Polycyclic aromatic hydrocarbons as skin carcinogens: Comparison of benzo[a]pyrene, dibenzo[def,p]chrysene and three environmental mixtures in the FVB/N mouse

Abstract

The polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), was compared to dibenzo[def,p]chrysene (DBC) and combinations of three environmental PAH mixtures (coal tar, diesel particulate and cigarette smoke condensate) using a two stage, FVB/N mouse skin tumor model. DBC (4 nmol) was most potent, reaching 100% tumor incidence with a shorter latency to tumor formation, less than 20 weeks of 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion compared to all other treatments. Multiplicity was 4 times greater than BaP (400 nmol). Both PAHs produced primarily papillomas followed by squamous cell carcinoma and carcinoma in situ. Diesel particulate extract (1 mg SRM 1650b; mix 1) did not differ from toluene controls and failed to elicit a carcinogenic response. Addition of coal tar extract (1 mg SRM 1597a; mix 2) produced a response similar to BaP. Further addition of 2 mg of cigarette smoke condensate (mix 3) did not alter the response with mix 2. PAH-DNA adducts measured in epidermis 12 h post initiation and analyzed by ³²P post‐labeling, did not correlate with tumor incidence. PAH‐dependent alteration in transcriptome of skin 12 h post initiation was assessed by microarray. Principal component analysis (sum of all treatments) of the 922 significantly altered genes (p < 0.05), showed DBC and BaP to cluster distinct from PAH mixtures and each other. BaP and mixtures up-regulated phase 1 and phase 2 metabolizing enzymes while DBC did not. The carcinogenicity with DBC and two of the mixtures was much greater than would be predicted based on published Relative Potency Factors (RPFs).

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are planar aromatic compounds with varying potencies of carcinogenicity defined by their individual structures (IARC, 2010). PAHs occur naturally in the environment in fossil fuels such as coal, oil, and tar and are considered environmental pollutants formed during incomplete combustion (coal, tobacco, diesel, asphalt, creosote, gasoline, wood smoke, etc.) leading to their presence in air, food and soils (Ding et al., 2011, Lewtas, 2007, Lijinsky, 1991, Weissenfels et al., 1992). PAHs occur in the environment typically as mixtures covering a spectrum from non‐toxic compounds to potent carcinogens (Allan et al., 2012, Baird et al., 2005, Mao et al., 2007, Wickramasinghe et al., 2012). Different types of combustion result in different compositions of PAHs both in relative amounts and individual PAHs present (Poster et al., 2000). Occupational exposure to PAH mixtures in aluminum production, iron and steel foundries, fossil fuel processing, wood impregnation, roofing and road sealing can pose risks for lung, skin, and bladder cancers (Boffetta et al., 1997, Cogliano et al., 2011, IARC, 2010). Epidemiological studies support a relationship between dermal exposure to PAHs and skin cancers (Boffetta et al., 1997, IARC, 2010, Marczynski et al., 2009). One of the most common cancers in Caucasian populations is non-melanoma skin cancer, recently reported to be on the rise throughout the world (Lomas et al., 2012). Benzo[a]pyrene (BaP), the most extensively studied carcinogenic PAH, is classified by IARC as a Group 1 or known human carcinogen (IARC, 2010). Four of the top ten priority pollutants, designated by the Agency for Toxic Substances and Disease Registry (ATSDR) in 2011, are single PAHs or PAH mixtures (PAHs, BaP, benzo[b]fluoranthene, and dibenzo[a,h]anthracene) (ATSDR, 2011).

PAHs are carcinogenic in a number of animal models with multiple targets, including skin (Arif et al., 1999, Courter et al., 2008, Darwiche et al., 2007, IARC, 2010, Nesnow et al., 1998, Wester et al., 2011). Our laboratories have documented that dibenzo[def,p]chrysene (DBC), formerly referred to as dibenzo[a,l]pyrene, is a potent carcinogen in mice (Castro et al., 2008a, Mahadevan et al., 2007a, Marston et al., 2001, Yu et al., 2006a, Yu et al., 2006b). Oral administration results in tumors of the liver, lung, breast, ovaries and hematopoietic tissue. DBC can also be an effective transplacental carcinogen (Castro et al., 2008a, Castro et al., 2008b, Chen et al., 2012, Guttenplan et al., 2011, Shorey et al., 2012, Yu et al., 2006a, Yu et al., 2006b).

PAHs require bioactivation through metabolism in order to be mutagenic, carcinogenic or teratogenic to target cellular macromolecules (Baird and Mahadevan, 2004, IARC, 2010). With higher molecular weight PAHs, such as BaP and DBC containing a “bay” and/or “fjord” region, respectively, the most well characterized bioactivation pathway has been cytochrome P450 (CYP)‐dependent epoxygenation, hydrolysis by epoxide hydrolase and a second CYP epoxygenation to the 7,8-dihydrodiol-9,10 epoxide (BPDE) in the case of BaP, and to the 11,12-dihydrodiol-13,14 epoxide (DBCDE) in the case of DBC (Shimada, 2006, Shou et al., 1996, Xue and Warshawsky, 2005). Hydrolysis of the initial epoxide produces two trans stereoisomers and the second epoxygenation can be above or below the plane of the ring; thus, four possible BPDEs or DBCDEs are produced (Fig. 1). With BaP, the most mutagenic and carcinogenic BPDE is thought to be (+)-7,8-anti-9,10-BPDE. PAHs such as BaP and DBC can also be bioactivated through 1-electron oxidations (peroxidases) producing radical cations (Cavalieri and Rogan, 1992, Cavelieri and Rogan, 1995), predominantly at the 1,6- and 3,6-positions. Once formed these radical cations may bind to DNA. The role of aldo–keto reductases (AKRs) in bioactivation of PAHs has also been demonstrated (Palackal et al., 2001, Palackal et al., 2002, Penning et al., 1996). AKRs effectively convert the PAH dihydrodiol to a catechol. As with other catechols, a redox-cycling can then occur through 1-electron reactions to the semi-quinone and quinone. These reversible reactions generate superoxide anion radical and other reactive oxygen species (ROS) and can also directly react with nucleophilic sites on DNA. The metabolism of PAHs through peroxidative and AKR-mediated pathways is consistent with oxidative stress‐associated PAH toxicity (Kumar et al., 2012).

The most important CYPs in PAH metabolism are CYP1A1, CYP1A2, CYP1B1, and to a lesser extent CYP2C9 and CYP3A4 (Shimada, 2006). Recent evidence from our laboratories and others has suggested that CYP1B1 plays a predominant role in the toxicity and carcinogenicity of both BaP and DBC in the mouse (Castro et al., 2008a, Uno et al., 2006).

The murine two-stage skin tumor model has been used extensively to investigate mechanisms of carcinogenesis (Cavalieri et al., 1991, Higginbotham et al., 1993) and the inbred FVB strain has been shown to be suitable for initiation/promotion studies (Hennings et al., 1993). This model is a powerful tool for studying early indicators of “high risk” papillomas that can develop into invasive squamous cell carcinomas (Glick et al., 2007). The vast majority of cancer studies in animal models have tested single PAHs. Unfortunately this is incongruous with the complex mixtures of PAHs to which human populations are exposed. In this study we sought to examine the relative potency of BaP and DBC, compared to combinations of some environmentally relevant PAH mixtures. We hypothesized that early PAH-dependent alterations in the transcriptome of mouse epidermis following initiation could be correlated with DNA adduct formation at the same time point and predict probable tumor outcomes. The EPA is currently evaluating the potential of a Relative Potency Factor (RPF) approach in estimating risk for exposure to PAH mixtures (U.S. EPA, 2010). Our results demonstrate that, at least with respect to skin cancer following dermal exposures, the RPF markedly underestimates DBC and PAH mixture potencies. Furthermore, alterations in gene expression 12 h post-initiation suggest the strong possibility that these PAH treatments are acting through multiple and distinct mechanisms.