Elsevier

Toxicology Letters

Volume 165, Issue 2, 20 August 2006, Pages 182-194
Toxicology Letters

Cigarette smoke condensate induces cytochromes P450 and aldo-keto reductases in oral cancer cells

https://doi.org/10.1016/j.toxlet.2006.03.008Get rights and content

Abstract

Our objective is to identify molecular factors which contribute to the increased risk of smokers for oral squamous cell carcinoma (OSCC). In the present study, we investigated the effects of cigarette smoke condensate (CSC) on gene expression profiles in different human oral cell phenotypes: normal epidermal keratinocytes (NHEK), oral dysplasia cell lines (Leuk1 and Leuk2), and a primary oral carcinoma cell line (101A). We determined differential gene expression patterns in CSC-exposed versus non-exposed cells using high-density microarray RNA expression profiling and validation by quantitative real-time RT-PCR. A set of 35 genes was specifically up- or down-regulated following CSC treatment (25 μg/ml for 24 h) by at least 2-fold in any one cell type. Notably, five genes of the cytochrome P450 (CYP1A1, CYP1B1) and aldo-keto reductase (AKR1C1, AKR1C3, AKR1B10) families were highly increased in expression, some of them 15- to 30-fold. The timing and extent of induction for these genes differed among the four cell phenotypes. A potential biological interaction network for the CSC response in oral cells was derived from these data, proposing novel putative response pathways. These CSC-responsive genes presumably participate in the prevention or repair of carcinogen-induced DNA damage in tobacco-related oral carcinogenesis, and may potentially be exploited for determining the severity of exposure and for correcting mutagenic damage in exposed tissues of the oral cavity.

Introduction

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the head and neck, with a worldwide incidence of 300,000 new cases annually. The major inducer of OSCC is exposure to tobacco, considered to be responsible for 50–90% of cases worldwide, and the incidence of OSCC in cigarette smokers is 7–10 times higher than for never smokers (Sudbo and Reith, 2005, Warnakulasuriya et al., 2005). Many of the chemical carcinogens contained in cigarette smoke are polycyclic aromatic hydrocarbons (PAH), a family of ubiquitous environmental carcinogens that are known to have mutagenic and carcinogenic effects. A variety of PAHs have been shown to cause cellular transformations only after metabolic activation by drug-metabolizing enzymes, such as cytochromes P450 (CYP) and aldo-keto reductases (AKRs) that produce highly reactive carcinogenic electrophiles (Pelkonen and Nebert, 1982, Rubin, 2001, Palackal et al., 2002, Mahadevan et al., 2005).

PAHs are activated to genotoxic intermediates through at least three primary pathways, all of which can lead to the production of G to T transversions (Palackal et al., 2002). In most cases, oxidation of PAHs by CYP enzymes is an initial step in the activation process. Among the various forms of CYPs determined so far, CYP1A1 and CYP1B1 have been shown to be the most important human CYP enzymes in the metabolic activation of PAHs and PAH dihydrodiols (Kim et al., 1998, Shimada et al., 1999, Nebert et al., 2004). It has been hypothesized that genetic variances in CYP expression, inducibility, or activity are responsible for individual susceptibility to cancer (Nebert, 1991, Alexandrie et al., 1994).

The human enzyme CYP1A1 is the most active among the CYPs in metabolizing procarcinogens, like PAHs and aromatic amines, into active species forming DNA adducts (Roberts-Thompson et al., 1993). CYP1A1 variants and cancer risk have been investigated in several studies (Bartsch et al., 2000). CYP1B1 also contributes to aromatic hydrocarbon hydroxylase activity, and interindividual variation in the expression of CYP1B1 has been observed (Shehin et al., 2000, Murray et al., 2001). Human CYP1B1 catalyzes the oxidation of polycyclic aromatic hydrocarbons to yield electrophilic intermediates capable of binding covalently to DNA (Shimada et al., 1996, Crofts et al., 1997), a step believed to be important in the initiation of carcinogenesis. Both CYP1A1 and CYP1B1 may play important roles in the bioactivation of chemically diverse tobacco smoke procarcinogens to reactive metabolites (Kim et al., 2004). Thus, the constitutive and inducible expression of CYP1A1 and CYP1B1 are considered to be important determinants of carcinogenesis, although the exact relationship between CYP1 expression and chemically induced carcinogenesis remains to be established.

One of the pathways involves dihydrodiol dehydrogenases, members of the aldo-keto reductase (AKR) superfamily that includes AKR1A1, AKR1C1, AKR1C2, AKR1C3 and AKR1C4 (Penning et al., 2000, Palackal et al., 2002, Yu et al., 2002). This pathway has been shown experimentally to produce PAH metabolites that form DNA adducts or reactive oxygen species (ROS) leading to oxidative DNA damage (Palackal et al., 2002). AKR1A1 is widely distributed among all mammalian species and can catalyze the reduction of a variety of aromatic and medium-chain aliphatic aldehydes to their corresponding alcohols (Hyndman and Flynn, 1999). AKR1C3 is particularly important in metabolizing potent trans-dihydrodiols containing more than two aromatic rings (Palackal et al., 2002). AKR1B10 is related to human aldose reductase AKR1B1; yet it remains to be determined whether AKR1B10 is induced by tobacco carcinogens or is involved in their metabolism (Penning, 2005).

Identification of genes whose expression is specifically modified by exposure to cigarette smoke will provide a better understanding of their mechanisms of action, and allow the development of sensitive and specific biomarkers for both exposure and susceptibility. Yet the mechanisms and key participants for this process are very poorly understood. The purpose of the present study was to identify molecular factors that may contribute to the pronounced increased risk of smokers for OSCC. We used high-density oligonucleotide microarray expression profiling to determine the effects of the cigarette smoke condensate (CSC) on gene expression in human keratinocytes and OSCC cells in vitro. We focused our analysis on CYPs (1A1 and 1B1) and AKRs (1A1, 1C3, and 1B10), which account for PAH metabolism, and confirmed by microarray and qRT-PCR that these relevant genes have highly induced expression in CSC-exposed cells. Extraction of potential biological interaction networks among those identified and validated genes also suggests novel mechanistic pathways to rationalize the CSC response in the target cells.

Section snippets

Materials

Cell culture reagents were obtained form the following suppliers: Dulbecco modified Eagle's medium (DMEM), trypsin (0.25%)–EDTA, penicillin–streptomycin (10 mg/ml), amphotericin B (0.25 mg/ml), HEPES buffer IM, l-glutamine (200 mM) from Cellgro (Herndorn, VA); KGM-2 medium from Cambrex (East Rutherford, NJ); fetal bovine serum (Premium) from Atlanta Biologicals (Atlanta, GA). Dimethyl sulfoxide was from Fisher Biotech (Fair Lawn, NJ), and DEPC-treated water from Ambion (Austin, TX). MTT

Effects of CSC treatment on cell viability

To address whether CSC affected the growth of normal, dysplastic, and tumor cells, the NHEK, Leuk1, Leuk2, and 101A cells were treated with different concentration of CSC (from 0 to 100 μg/ml) for time periods up to 24 h, and viability examined by MTT dye reduction assay (Fig. 1). We observed decreasing cell viability with increasing concentration of CSC, and with increasing exposure time at each dose. Based on these data, we used 25 μg/ml of CSC for microarray and quantitative real-time RT-PCR

Discussion

Tobacco use is a major independent risk factor for the development of oral and pharyngeal cancer and other malignancies of the upper aerodigestive tract (Warnakulasuriya et al., 2005, Almahmeed et al., 2004). We have documented here the effects of CSC, a complex mixture of carcinogens, on gene expression profiles in different human cell lines ranging from normal keratinocytes to pre-malignant oral dysplasia and OSCC tumor cells. We particularly focused on genes known to be involved in the

Acknowledgements

This work was supported by NIH grant DE13150 and by Philip Morris USA Inc. and Philip Morris International (W.Z.). The Leuk1 and Leuk2 cells were kind gifts from Dr. P. Sacks, New York University, New York, NY; 101A cells were kindly provided by Dr. T. Carey, University of Michigan, Ann Arbor, MI.

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