Formation and Subsequent Excision of O⁶-Ethylguanine from DNA of Rat Liver following Administration of Diethylnitrosamine¹

The alkylation of rat liver DNA at short times after administration of low doses (0.5 to 4 mg/kg) of diethylnitrosamine was studied and compared to alkylation after higher doses (10 to 100 mg/kg). Ethylation of DNA as measured by the production of 7-ethylguanine was virtually complete within 30 min after i.p. injection of 0.5-mg/kg doses of the carcinogen, within 90 min after 2 mg/kg, and within 120 min after 4 mg/kg. 7-Ethylguanine levels declined only very slightly over a further 3- to 6-hr period. O⁶-Ethylguanine produced by these doses of diethylnitrosamine was removed rapidly from the DNA by an enzymatic process as indicated by a loss of from 40 to 60% of the initial O⁶-ethylguanine content within 4 hr. For this reason, a ratio of O⁶-ethylguanine to 7-ethylguanine in the ethylated DNA close to 0.71 (which was that found by reaction of DNA in vitro with N-ethyl-N-nitrosourea) was observed only at the earliest time points measured. These results indicate that the higher O⁶-ethylguanine:7-ethylguanine ratio observed at higher exposures to diethylnitrosamine in the present experiments and those of Scherer et al. (Chem-Biol. Interact., 19: 1–11, 1977) were due to rapid excision of smaller amounts of O⁶-ethylguanine from the DNA and not due to a facilitation of O⁶-ethylguanine production by a dose-dependent process as postulated by Scherer et al.

The rate of loss of O⁶-ethylguanine from liver DNA after administration of low doses of diethylnitrosamine showed complex kinetics, and the half-life depended on the extent of alkylation. However, the rate of loss was comparable to that of O⁶-methylguanine from the hepatic DNA after production of similar extents of alkylation by dimethylnitrosamine. A cell-free extract was isolated from rat liver which catalyzed the removal of O⁶-ethylguanine from DNA. The activity of this enzyme was sufficient to account for the rate observed in vivo after doses of up to 4 mg of diethylnitrosamine per kg. The enzyme catalyzed the loss of O⁶-[³H]ethylguanine from the acid-precipitable substrate DNA but did not lead to release of 7-ethylguanine or guanine, indicating that it was specific for the O⁶ product. The same preparation produced approximately equal rates of removal of O⁶-methylguanine from methylated DNA and O⁶-ethylguanine from ethylated DNA when the substrate concentrations were similar. The loss of O⁶-ethylguanine from the acid-precipitable DNA was not accompanied by the appearance in the supernatant of either the free base or an oligonucleotide containing the O⁶ derivative. It appeared that the material removed from the DNA became bound to a macromolecule in a form which could ultimately lead to the conversion of the O⁶-alkyl group to the corresponding alcohol. All of the experiments were consistent with the hypothesis that the same enzyme system is responsible for the rapid removal of both O⁶-ethylguanine and O⁶-methylguanine after administration of low doses of the corresponding dialkylnitrosamines.