (O2-EtThy) and 14 days for O4-EtThy were calculated, whereas the corresponding values for O2-methylthymine (O2-MeThy) and O4-MeThy were 12 h and less than 4 h, respectively. Substantial, although smaller differences were also found for O2-alkylCyt (86 versus less than 4 h), 3-alkylGua (104 versus 17 h) and dTp(alkyl)dT (32 versus 7 days), whereas the rates of removal of 7-EtGua and 7-MeGua differed by 2.5-5 times (depending on the period compared). At 56 days after ENU the two major lesions were the ethylphosphotriester and O2-EtThy, whereas only traces of 7-EtGua were observed. Parenchymal and non-parenchymal liver cells of some DMN-treated rats were separated before DNA isolation. It was found that the extents of DNA alkylation at 6 h after DMN administration were almost identical, indicating that DMN activation is the same for both cell types. The results are discussed in relation to the carcinogenic effects of methylating and ethylating agents.

Male Wistar rats received a single i.p. injection of [3H]ethylnitrosourea (140 mg/kg) and were killed after 2 h, 1, 3, 6, 28 or 56 days. DNA of the following organs was isolated and analysed for the presence of 12 different ethylated bases and ethylphosphotriesters: brain, lung, liver, spleen, kidney, intestine, testis and bone marrow. At 2 h after injection the extent of DNA ethylation was found to be heterogeneous: highest in liver and lowest (3-4 times lower) in testis and brain. The rates at which O2- and O4-ethylthymine and the ethylphosphotriester dTp(Et)dT were lost, were very low in all organs except intestine and spleen. Most likely, loss in the latter organs is exclusively due to cell turnover. The rate of O6-ethylguanine repair strongly varied from organ to organ: high in liver, very low in testis and brain and intermediate in the other organs. In none of the DNAs were significant amounts of imidazole ring-opened 7-ethylguanine found. Our results strengthen the notion that a substantial part of carcinogen-induced DNA damage is of a highly persistent nature and might contribute to the carcinogenic process long after the original exposure has occurred.

No abstract

Male Wistar rats received a single injection of ethylnitrosourea (ENU; 140 mg/kg), dimethylnitrosamine (DMN; 10 mg/kg) or methyl methanesulphonate (MMS; 80 mg/kg). After 1, 6, 28 and 56 days liver cells were assayed for the presence of preclastogenic lesions. This was done by analysis of micronuclei in hepatocytes isolated at 2, 3 and 4 days after partial hepatectomy. Treatment with MMS did not give rise to a statistically significant increased micronucleus frequency after the two time intervals tested (1 and 6 days). In contrast, frequencies of micronuclei were significantly enhanced at all time intervals after treatment with either ENU or DMN. These results support our previous conclusion that only those alkylating agents which give rise to substantial DNA O-alkylation are able to induce long-lived preclastogenic damage in rat liver cells. Both after ENU and DMN treatment, frequencies of micronuclei were significantly higher at day 6 than at days 1, 28 and 56. It is postulated that part of the primary preclastogenic lesions (supposed to be DNA adducts) are converted into secondary preclastogenic lesions during the first period after exposure to the chemicals. Apparently, both primary and secondary preclastogenic lesions are gradually lost after day 6. This loss is rather low (less than 25%) after ENU, but more impressive after DMN (approximately 60%). The persistent nature of preclastogenic damage induced by diethylnitrosamine was demonstrated in an experiment which showed that a single dose of this agent (50 mg/kg) had the same clastogenic effect as a fractionated dose (five weekly injections of 10 mg/kg). This result also implies that a threshold dose for induction of micronuclei, if present, must be very low, appreciably below 10 mg/kg.

Antibodies raised in rabbits against the bovine serum albumin conjugates of O6-ethylguanosine (O6-EtGuo) and N-(guanosin-8-yl)-N-acetyl-2-aminofluorene (Guo-8-AAF) have been used in a double peroxidase-antiperoxidase staining assay to visualize the localization of DNA-O6-ethyldeoxyguanosine (O6-EtdGuo) and some of the interaction products of N-acetyl-2-aminofluorene (AAF) with DNA guanine in liver sections of rats treated with diethylnitrosamine (DEN), ethylnitrosourea (ENU), or AAF respectively. O6-EtdGuo could be detected in nuclei of parenchymal cells after injection of DEN (12-50 mg/kg) or ENU (140 mg/kg). Clear time and dose dependencies were observed. The lowest dose of DEN which, at 5 h after injection, resulted in immunohistochemically detectable levels of O6-EtdGuo, was 12 mg/kg; 5 h after 6 mg/kg no consistent difference between treated and untreated rats could be observed. A striking heterogeneity in staining pattern was observed after DEN: centrilobular regions were stained much more than peripheral zones. At 7 days after a single DEN injection of 50 mg/kg small rims of significantly stained hepatocytes could still be observed around the central veins. No heterogeneity of staining pattern was observed 2 h after ENU. ENU, in contrast with DEN, also resulted in a significant staining of nonparenchymal cells. At 24 h after ENU no significant staining of hepatocytes could be detected, but positive staining was still present in bile duct cells, vascular endothelial cells and sinusoidal cells. The results indicate a detection level of approximately 5 mumol O6-EtdGuo/mol DNA-P, i.e., 5 X 10(4) O6-EtdGuo residues per diploid genome. Using the anti-Guo-8-AAF antiserum, positive results were obtained 6 days after a single AAF dose of 0.5-10 mg/kg, corresponding to a detection limit of less than or equal to 0.4 mumol dGuo-8-(A)AF/mol DNA-P. Staining was rather homogenously distributed over the liver lobules. Persistency of the AAF-DNA interaction products was investigated both after 10 and 2 mg/kg AAF. Increased nuclear staining could be observed up to 8 weeks after 10 mg/kg and 4 weeks after 2 mg/kg.