Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling

Abstract

Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kg body weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por.

Fig. 1.

Quantitative TLC ³²P-postlabelling analysis of dG-N²-BPDE adducts in organs of HRN and WT mice treated i.p. (A and C) or orally (B and D) with 12.5 (C and D) or 125 mg/kg bw BaP (A and B) for 24h. F = fold increase in DNA binding in HRN mice compared to WT mice. Values are given as means ± SD (n = 3); each DNA sample was determined by two postlabelled analyses. Comparison was performed by t-test analysis: *P <0.01 different from WT. RAL, relative adduct labelling.

Fig. 2.

Quantitative TLC ³²P-postlabelling analysis of dG-N²-BPDE adducts in organs of RCN mice treated i.p. with 125 mg/kg bw BaP for 24h without and with 3-MC pretreatment (single dose of 40 mg/kg bw 3-MC 14 days before BaP treatment. F = fold increase in DNA binding in RCN mice treated with BaP compared with RCN mice treated with BaP and inducer (i.e. 3-MC). Values are given as means ± SD (n = 3); each DNA sample was determined by two postlabelled analyses. Comparison was performed by t-test analysis: *P <0.01 different from RCN mice treated with BaP but without inducer. RAL, relative adduct labelling. Inset: Typical autoradiographic profile of BaP-derived DNA adducts obtained by ³²P-postlabelling; solvent conditions for the resolution of ³²P-labelled adducts on polyethyleneimine-cellulose thin-layer chromatography were: D1, 1.0M sodium phosphate, pH 6; D3, 4.0M lithium formate, 7.0M urea, pH 3.5; D4, 0.8M LiCl, 0.5M Tris, 8.5M urea, pH 8. The arrow indicates the position of the 5’-³²P-labelled biphosphate dG-N²-BPDE adduct. ##, not determined (DNA sample lost).

Fig.3.

Immunostaining (20×) for BPDE-DNA adducts in hepatic tissue sections of HRN and WT mice treated i.p. with 125 mg/kg bw BaP for 24h. Staining of BaP-treated mice (A and D) with specific BPDE-DNA antiserum; the arrows indicate stained nuclei detecting dG-N²-BPDE adducts. Staining of BaP-treated mice (B and E) with BPDE-DNA-absorbed BPDE-DNA antiserum (background). No staining was observed in control (untreated) animals (C and F).

Fig. 4.

Expression of mEH and cytochrome b₅ (Cyt b₅) in livers of HRN and WT mice, control (untreated) mice or mice treated i.p. daily with 125 mg/kg bw BaP for 5 days. Pooled hepatic microsomal samples were used for analyses as described in Section 2. Values are given as means ± SD of 3 determinations.