Elsevier

Toxicology Letters

Volume 167, Issue 3, 15 December 2006, Pages 173-182
Toxicology Letters

Quantitative analysis of benzo[a]pyrene biotransformation and adduct formation in Ahr knockout mice

https://doi.org/10.1016/j.toxlet.2006.09.005Get rights and content

Abstract

Benzo[a]pyrene (BP) is an ubiquitous environmental pollutant with potent mutagenic and carcinogenic properties. The Ah receptor (Ahr) is involved in the metabolic activation of BP and is therefore important in the induction of chemical carcinogenesis. In this study, the relationship between Ahr genotype and biotransformation of BP in internal organs was investigated in Ahr (+/+), Ahr (+/−) and Ahr (−/−) mice. The mice were treated with BP (100 mg/kg) by gavage. Gene expression was measured after 24 h by real-time RT-PCR and showed induction of Cyp1a1 in liver and lung, and Cyp1b1 in lung in both Ahr (+/+) and Ahr (+/−). No induction of the Cyp genes was observed in the Ahr (−/−). There was a significant basal expression of Cyp1b1 in the liver of all genotypes, and this expression was independent of the BP exposure. Analyzed by HPLC-fluorescence, there were increased levels of protein and DNA adducts, metabolites, conjugates and unmetabolized BP in the internal organs of Ahr (−/−) as compared to Ahr (+/+) and Ahr (+/−) mice. This may be partly explained by a delayed bioactivation of BP in the Ahr deficient mice. The BP metabolism observed in the Ahr (−/−) mice is also evidence of an Ahr independent biotransformation of BP.

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.

References (32)

  • A.C. Beach et al.

    Human biomonitoring and the 32P-postlabelling assay

    Carcinogenesis

    (1992)
  • G. Berge et al.

    Analysis of resveratrol as a lung cancer chemopreventive agent in A/J mice exposed to benzo[a]pyrene

    Br. J. Cancer

    (2004)
  • A.H. Conney

    Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: G.H.A. Clowes Memorial Lecture

    Cancer Res.

    (1982)
  • A. Dipple

    DNA adducts of chemical carcinogens

    Carcinogenesis

    (1995)
  • B. Ebert et al.

    Identification of BCRP as transporter of benzo[a]pyrene conjugates metabolically formed in Caco-2 cells and its induction by Ah-receptor agonists

    Carcinogenesis

    (2005)
  • H.L. Eriksson et al.

    32P-postlabeling of DNA adducts arising from complex mixtures: HPLC versus TLC separation applied to adducts from petroleum products

    Arch. Toxicol.

    (2004)
  • Cited by (39)

    • Cigarette smoking and reproductive and developmental toxicity

      2017, Reproductive and Developmental Toxicology
    • Toxicological issues associated with production and processing of meat

      2013, Meat Science
      Citation Excerpt :

      PAH molecules have a capability of self-induction of their enzymatic activation by receptor mechanism over AhR. Very often amplification proceeds in cooperation with dioxins (Sagredo et al., 2006). Meat gets contaminated with PAHs mainly at smoking or barbecuing over open horizontal coals.

    • Aryl hydrocarbon receptor-mediated Cyp1a1 expression is modulated in a CLOCK-dependent circadian manner

      2011, Toxicology
      Citation Excerpt :

      These results suggest that the expression of AhR is positively regulated by CLOCK protein. Benzo[α]pyrene, an exogenous ligand of AhR, potently induces the expression of Cyp1a1 (Sagredo et al., 2006). In the lungs of wild-type mice, mRNA levels of Cyp1a1 at 2-h after benzo[α]pyrene injection significantly varied according to its dosing time (p < 0.05; Fig. 4A).

    • Ganoderma applanatum terpenes protect mouse liver against benzo(α)pyren-induced oxidative stress and inflammation

      2011, Environmental Toxicology and Pharmacology
      Citation Excerpt :

      Most humans are more or less constantly exposed to BaP (Takaishi et al., 2009). Although the precise mechanism of genotoxic effects caused by BaP is not clear, there is evidence that BaP causes generation of reactive oxygen metabolites, inhibits the activity of antioxidant enzymes, suppresses immune responses and induces inflammation in tissue (Park et al., 2010; Kim et al., 2008; Kim and Lee, 1997; Sagredo et al., 2006; Takaishi et al., 2009; Lakshmi et al., 2006; Umannová et al., 2008; Khalil et al., 2010; Marie-Desvergne et al., 2010). Many studies had shown that several radical scavengers and antioxidants exhibited protective effects against BaP-induced oxidative stress (Park et al., 2010; Kim et al., 2008; Lakshmi et al., 2006).

    View all citing articles on Scopus
    View full text