Formation of 5-hydroxymethyl-2′-deoxyuridine in hepatic DNA of rats treated with γ-irradiation, diethylnitrosamine, 2-acetylaminofluorene or the peroxisome proliferator ciprofibrate

Abstract

The peroxisome proliferator ciprofibrate was tested for its ability to induce DNA damage in the form of 5-hydroxymethyl-2'-deoxyuridine (HMdU), an adduct that results from the reaction of thymine in DNA with hydroxyl radicals. In order to quantify HMdU, DNA containing [3H]thymidine of high specific activity had to be obtained. Since hepatocytes normally have a very low rate of DNA synthesis, rats were subjected to partial hepatectomy to stimulate DNA synthesis and then were administered [methyl-3H]thymidine by three p.o., i.p. or i.v. injections 20, 22 and 24 h after partial hepatectomy; or by slow infusion through the portal vein, starting 20 h after partial hepatectomy for 4 h. The specific activity of DNA in rats receiving [3H]thymidine through the portal vein was considerably higher than in rats receiving p.o., i.p. or i.v. injections. Rats were then exposed to various doses of gamma-irradiation after partial hepatectomy and infusion of [6-3H]thymidine through the portal vein. DNA from the liver was extracted, enzymatically hydrolyzed and analyzed by HPLC. The percentage of HMdU in DNA increased in a dose-dependent manner. Rats were then treated with the carcinogens 2-acetylaminofluorene (AAF) or diethylnitrosamine (DEN) in conjunction with partial hepatectomy and infusion of [methyl-3H]thymidine. There was an increase in HMdU formation after a single administration of DEN or AAF. Another group of rats was fed a diet containing the peroxisome proliferator ciprofibrate for 3 weeks. After partial hepatectomy and infusion of [6-3H]thymidine, these rats were fed the same ciprofibrate-containing diet for 2-4 more weeks. HMdU was detected in DNA at 2-4 weeks after [6-3H]thymidine infusion, but the level at 4 weeks was nearly 50% less than at 2 weeks. This study shows that oxidative DNA damage in the form of HMdU is induced in the liver by gamma-irradiation, DEN, AAF and peroxisome proliferation.