Quantitative analysis of benzo[a]pyrene biotransformation and adduct formation in Ahr knockout mice
Introduction
Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of compounds formed during incomplete combustion of organic matter and fossil fuels in industrial processes, automobile exhaust, cigarette smoke and charbroiled food (IARC Monographs, 1983a, IARC Monographs, 1983b). Exposure to PAHs is high in certain occupational environments. Several of the PAH congeners are classified as carcinogens. Benzo[a]pyrene (BP) is a well-studied member of the PAH family and has served as a model for the biotransformation and carcinogenic effects of PAHs (Conney, 1982, Dipple, 1995, Harvey and Geacintov, 1988, Hogan et al., 1981, Stowers and Anderson, 1985). BP and other PAHs are primarily activated by P450 enzymes regulated by the aryl hydrocarbon receptor (Ahr) pathway (Whitlock, 1999). The Ahr also plays an important role in the regulation of cell growth and differentiation. The discovery of the Ahr originated from studies with Ah responsive/non-responsive mouse models (Nebert, 1989). The importance of the Ahr in the activation of PAH has then led to several Ahr and cytochrome P450 knockout mouse models (Kondraganti et al., 2003, McFadyen et al., 2003, Nakatsuru et al., 2004, Shimizu et al., 2000, Uno et al., 2004, Uno et al., 2006).
BP acts as a ligand and binds to the Ahr in the cytoplasm. The liganded Ahr is then translocated to the nucleus where it forms a heterodimer with the Ahr-nuclear translocator (Arnt). The Ahr/Arnt heterodimer recognize and binds to xenobiotic responsive element (XRE) sequences located in the promoter region of several genes such as cytochrome P450 (Cyp)1a1, Cyp1a2, Cyp1b1, glutathione S-transferases (Gst), and UDP-glucoronosyl-transferases (Ugt) (Nebert et al., 2000, Whitlock, 1999). The binding results in transcriptional activation of the genes and induction of phases I and II metabolizing enzymes as well as phase III transporter proteins (Klaassen, 2002, Xu et al., 2005). The encoded cytochrome P450 enzymes will then transform PAH to hydroxyl containing metabolites that are rapidly conjugated to glucoronides and sulphates by phase II enzymes. The bioactivation of BP goes through reactive intermediates, like epoxides, that may produce DNA and protein adducts (Fig. 1). The formation of covalent DNA adducts is an important first step in the initiation of PAH induced carcinogenesis (Hogan et al., 1981, Stowers and Anderson, 1985), and it has been suggested that increased adduct levels may be predictive of cancer risk (Veglia et al., 2003).
Shimizu et al. (2000) found that BP carcinogenicity was lost in mice lacking the Ahr. The mice received topical application and subcutaneous injection of the PAH, and only the Ahr (+/+) and Ahr (+/−) mice developed tumors. Kondraganti et al. (2003) found that total hepatic BP-DNA adduct levels were almost equal in Ahr (−/−) and Ahr (+/+) mice after a single i.p. dose of BP. In the knockout studies by Uno et al., 2004, Uno et al., 2006, it was shown that BP-DNA adducts and genotoxicity increased in the absence of the Cyp 1a1 and Cyp 1b1 genes.
To gain further insight in the role of the Ahr in the metabolic activation and detoxication of PAH, we have studied the relationship between Ahr genotype and bioactivation and biotransformation of BP in internal organs. In contrast to previous studies we have treated the animals with a single dose of BP by gavage. In the present report, we have quantitated protein and DNA adducts and metabolites. By the use of a highly specific HPLC-fluorescence method, we find increased levels of protein and DNA adducts, metabolites and unmetabolized BP in the Ahr (−/−) as compared to Ahr (+/+) mice. Gene expression has been measured by quantitative real-time RT-PCR.
Section snippets
Chemicals and standards
(±)-Benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol (BP-tetrol I-1), (±)-benzo[a]pyrene-r-7,t-8,t-9,10-tetrahydrotetrol (BP-tetrol I-2), (±)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol (BP-tetrol II-1), (±)-benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol (BP-tetrol II-2), benzo[a]pyrene-4,5-dihydrodiol, benzo[a]pyrene-7,8-dihydrodiol, benzo[a]pyrene-9,10-dihydrodiol, benzo[a]pyrene-3-phenol and benzo[a]pyrene-9-phenol were purchased from the National Cancer Institute (NCI), Chemical
Result
In animals exposed to BP, real-time RT-PCR analysis showed induction of Cyp1a1 in liver and lung in both Ahr (+/+) and Ahr (+/−) but no induction in the Ahr (−/−) (Fig. 2A). There was also an induction of Cyp1b1 in the lung of both Ahr (+/+) and Ahr (+/−), but no induction in the Ahr (−/−) (Fig. 2B). There was a significant basal expression of Cyp1b1 in the liver of all genotypes, and this expression was independent of the BP exposure. Constitutive Cyp1a1 expression level showed an Ahr
Discussion
In the present study, the metabolism of BP given by gavage in Ahr knockout, heterozygotes and wild type mice has been compared. A significant accumulation of unmetabolized BP and increased levels of adducts and metabolites were found in Ahr (−/−) as compared to Ahr (+/−) and Ahr (+/+) mice. The BP-tetrol levels showed an inverse relationship compared to the Ahr gene–dose. In the liver of the Ahr (−/−) mice, the levels of BP-tetrol II-2 were higher than BP-tetrol I-1. These results indicate that
Acknowledgements
The authors wish to thank Einar Eilertsen for advice and help in animal treatment. This study was supported by the Norwegian Research Council and the Norwegian Cancer Society.
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