Initiation of hepatocarcinogenesis in infant male B6C3F1 mice by N-hydroxy-2-aminofluorene or N-hydroxy-2-acetylaminofluorene depends primarily on metabolism to N-sulfooxy-2-aminofluorene and formation of DNA-(deoxyguanosin-8-yl)-2-aminofluorene adducts.

Abstract

Previous work from this laboratory provided strong evidence that N-sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) in the livers of infant male B6C3F1 (C57BL/6J X C3H/HeJ F1) mice. Over 90% of the hepatic DNA adducts in these mice consisted of N-(deoxyguanosin-8-yl)-2-aminofluorene [N-(dGuo-8-yl)-AF] and less than 10% were deoxyguanosinyl adducts containing 2-acetylaminofluorene (AAF) residues. In the present study hepatic DNA adduct formation and tumor initiation by N-hydroxy-2-aminofluorene (N-hydroxy-AF) were examined in these mice. N-(dGuo-8-yl)-AF was the only adduct detected in the hepatic DNA; the level at 9 h after a single i.p. dose of 0.04 or 0.06 mumol/g body wt of [3H]N-hydroxy-AF was 1.0 or 1.7 pmol/mg DNA. Pretreatment with a single i.p. dose (0.04 mumol/g body wt) of the sulfotransferase inhibitor pentachlorophenol (PCP) decreased the DNA adduct level by greater than 80%. Similar levels of this adduct were found by 32P-postlabeling analysis of DNA from mice treated with unlabeled N-hydroxy-AF. The liver DNA of infant male brachymorphic B6C3F2 mice [deficient in 3'-phosphoadenosine-5'-phosphosulfate (PAPS)] contained only 0.3 pmol/mg DNA of N-(dGuo-8-yl)-AF after an i.p. dose of 0.06 mumol of N-hydroxy-AF/g body wt, while their phenotypically normal (PAPS-sufficient) male littermates had 1.9 pmol/mg DNA. A single i.p. dose of 0, 0.015, 0.03, 0.06 or 0.12 mumol/g body wt of N-hydroxy-AF in infant male B6C3F1 mice induced by 10 months an average of 0.2, 2.5, 7, 11 or 14 hepatomas/mouse. Pretreatment with PCP reduced the liver tumor multiplicity at each dose level by greater than 80%. Essentially the same average tumor multiplicities and inhibitions of tumor formation by PCP pretreatment were obtained following injections of N-hydroxy-AF or N-hydroxy-AAF at the three lower dose levels. Collectively these data strongly indicated that N-sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxy-AF in the livers of infant male B6C3F1 mice. Furthermore, since only N-(dGuo-8-yl)-AF adducts were found in the hepatic DNA these lesions appear to be critical in the initiation of hepatocarcinogenesis in these mice by N-hydroxy-AF.