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

Research output: Contribution to journalArticle

44 Scopus citations

Abstract

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-Ethylgua-nine levels declined only very slightly over a further 3- to 6-hr period. O6-Ethylguanine produced by these doses of diethyl-nitrosamine 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-ethylgua-nine from the DNA and not due to a facilitation of O6-ethylgua-nine 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-precip-itable substrate DNA but did not lead to release of 7-ethylgua-nine or guanine, indicating that it was specific for the O6product. The same preparation produced approximately equal rates of removal of O6-methylguanine from methylated DNA and O8-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 conver.

Original languageEnglish (US)
Pages (from-to)5003-5009
Number of pages7
JournalCancer Research
Volume39
Issue number12
Publication statusPublished - Dec 1 1979

    Fingerprint

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

Cite this