Differential toxicity of heterocyclic aromatic amines and their mixture in metabolically competent HepaRG cells
Introduction
Humans are presumed to be simultaneously and continuously exposed to various dietary food contaminants, such as heterocyclic aromatic amines (HAA), polycyclic aromatic hydrocarbons, N-nitroso compounds and lipid peroxides. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) are two of the most abundant HAA that are formed in fried or grilled meat fish and poultry. Both have been found to be genotoxic and carcinogenic at multiple tissue sites and in multiple species. However, although chemically similar, these two HAA produced, in general, different tumor profiles in rodents and only MeIQx was found to induce liver tumors (Hasegawa et al., 1993, Kato et al., 1988). Some epidemiological studies have also implicated grilled foods in the etiology of human cancer (Willett, 1995).
In humans, bioactivation of both PhIP and MeIQx occurs through N-oxidation of the exocyclic amine group to produce N-hydroxy-derivatives that are mutagenic (Fig. 1). The genotoxic N-hydroxylation pathway of these HAA involves primarily cytochrome P450 1A2 (CYP1A2) in the liver and CYP1A1 and CYP1B1 in extrahepatic tissues (Crofts et al., 1998). CYP1A2 also catalyzes the detoxication of MeIQx through 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH) formation (Turesky et al., 2002). In addition, both parent HAA and their primary oxidative metabolites can be further converted to a variety of phase II metabolites including glucuronides, sulfate esters, and acetylated products, some known as genotoxic species (Buonarati et al., 1990). The liver is recognized as the most metabolically active tissue in the biotransformation of PhIP and MeIQx in both rodents and humans (Turesky et al., 2002). However, interspecies differences have been shown in both catalytic activity and regioselectivity of liver enzymes involved in metabolism of PhIP and MeIQx (Langouet et al., 2002), potentially affecting their biological activities and making questionable the use of animal models for estimating risks of HAA for human health.
Little information exists on HAA liver toxicity in humans. PhIP genotoxicity has been investigated using human in vitro models (Winter et al., 2008, Majer et al., 2004, Knasmuller et al., 1999, Wilkening et al., 2003) but very few data are available regarding MeIQx (Knasmuller et al., 1999) despite its potential to induce liver tumors in rodents (Hasegawa et al., 1993, Kato et al., 1988). Moreover, except several studies performed in rat (Tsuda et al., 1999, Hirata et al., 2008), there is no data on liver toxicity of HAA mixtures.
In the present study, we used the human hepatoma HepaRG cell line (Gripon et al., 2002) to estimate the toxicity of HAA in human liver. Contrary to other liver cell lines (Guillouzo, 1998), HepaRG cells possess the unique properties of being metabolically competent, expressing functional phase I and phase II metabolizing enzymes at levels comparable to those usually found in primary human hepatocytes (Aninat et al., 2006, Josse et al., 2008, Turpeinen et al., 2009). In addition, a recent transcriptome analysis has shown that most of the genes modulated by phenobarbital were in common between HepaRG cells and cultured primary human hepatocytes, providing further support for a relative similarity between both cell types (Lambert et al., 2009). We investigated the cytotoxic and genotoxic effects of PhIP and MeIQx individually and in binary mixture. Cytotoxic effects were estimated by measuring cell viability, apoptosis, intracellular GSH content and expression of oxidative stress-related genes while genotoxic effects were assessed by using the comet assay. Moreover, in order to explore the underlying mechanism of interaction between both HAA, we measured the effects of PhIP and/or MeIQx on mRNA expression and activity of enzymes involved in their activation and detoxification pathways.
Section snippets
Chemicals
Cell culture tested dimethylsulfoxide (DMSO), 3-methylcholanthrene (3-MC) and neutral red (NR) were purchased from Sigma-Aldrich (St. Quentin-Fallavier, France). Williams' E medium was supplied by Eurobio (Les Ulis, France) and fetal calf serum (FCS) by Perbio (Brebières, France). PhIP and MeIQx were obtained from Toronto Research Chemicals (Toronto, Canada) and methylmethane sulfonate (MMS) from Acros Organics (Geel, Belgium). All other chemicals were of the highest quality available.
Cell culture and HAA treatments
Human
Toxicity of PhIP and MeIQx alone or in combination at various concentrations after a 24 h exposure
Both cytotoxic and genotoxic effects were evaluated. Cell viability was estimated after a 24 h exposure to various concentrations from 10 µM up to the maximum soluble concentration of each HAA. Data displayed in Fig. 2A show that MeIQx induced a 33% decrease in cell viability at the highest concentration tested (1 mM) using the ATP content assay. PhIP and [P+M] did not cause any change in cell viability and morphology at concentrations up to 150 and 100 µM, respectively. Similar results were
Discussion
In the present study, we showed that PhIP and MeIQx induced different toxic effects in metabolically competent HepaRG cells when used individually or in binary mixture. The interaction between both HAA was shown to be dependent on the endpoint measured and the mixture concentration. PhIP and MeIQx in binary combination were shown to exert synergistic effects on apoptosis and inhibitory effects on DNA damage. We propose that one of the mechanisms underlying this inhibitory effect is a modulation
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by grants from ANR (contract 06SEST17) and the Ligue 35 contre le Cancer. We thank Dr B. Fromenty for helpful comments and Dr David Steen for critical reading of the manuscript. We acknowledge the ImPACcell platform (Rennes) for help with some experiments.
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2019, Toxicology in VitroCitation Excerpt :Using these data, we calculated the benchmark dose (BMD), the no observed adverse effect level (NOAEL), and the lowest observed adverse effect level (LOAEL; Table 3), which were similar or slightly higher for MeIQx-M1 when compared to the values for MeIQx. The cytotoxicity data for the parent compound is consistent with data reported for MeIQx in HepaRG cells (Dumont et al., 2010), human liver cancer cells (Hep G2; Pezdirc et al., 2013), and non-malignantly transformed human colon epithelial cells (HCECs; Zhang et al., 2017). The food carcinogen MeIQx can be metabolized to MeIQx-M1 by the microbial community in the human intestine.
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Present address: Inserm UMR 744 – Université Lille Nord de France – Institut Pasteur de Lille, BP 245, 1, rue du professeur Calmette, F-59019 Lille cedex, France.