Metabolic activation of benzo[a]pyrene in vitro by hepatic cytochrome P450 contrasts with detoxification in vivo: experiments with hepatic cytochrome P450 reductase null mice

B18

Hepatic cytochrome P450 (CYP) enzymes play a pivotal role in the metabolism of many drugs and carcinogens. Much of the work carried out on the role of hepatic CYPs in xenobiotic metabolism has been done in vitro. However, additional factors such as route of administration, absorption, renal clearance and extra-hepatic CYP expression, make it difficult to extrapolate from in-vitro data to in-vivo pharmacokinetics. Moreover, functional redundancy inevitably found in the large CYP family of isoenzymes make it difficult to determine the role of CYPs in metabolism as a whole. To overcome these limitations a mouse line, HRN (Hepatic Cytochrome P450 Reductase Null), has been developed in which cytochrome P450 oxidoreductase (POR), the unique electron donor to CYPs is deleted specifically in the liver, resulting in the loss of essentially all hepatic P450 function. CYP1A1 or CYP1B1 activate many polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP). As a result, it is widely accepted that CYP1 potentiates BaP genotoxicity. We used the HRN model to evaluate the role of hepatic versus extrahepatic metabolism and disposition of BaP. HRN and wild-type (WT) mice were treated with 125 mg/kg body weight (bw) BaP once daily for up to five days by intraperitoneal injection. DNA adduct levels measured by ³²P-postlabelling at day 1 and 5 were up to 13-fold higher in livers of HRN than in WT mice. On day 1 sequential blood samples were obtained and analysed by HPLC with fluorescence detection for BaP clearance. Pharmacokinetic analysis of BaP in blood revealed no difference in e.g. clearance, terminal half-life, and AUC in HRN relative to WT mice. When hepatic microsomal fractions from HRN and WT mice were incubated with BaP, DNA adduct formation was 7-fold higher in WT than in HRN fractions. Most of the hepatic microsomal activation of BaP in vitro was attributable to CYP1A enzyme activity. These data reveal an apparent paradox, whereby CYP enzyme activity appears to be more important for detoxification of BaP in vivo, despite being essential for its metabolic activation in vitro.