The genotoxicity of aromatic amines in primary hepatocytes isolated from C57BL/6 and DBA/2 mice.

Abstract

The capacity of the chemical carcinogen 2-acetylaminofluorene (AAF) and its derivates to cause DNA damage in primary mouse hepatocytes from aryl-hydrocarbon responsive C57BL/6 and non-responsive DBA/2 mice was studied using the alkaline elution technique. Low levels of DNA damage were observed after exposure of hepatocytes to either AAF or 2-aminofluorene (AF) (50-100 microM). Quantitation of metabolites produced from AAF in hepatocytes from untreated C57BL/6 and DBA/2 mice using h.p.l.c. showed a similar metabolic profile with respect to C- and N-hydroxylations. After in vivo pretreatment with the potent monooxygenase inducer TCDD (50 micrograms/kg), N-hydroxylation in the C57BL/6- and DBA/2-derived hepatocytes increased 25- and 5-fold, respectively. However, the C-hydroxylation pathways were still responsible for approximately 90% of the metabolism in cells from both strains. This may explain why only a slight increase in the DNA damage was observed in C57BL/6 mouse hepatocytes after incubation with AF or AAF and no increase in DNA damage was seen in the DBA/2 hepatocytes isolated from TCDD treated animals. Both N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene (N- OAc -AAF) caused clear dose-dependent increases in DNA strand breaks (5-100 microM), suggesting that N-hydroxylation was the rate limiting step in the activation process of AAF leading to the DNA damage. Treatment of hepatocytes with paraoxon, an inhibitor of microsomal deacetylase activity, prior to exposure to either N-OH-AAF or N- OAc -AAF completely inhibited the damage caused by N-OH-AAF, while the damage caused by N- OAc -AAF was only partially inhibited. This suggests that these compounds are causing genotoxic effects after deacetylation. In accordance with this, N-hydroxy-2-aminofluorene (N-OH-AF), the deacetylated metabolite of N-OH-AAF, was an effective genotoxic agent, causing DNA strand breaks at low doses. Depletion of cellular glutathione by pretreatment with diethyl maleate, increased the sensitivity of the cells to the damage induced by N-OH-AF. These data indicate that glutathione may play an important role in the detoxification of N-OH-AF in mouse hepatocytes.