Association between DNA strand breaks and specific DNA adducts in murine hepatocytes following in vivo and in vitro exposure to N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene

Abstract

N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene (N-OAc-AAF) have previously been shown to induce dose-dependent DNA strand breaks in primary hepatocytes from mice and rats. In an attempt to determine the relationship between the extent of DNA strand breaks and the formation of specific DNA-carcinogen bound adducts in murine liver, the capability of N-OH-AAF and N-OAc-AAF to induce both DNA single strand breaks and adduct formation in in vivo and in primary hepatocytes was measured. N-OH-AAF induced a low level of DNA damage in F344 rats (10 mg/kg, i.p.) and in B6 mice (40 mg/kg, i.p.) 4 h after treatment. The DNA adducts identified in vivo were N-(guanin-8-yl)-2-acetylaminofluorene (Gua-C8-AAF) 55% versus 11%, N-(guanin-8-yl)-2-aminofluorene (Gua-C8-AF) 34% versus 67% and 3-(guanin-N2-yl)-2-acetylaminofluorene (Gua-N2-AAF) 11% versus 10%, respectively, for rat and mouse liver. An additional unknown adduct (12%) was detected in mouse liver. Dose dependent DNA binding and formation of individual DNA adducts were observed in rat and mouse primary hepatocytes following 1 h exposure to [ring-3H]-N-OH-AAF (0.1-20 microM) and [ring-3H]-N-OAc-AAF (5-20 microM). The patterns of DNA adducts in mouse and rat primary hepatocytes exposed to N-OH-AAF and N-OAc-AF were similar to those obtained in liver following in vivo treatment with N-OH-AAF. The deacetylase inhibitor, paraoxon (10(-4) M) completely inhibited DNA damage induced by N-OH-AAF in mouse and partially in rat hepatocytes while DNA damage caused by N-OAc-AAF was only partially inhibited by paraoxon (10(-4) M) in both species. Parallel experiments showed that paraoxon, at low concentration (10(-6) M), did not alter either the level of DNA binding or the pattern of adduct formation in rat hepatocytes treated with N-OH-AAF (20 microM). However, at 10(-4) M paraoxon partially blocked DNA binding (60%) and the formation of Gua-C8-AAF (95%) and Gua-N2-AAF (80%) while Gua-C8-AF was increased two-fold. In mouse hepatocytes paraoxon pretreatment (10(-4) M) inhibited the formation of Gua-C8-AF by 70% following exposure to N-OH-AAF (20 microM). Gua-C8-AAF and Gua-N2-AAF were also inhibited but only at 10(-4) M paraoxon.(ABSTRACT TRUNCATED AT 400 WORDS)