Academic journal article Environmental Health Perspectives

Synergistic and Antagonistic Mutation Responses of Human MCL-5 Cells to Mixtures of Benzo[a]pyrene and 2-Amino-1-Methyl-6-Phenylimidazo[4,5-B]pyridine: Dose-Related Variation in the Joint Effects of Common Dietary Carcinogens

Academic journal article Environmental Health Perspectives

Synergistic and Antagonistic Mutation Responses of Human MCL-5 Cells to Mixtures of Benzo[a]pyrene and 2-Amino-1-Methyl-6-Phenylimidazo[4,5-B]pyridine: Dose-Related Variation in the Joint Effects of Common Dietary Carcinogens

Article excerpt

Introduction

Consumption of red meat is positively correlated with some human cancers, and cooking meat produces heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs) (Sinha et al. 2005). The HCA 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) is bioavailable to humans who consume cooked meat (ingesting 0.1-15 pg PhIP per day) (Felton et al. 2002). PhIP is a rodent carcinogen (Sugimura 1997), inducing cancer in the prostate, colon, and mammary gland of rats (Crofts et al. 1997; Ito et al. 1991). The activation of PhIP to DNA-damaging species occurs via A-hydroxylation catalyzed by cytochromes P450 (CYP) 1A1 and 1A2 (Crofts et al. 1997; Zhao et al. 1994), thereby forming promutagenic adducts at C8 of guanine and resulting in GC:TA transversions and deletions (Boyce et al. 2014; Lynch et al. 1998). Benzo[a]pyrene (BaP) is a carcinogen that is generated by incomplete combustion of organic substances, leading to the contamination of numerous foodstuffs (Lijinski and Shubik 1964). BaP is metabolized by enzymes from the CYP1A family to epoxide derivatives that form DNA adducts and result in mutation and tumors [International Agency for Research on Cancer (IARC) 2010]. Through the consumption of contaminated food, the average human daily exposure to BaP is estimated to be 1-500 ng (IARC 2010). Experimental studies suggest a positive link between exposure to BaP and cancer in animals and in humans (Sinha et al. 2005).

Published assessment of genotoxic carcinogens, particularly dietary carcinogens, in mixtures is limited. Current approaches to mixtures risk assessment include whole-mixture- and component-based methods [Agency for Toxic Substances and Disease Registry (ATSDR) 2004; U.S. Environmental Protection Agency (EPA) 2000]; wholemixture approaches are preferred because they account for unidentified components and interactions between chemicals. However, the complexity and variability of mixtures makes this approach difficult, and component-based methods such as dose or response additivity are often used (Boobis et al. 2011; COT 2002; Lutz et al. 2005).

Synergistic effects from interactions between PAHs on DNA adduct levels have been reported (Staal et al. 2007; Tarantini et al. 2009, 2011), and prolonged activation of DNA damage signaling suggestive of persistent DNA damage by mixtures of PAHs has been observed (Jarvis et al. 2013; Mattsson et al. 2009; Niziolek-Kierecka et al. 2012), suggesting that the present risk assessment strategies may underestimate risk. Contrasting studies showing antagonistic or additive effects from mixtures of PAHs (Courter et al. 2008; Mahadevan et al. 2007; Marston et al. 2001; Staal et al. 2008; White 2002) or of heterocyclic aromatic amines (Dumont et al. 2010) have been published. To our knowledge, there is no information regarding mixtures of PAHs with HCAs at concentrations that are relevant to human exposure [micromolar to subnanomolar concentrations, with the highest concentrations in gastrointestinal (GI) microenvironments]. Such information is important for risk assessment of food-borne chemical carcinogens. Thus, our aim was to determine the mutagenic response to mixtures of BaP and PhIP at concentrations relevant to human exposure.

Methods

Materials. RPMI-1640 medium (with phenol red, without L-glutamine and histidine), heat-inactivated horse serum (HIHS), L-glutamine, penicillin/streptomycin, and hygromycin B were obtained from Life Technologies (Paisley, UK). All other chemicals were purchased from Sigma-Aldrich (Poole, UK).

Cell culture. MCL-5 is a human B lymphoblastoid cell line that constitutively expresses CYP1A1 (Crespi et al. 1991) and stably expresses transfected CYP3A4, CYP2E1, CYP1A2, CYP2A6, and microsomal epoxide hydrolase (Crespi et al. 1991). Thus, this cell line can activate BaP and PhIP to DNA-damaging species without the need for exogenous activation systems (S9 fraction). Moreover, these cells are relevant to the human exposure route (the diet) because CYP1A1 is expressed in the GI tract (Paine et al. …

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