Inhibition of DNA synthesis in primary cultures of adult rat hepatocytes by benzo[a]pyrene and related aromatic hydrocarbons: Role of Ah receptor-dependent events

Studies were conducted to examine the effects of benzo[a]pyrene (BaP) and related-aromatic hydrocarbons (AHs) on the DNA synthetic profiles of adult rat hepatocytes in primary culture. Scheduled DNA synthesis in control cultures peaked at 64 h and was negligible by 72 h after initial seeding of freshly isolated hepatocytes. A concentration-dependent inhibition of DNA synthesis was observed in 1-day old hepatocyte cultures treated with BaP (0.3-30 μM) for up to 28 h. Comparable inhibitory responses were observed in cultures treated for 24 h with 2,3,7,8-tetrachlorodi-benzo-p-dioxin (TCDD, 0.01-1 nM) or 2,3,7,8-tetrachlorodibenzofuran (TCDF, 0.01-1 nM), but not in cultures treated with perylene (0.01-100 nM) or benzo[e])pyrene (1-1000 nM). Ethoxyresorufin-O-deethylase (EROD) activity was highly inducible in hepatocytes challenged for 24 h with BaP (0.3-3 μM) or TCDD (0.1-100 nM) with peak induction observed at 12 or 36 h after chemical challenge, respectively. To determine if DNA synthesis inhibition by these agents involved aryl hydrocarbon receptor (AhR)-related events, subsequent experiments were conducted to examine the interactions of α-naphthoflavone (α-NF) and ellipticine (ET) with BaP and TCDD in this cell system. Pretreatment with α-NF (10 nM) for 24 h prevented the inhibitory effects of both BaP (3 μM) and TCDD (1 nM), while ET (0.01 nM) pretreatment selectively antagonized the effects of BaP (3 μM). Pretreatment of hepatocytes with TCDD or TCDF (1 nM) for 24 h before the onset of DNA synthesis followed by challenge with BaP (3 μM) partially antagonized the inhibitory response to BaP. These data implicate AhR-related signal transduction in the inhibition of hepatocyte DNA synthesis by BaP and related AHs and suggest that in the case of BaP, metabolism by cytochrome P450 to toxic intermediates contributes to the inhibitory response.