Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. In this study human recombinant CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5) were expressed in Supersomes™ together with their reductases, NADPH:CYP oxidoreductase, epoxide hydrolase and cytochrome b₅, to investigate BaP metabolism. Human CYPs produced up to eight BaP metabolites. Among these, BaP‐7,8‐dihydrodiol and BaP‐9‐ol, which are intermediates in BaP‐derived DNA adduct formation, were mainly formed by CYP1A1 and 1B1, and to a lesser extent by CYP2C19 and 3A4. BaP‐3‐ol, a metabolite that is a ‘detoxified’ product of BaP, was formed by most human CYPs tested, although CYP1A1 and 1B1 produced it the most efficiently. Based on the amounts of the individual BaP metabolites formed by these CYPs and their expression levels in human liver, we determined their contributions to BaP metabolite formation in this organ. Our results indicate that hepatic CYP1A1 and CYP2C19 are most important in the activation of BaP to BaP‐7,8‐dihydrodiol, whereas CYP2C19, 3A4, and 1A1 are the major enzymes contributing to the formation of BaP‐9‐ol. BaP‐3‐ol is predominantly formed by hepatic CYP3A4, while CYP1A1 and 2C19 are less active. Environ. Mol. Mutagen. 57:229–235, 2016. © 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.

The tumour suppressor p53 is one of the most important cancer genes. Previous findings have shown that p53 expression can influence DNA adduct formation of the environmental carcinogen benzo[a]pyrene (BaP) in human cells, indicating a role for p53 in the cytochrome P450 (CYP) 1A1-mediated biotransformation of BaP in vitro. We investigated the potential role of p53 in xenobiotic metabolism in vivo by treating Trp53(+/+), Trp53(+/–) and Trp53(−/−) mice with BaP. BaP-DNA adduct levels, as measured by ³²P-postlabelling analysis, were significantly higher in liver and kidney of Trp53(−/−) mice than of Trp53(+/+) mice. Complementarily, significantly higher amounts of BaP metabolites were also formed ex vivo in hepatic microsomes from BaP-pretreated Trp53(−/−) mice. Bypass of the need for metabolic activation by treating mice with BaP-7,8-dihydrodiol-9,10-epoxide resulted in similar adduct levels in liver and kidney in all mouse lines, confirming that the influence of p53 is on the biotransformation of the parent compound. Higher BaP-DNA adduct levels in the livers of Trp53(−/−) mice correlated with higher CYP1A protein levels and increased CYP1A enzyme activity in these animals. Our study demonstrates a role for p53 in the metabolism of BaP in vivo, confirming previous in vitro results on a novel role for p53 in CYP1A1-mediated BaP metabolism. However, our results also suggest that the mechanisms involved in the altered expression and activity of the CYP1A1 enzyme by p53 in vitro and in vivo are different.

Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b₅ reductase (CBR) in the presence of cytochrome b₅ can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b₅ in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b₅ in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by ³²P-postlabeling was characterized as 10-(deoxyguanosin-N²-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b₅ plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b₅/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP.

Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. The tumour suppressorTP53is a critical gene in carcinogenesis and frequently mutated in AA-induced urothelial tumours. We investigated the impact of p53 on AAI-induced nephrotoxicity and DNA damage in vivo by treatingTrp53(+/+),Trp53(+/−) andTrp53(−/−) mice with 3.5 mg/kg body weight (bw) AAI daily for 2 or 6 days. Renal histopathology showed a gradient of intensity in proximal tubular injury fromTrp53(+/+) toTrp53(−/−) mice, especially after 6 days. The observed renal injury was supported by nuclear magnetic resonance (NMR)-based metabonomic measurements, where a consistentTrp53genotype-dependent trend was observed for urinary metabolites that indicate aminoaciduria (i.e. alanine), lactic aciduria (i.e. lactate) and glycosuria (i.e. glucose). However,Trp53genotype had no impact on AAI-DNA adduct levels, as measured by³²P-postlabelling, in either target (kidney and bladder) or non-target (liver) tissues, indicating that the underlying mechanisms of p53-related AAI-induced nephrotoxicity cannot be explained by differences in AAI genotoxicity. Performing gas chromatography–mass spectrometry (GC–MS) on kidney tissues showed metabolic pathways affected by AAI treatment, but againTrp53status did not clearly impact on such metabolic profiles. We also cultured primary mouse embryonic fibroblasts (MEFs) derived fromTrp53(+/+),Trp53(+/−) andTrp53(−/−) mice and exposed them to AAI in vitro (50 µM for up to 48 h). We found thatTrp53genotype impacted on the expression of NAD(P)H:quinone oxidoreductase (Nqo1), a key enzyme involved in AAI bioactivation. Nqo1 induction was highest inTrp53(+/+) MEFs and lowest inTrp53(−/−) MEFs; and it correlated with AAI-DNA adduct formation, with lowest adduct levels being observed in AAI-exposedTrp53(−/−) MEFs. Overall, our results clearly demonstrate that p53 status impacts on AAI-induced renal injury, but the underlying mechanism(s) involved remain to be further explored. Despite the impact of p53 on AAI bioactivation and DNA damage in vitro, such effects were not observed in vivo.