Formation and stability of alkylated pyrimidines and purines (including imidazole ring-opened 7-alkylguanine) and alkylphosphotriesters in liver DNA of adult rats treated with ethylnitrosourea or dimethylnitrosamine

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.