Laurence Ribonnet

Edwige Van Der Heiden

Ingrid Nobels

Agnès Chaumont

Anne-Sophie Remacle

Sarah De Saeger

Yves-Jacques Schneider

Marie-Louise Scippo

Ronny Blust

Luc Pussemier

A.-S Remacle

S De 4 Saeger

Y.-J Schneider

M.-L Scippo

Y Larondelle
email: yvan.larondelle@uclouvain.be

Potential of an in vitro toolbox combined with exposure data as a first step for the risk assessment of dietary chemical contaminants

Keywords: Food contaminants, intestine, stress genes, cytotoxicity, CYP, AhR, 16 estrogen, Caco-2 cells, ochratoxin A, deoxynivalenol, imazalil, benomyl 17 18

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

In vitro risk assessment of dietary contaminants has become a priority in human food safety. In the present paper, we propose an in vitro approach associating different complementary tools in an original toolbox aiming at improving the assessment of the toxicological impact of dietary contaminants at realistic human exposure levels, with a special focus on the intestinal compartment. The system is based on the use of four complementary cellular tools, namely stress gene induction in transgenic strains of Escherichia coli, modulation of the activity of key biotransformation enzymes (cytochrome P-450 (CYP) 1A1 and 3A4) in a human intestinal cell line, and activation of aryl hydrocarbon receptor (AhR) and estrogenic receptor (ER)-dependent genes in agonistic and antagonistic assays with luciferase reporter cells. It was applied to four chosen model molecules: ochratoxin A (OTA) and deoxynivalenol (DON), two common food-borne mycotoxins, and imazalil (IMA) and benomyl (BEN), two fungicides widely recovered in foodstuffs. All these assays were performed at or around a realistic intestinal concentration, determined through a deterministic approach based on the calculation of a theoretical maximum daily intake (TMDI). Using the four model molecules, we clearly highlighted that induction of CYP1A1 activity and inhibition of CYP3A4 activity occurred in Caco-2 cells at a realistic intestinal concentration of IMA. Furthermore, some bacterial stress genes were induced in a range of realistic concentrations, following exposure to DON and IMA. In addition, BEN clearly provoked an ER agonistic activity in a human estrogen sensitive reporter cell line. All these results are in accordance with literature, suggesting that our in vitro toolbox constitutes an interesting approach in order to obtain a first 'fingerprint' of dietary contaminants at realistic human exposure for further risk assessment.

Introduction 19

Nowadays, the chemical safety of food is of major concern since our diet contains a wide 20 range of undesired substances. These food contaminants include for example pesticides, 21 heavy metals and persistent organic pollutants (POPs), which are widespread in the 22 environment and readily enter the food chain. In addition, food contamination may also 23 occur during food processing and cooking (e.g. benzo[a]pyrene (B[a]P), acrylamide) or 24 through the release of chemicals from consumer products, such as baby toys, and 25 packaging (e.g. bisphenol A, phthalates). Furthermore, mycotoxins, secondary 26 metabolites produced by fungal species in high moisture conditions occurring in field or 27 during storage of cereals or other agricultural goods, are recovered in derived foodstuffs. [START_REF] Murray | Altered CYP expression and function in response to dietary factors: 29 potential roles in disease pathogenesis[END_REF] Due to the widespread of dietary contamination by mycotoxins, these substances are also 29 considered as an important hazard for human and animal health. Given the fact that the effects of food contaminants on human health are not always 1 thoroughly known, more investigations about their toxicity and more risk assessment 2 studies should be performed. The process of risk assessment generally integrates 3 information from both exposure assessment and hazard characterization. In order to be 4 relevant, the hazard must be characterized experimentally at realistic doses. 5 6 The dietary contaminants present in the gastro-intestinal tract should gain access to the 7 intestinal mucosa. There, the enterocytes, can be considered on one hand as the first 8 biological barrier encountered by the xenobiotics and, on the other hand, as a human 9 tissue submitted to high concentrations of these compounds. The large microbial 10 population present in the intestine and especially in the colon (e.g. Bacteroides, 11

Lactobacillus, Bifidobacterium, Escherichia) should also be submitted to most of the 12 dietary contaminants and could be affected. These microbes contribute to the normal 13 digestive function and, in some cases, to the transformation of dietary compounds or 14 drugs into often more readily absorbed and more or less toxic forms [START_REF] Sousa | The gastrointestinal microbiota as a site for the biotransformation of drugs[END_REF]15 Cani and Delzenne, 2009; [START_REF] Laparra | Interactions of gut microbiota with functional food 16 components and nutraceuticals[END_REF]. Toxicity on bacteria may thus have 16 an impact on the bioavailability and effects of ingested chemical compounds. 17 18 In order to estimate realistic concentrations at the intestinal level, one has to take into 19 account the dilution of the contaminant in the alimentary bolus. In addition, the question 20 of bioaccessibility should ideally be considered as well. For assessing realistic 21 concentrations at the hepatic level, absorption and intestinal first-pass metabolism of 22 dietary compounds are also of crucial importance. Recommendations for revising current 23 risk assessment models are often stated, with the objective to reduce animal testing, to 24 improve relevance of toxicodynamic data for human health and to focus on realistic 25 exposure and toxicokinetics. Consistent with these recommendations, a package of high 26 throughput in vitro experimental tools would be necessary in order to quickly assess some 27 important toxicological effects due to the dietary contaminants at realistic exposure 28 levels. In this paper, we therefore elaborated a model toolbox comprising a set of selected 29 complementary relevant in vitro assays in bacterial and eukaryotic cell types, allowing 30 taking a first 'fingerprint' of tested toxicants in realistic chronic exposure conditions, with 1 a special focus on the intestinal compartment. 2 3 In a first in vitro assay, we used reporter genes in genetically modified Escherichia coli 4 strains that respond to different types of stress [START_REF] Dardenne | Dose-response relationships 1 and statistical performance of a battery of bacterial gene profiling assays[END_REF]Dardenne et al., 5 2008), in order to assess acute toxicity and give an insight into the toxic mode of action 6 of dietary compounds. 7

8

A second in vitro assay focused on the cytochrome P-450 (CYP) biotransformation 9 enzymes present in enterocytes. These cells are indeed the first major site for metabolism 10 prior to systemic uptake of orally ingested xenobiotics and the major catalysts of first-11 pass metabolism in enterocytes are the CYP biotransformation enzymes, mainly the 12 CYP3A4 isoform and to a lesser extent, the CYP1A1 isoform (Prueksaritanont et al., 13 1996; [START_REF] Lown | Grapefruit juice increases felodipine 39 oral availability in humans by decreasing intestinal CYP3A protein expression[END_REF][START_REF] Lampen | 10 Catalytic activities, protein-and mRNA-expression of cytochrome P450 isoenzymes in 11 intestinal cell lines[END_REF][START_REF] Paine | Cytochrome P-450 1A1 18 expression in human small bowel: interindividual variation and inhibition by 19 ketoconazole[END_REF]Zhang et al., 1999;14 Paine et al., 2006). However, a great inter-individual variability in the expression of these 15 CYP isoforms may be observed in the human intestine [START_REF] Paine | Characterization of interintestinal and 15 intraintestinal variations in human CYP3A-dependent metabolism[END_REF]Paine et al., 16 1999; [START_REF] Lindell | 32 Variable expression of CYP and Pgp genes in the human small intestine[END_REF][START_REF] Paine | The human intestinal cytochrome P450 "pie[END_REF]. It has been established in enterocytes as 17 well as in the Caco-2 intestinal cell line, that CYP1A1 is likely to be induced rather than 18 constitutive [START_REF] Boulenc | Regulation of cytochrome P4501A1 gene expression in a human intestinal cell 21 line, Caco-2[END_REF][START_REF] Rosenberg | Regulation of cytochrome P450 in cultured human 1 colonic cells[END_REF][START_REF] Buchthal | Induction of cytochrome P4501A by smoking or omeprazole in comparison with 29 UDP-glucuronosyltransferase in biopsies of human duodenal mucosa[END_REF]19 Prueksaritanont et al., 1996), with diet clearly taking part in this induction (Fontana et al., 20 1999). Although most CYP oxidations are involved in detoxification pathways, CYP1A1 21 also catalyzes the production of reactive cytotoxic metabolites and the activation of pro-22 carcinogens, such as PAHs, like B[a]P, into carcinogenic DNA-binding species (Schwarz 23 et al., 2001;[START_REF] Shimada | Metabolic 42 activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes 43 P450 1A1 and P450 1B1 allelic variants and other human cytochromes P450 in 44 Salmonella typhimurium NM2009[END_REF]. Furthermore, some endogenous compounds are 24 metabolized by CYP1A1. It has been shown, for example, that CYP1A1 is implicated in 25 all-trans-retinoic acid (atRA) metabolism [START_REF] Lampen | Metabolism of vitamin A and its 13 active metabolite all-trans-retinoic acid in small intestinal enterocytes[END_REF][START_REF] Marill | Identification of human 6 cytochrome P450s involved in the formation of all-trans-retinoic acid principal 7 metabolites[END_REF][START_REF] Hou | Possible inhibitory mechanism of Curcuma drugs on CYP3A4 27 in 1α,25 dihydroxyvitamin D3 treated Caco-2 cells[END_REF] McSorley and Daly, 2000) and 17β-estradiol (E2) catabolism to weak estrogen (Lee et 27 al., 2003;[START_REF] Tsuchiya | Cytochrome P450-mediated 21 metabolism of estrogens and its regulation in human[END_REF]. Therefore, dietary regulation of CYP enzymes may 28 contribute to pathogenic mechanisms [START_REF] Murray | Altered CYP expression and function in response to dietary factors: 29 potential roles in disease pathogenesis[END_REF]. It is also well known that CYP3A4 29 is implicated in the metabolic processing of a large range of (pro-)drugs. The co-30 occurrence at the level of the intestinal epithelium of other compounds modulating the 31 CYP3A4 content, such as grapefruit juice, may be detrimental [START_REF] Guengerich | Role of cytochrome P450 enzymes in drug-drug interactions[END_REF]Lown 1 et al., 1997;[START_REF] Dresser | Pharmacokinetic-16 pharmacodynamic consequences and clinical relevance of cytochrome P450 3A4 17 inhibition[END_REF]. 2 3 The third in vitro assay focused on the aryl hydrocarbon nuclear receptor (AhR), a ligand-4 activated transcription factor of the Per-Arnt-Sim (PAS) superfamily, which is known to 5 regulate CYP1A1 transcription. It is located in its free state in the cytosol, as a complex 6 with the chaperone proteins Hsp90, p23 and immunophilins. Ligand binding to AhR 7 triggers its nuclear translocation and fixation to the aryl receptor nuclear translocator 8 (Arnt), resulting in binding of the complex to the dioxin-responsive elements (DREs) on 9 target genes [START_REF] Denison | Xenobiotic-inducible transcription of 9 cytochrome P450 genes[END_REF][START_REF] Delescluse | Is CYP1A1 induction 4 always related to AHR signaling pathway?[END_REF][START_REF] Ma | Induction of CYP1A1. The AhR/DRE Paradigm: transcription, Receptor 42 Regulation, and Expanding Biological Roles[END_REF]; Mimura 10 and Fujii-Kuriyama, 2003;[START_REF] Kawajiri | Cytochrome P450 gene regulation and 37 physiological functions mediated by the aryl hydrocarbon receptor[END_REF]. Several very toxic 11 dietary contaminants, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have been 12

reported to bind to AhR and to trigger downstream target gene transcription (Denison and 13 Nagy, 2003;[START_REF] Ashida | An update on the dietary ligands of the 6 AhR[END_REF]. It is now well established that, among other genes, the 14 transcription of CYP1A1, CYP1A2, UDP-glucuronosyltransferase (UGT) and 15 glutathione-S-transferase (GST) is mediated by the AhR. 16

17

A fourth and last in vitro assay focused on the estrogen receptor (ER), another ligand-18 activated transcription factor, belonging to the superfamily of steroid hormone nuclear 19 receptors. ER needs ligand binding in the nucleus to trigger conformational changes and 20 to subsequently homodimerize, bind to an estrogen response element (ERE) and activate 21 transcription of its target genes [START_REF] Nilsson | Mechanisms of estrogen 41 action[END_REF]. Furthermore, a cross-talk between 22 AhR and ER pathways has been established on the basis of anti-estrogenic effects of AhR 23 ligands, such as TCDD [START_REF] Nilsson | Mechanisms of estrogen 41 action[END_REF][START_REF] Ohtake | Intrinsic AhR function 1 underlies cross-talk of dioxins with sex hormone signalings[END_REF][START_REF] Ohtake | AhR acts as an E3 ubiquitin ligase to 4 modulate steroid receptor functions[END_REF]. 24 Since (i) the toxic effects of TCDD are known to be in part mediated by the AhR 25 [START_REF] Mimura | Functional role of AhR in the expression of 22 toxic effects by TCDD[END_REF] OTA is produced by fungal species from the genera Penicillium and Aspergillus and 13 occurs in cereals, but also in nuts, pulses, beans, spices, dried fruits, grape(s) (juice), 14 wine, raisins, beer, cocoa products or coffee [START_REF] Efsa | Opinion of the Scientific Panel on Contaminants in the Food Chain on a 28 request from the commission related to ochratoxin A in food[END_REF]. Furthermore, the carry-over 15 to animal offal might be important, especially in the kidney for which however the human 16 consumption is relatively limited [START_REF] Efsa | Opinion of the scientific panel on contaminants in the food chain on a 25 request from the commission related to ochratoxin A as undesirable substance in animal 26 feed[END_REF]. It is mainly nephrotoxic in mammalian 17 species, but also immunotoxic, neurotoxic and teratogenic [START_REF] Efsa | Opinion of the Scientific Panel on Contaminants in the Food Chain on a 28 request from the commission related to ochratoxin A in food[END_REF]. The occurrence 18 of OTA in food and human blood samples has been associated with human endemic 19 nephropathy and tumours of the upper urinary tract, in different countries (Maaroufi et 20 al., 1995;[START_REF] Radic | Ochratoxin A in human sera in 39 the area with endemic nephropathy in Croatia[END_REF][START_REF] Stoev | The role of ochratoxin A as a possible cause of Balkan endemic 8 nephropathy and its risk evaluation[END_REF][START_REF] Vrabcheva | Co-occurrence of 42 ochratoxin A and citrinin in cereals from Bulgarian villages with a history of Balkan 43 endemic nephropathy[END_REF]. However, due to 21 inadequate evidence in humans, OTA has been classified as possible human carcinogen 22 (group 2B) by the International Agency for Research on Cancer (IARC, 1993). One 23 debated hypothesis for the carcinogenicity of OTA in kidney implies covalent DNA 24 adduct formation after bioactivation by biotransformation enzymes (for a review, see 25

Pfohl-Leszkowicz and Manderville (2007)). DON is a trichothecene also called 26 vomitoxin, which is produced by strains of Fusarium. It is often reported in wheat, 27 barley, maize, oat, rice, rye, sorghum and triticale but also in processed food products 28 such as wheat flour, bread, beer or noodles. In humans, primary targets of DON are the 29 immune function [START_REF] Pestka | Deoxynivalenol: toxicology and potential 23 effects on humans[END_REF] and the mucosa of the gastro-intestinal 30 tract, which may be damaged, often resulting in gastroenteritis symptoms after acute 31 exposure. Furthermore, genotoxic potential of DON and association with esophageal 1 cancer have been suggested [START_REF] Rotter | Toxicology of deoxynivalenol 3 (vomitoxin)[END_REF][START_REF] Bony | Genotoxicity assessment of 17 deoxynivalenol in the Caco-2 cell line model using the Comet assay[END_REF]. 2 3 Pesticides may also be daily ingested, mainly through fruit and vegetable consumption. 4

IMA is a post-harvest imidazole fungicide mainly applied on citrus fruits. This chemical 5 is also used as broad-spectrum veterinary topical antimycotic agent under the name of 6 enilconazole [START_REF] Vanden Bossche | Antifungal agents of use in 34 animal health -chemical, biochemical and pharmacological aspects[END_REF]. IMA belongs to the group of the 10 7 pesticides the most frequently reported in fruits and vegetables by European Member 8 States [START_REF] Poulsen | Results from the monitoring of pesticide 31 residues in fruit and vegetables on the Danish market, 2000-01[END_REF]. The mode of action of IMA is based on the 9 inhibition of sterol demethylation, which affects membrane integrity in fungal cells 10 (Ghannoum and Rice, 1999; EFSA, 2007). Little information is available regarding the 11 toxic effects of IMA on human health. However, it seems that the liver is the primary 12 target for IMA in experimental animals (EFSA, 2007) even if IMA has also been shown 13 to act on cytochrome P450 (CYP) activity in the intestinal Caco-2 cell line (Sergent et al., 14 2009). IMA is an inhibitor of the activity of human aromatase (CYP19), an enzyme that 15 catalyzes the conversion of androgens to estrogens; it appears to be as potent as the 16 aromatase inhibiting antiestrogen drugs used to treat breast cancer (Vinggaard et al., 17 2000;[START_REF] Mckinlay | Calculating human 13 exposure to endocrine disrupting pesticides via agricultural and non-agricultural exposure 14 routes[END_REF]. BEN is another widely used fungicide, belonging to the 18 imidazole family. Residues of carbendazim and n-butylisocyanate, two degradation 19 products of BEN in soils and aqueous solutions, are commonly reported in fruits and 20 vegetables. Hepatotoxicity, teratogenicity, male reproductive toxicity and carcinogenicity 21 have been reported following exposure to BEN [START_REF] Igbedioh | Effect of benomyl toxicity on some 32 constituents of albino rats[END_REF]McCarroll 22 et al., 2002;[START_REF] Hewitt | Potential teratogenic effects of 24 benomyl in rat embryos cultured in vitro[END_REF]. 23

24

Practically, the proposed in vitro toolbox to be evaluated has been applied through the 25 following procedure to OTA, DON, IMA and BEN toxicity assessment at dietary realistic 26 exposure levels. At first, realistic concentrations of these contaminants occurring in the 27 gastro-intestinal tract have been calculated and cytotoxicity assessed. Secondly, the 28 effects of OTA, DON, IMA and BEN on stress genes from an intestinal bacterium and on 29 the activity of key intestinal biotransformation enzymes (CYP1A1 and CYP3A4) have 30 been investigated at realistic experimental concentrations. Since induction of CYP1A1 31 activity is mainly mediated by the AhR pathway and considering that some toxic effects 1 may be mediated by this receptor (e.g. TCDD), we also investigated dose-dependent AhR 2 agonistic and antagonistic activities in luciferase reporter cells. Furthermore, still using 3 luciferase reporter cells, we assessed the dose-dependent agonistic and antagonistic 4 activities on the human estrogenic receptor (hER), in order to detect potential estrogenic 5 disruption by dietary contaminants mimicking estrogens. Results were systematically 6 compared with the existing literature in the discussion part, in order to validate our 7 toolbox and to propose it as an efficient tool for the screening of toxicological properties 8 of less studied dietary contaminants. 9 10

Materials and methods

11

Determination of realistic intestinal concentrations 12

In order to work with realistic intestinal concentrations, we have considered that the daily 13 intake of a contaminant, which is the sum of the quantity ingested through the 14 consumption of the different foodstuffs containing the substance, is diluted in the 15 alimentary bolus. This quantity ingested is obtained through the multiplication of 16 consumption and contamination data for each foodstuff susceptible to contain the 17 substance. 18 19 In the present work, we have used European consumption data from the GEMS/FOOD 20 Regional Diets (WHO, 2003). These diets, based on food balance sheet data compiled by 21 the FAO, give regional per capita average consumption [g/person/day] of raw and semi-22 processed agricultural commodities. We have also used the European maximum (residue) 23 main consumed foodstuffs as contamination data. We have then applied the concept of 28 theoretical maximum daily intake (TMDI), which assumes that a dietary chemical occurs 29 in food at the M(R)L legally authorized and that the average daily consumption of 30 foodstuffs per person corresponds to the published values from GEMS/FOOD Regional 31 Diets. The daily intake estimated through that approach is representative of a long-term 1 exposure (WHO, 1997) (Formula 1). 2

TMDI [ng / person /day] = (Q E i i ∑ ⋅ M(R)L i )
3 -Q Ei : consumed quantity of foodstuff i in the European diet from 4 GEMS/FOOD Regional Diets [g/person/day] 5 -M(R)L i : Maximum (residue) limit of contaminant in foodstuff i [µg/kg] 6

Formula 1: Theoretical maximum daily intake (TMDI) of a dietary chemical 7 contaminant 8

Dilution hypothesis of the calculated daily intake is based on the assumption that, in the 9 worst case, the dilution occurs in 1L of gastro-intestinal secretions per meal, with one 10 meal per day and gives a maximal realistic intestinal concentration (C) (Formula 2). 11 Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% heat-1 inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific Inc., Waltham, MA), 0.1 2 mM non-essential amino acids (Lonza, Walkersville, MD) and 2mM L-glutamine 3 (Lonza). Cells were incubated at 37°C in a humidified chamber with 5% CO 2 , with 4 medium change every 2 days. 5

C [ng / ml] = TMDI V ⋅ n⋅

Stable reporter cell lines 6

Rat hepatoma H4IIE (DR-CALUX ® ) (Biodetection System, NL), and human hepatoma 7

HepG2-Luc were used as dioxin sensitive reporter cells containing AhR, and human 8 breast cancer MMV-Luc cells (originating from MCF-7 cells) as estrogen sensitive 9 reporter cells containing mainly ER-α. These three cell lines were the result of a stable 10 transformation with an inducible firefly luciferase reporter gene [START_REF] Aarts | Ah receptor-mediated luciferase expression: a tool for monitoring dioxin-like 4 toxicity[END_REF]11 Willemsen et al., 2004;[START_REF] Van Der Heiden | Food flavonoid aryl hydrocarbon 31 receptor-mediated agonistic/antagonistic/synergic activities in human and rat reporter 32 gene assays[END_REF]. H4IIE (DR-CALUX ® ), HepG2-Luc 12 and MMV-Luc cells were grown respectively in Minimum Essential Medium (MEM) 13 alpha, MEM and DMEM (all from Thermo Fisher Scientific Inc.), supplemented with 14 10% heat-inactivated FBS (AE scientific, Marcq, BE). DMEM was further supplemented 15 with 100 U/ml of penicillin G, 100 µg/ml of streptomycin (Thermo Fisher Scientific Inc.) 16 and 1 µg/ml of bovine pancreas insulin (Sigma-Aldrich). Cells were grown at 37°C in 5% 17 SfiA strain is part of the SOS chromotest derived from E. coli GC4436 with a deletion in 28 the lac operon carrying a sfiA::lacZ fusion, so that responses to DNA damaging agents 29 can be measured [START_REF] Quillardet | The SOS Chromotest, a colorimetric bacterial 37 assay for genotoxins-procedures[END_REF]. 30 metabolic activity as a way to assess the effect of dietary contaminants on Caco-2 cell 8 proliferation. Cells were seeded in 96-well plates at a density of 1.10 4 cells/cm 2 and were 9 incubated during 48h, 24h after seeding, with OTA, DON, IMA or BEN diluted in 0.5% 10 FBS-enriched culture medium at a final concentration of respectively 1 ng/ml, 250 ng/ml, 11 2500 ng/ml and 250 ng/ml. Control vehicles corresponding to 0.0002% (v/v) DMSO for 12 OTA, 0.05% (v/v) ethanol for DON, and 0.01% (v/v) DMSO for IMA and BEN as well 13 as negative control, consisting in 0.5% FBS-enriched culture medium alone, were also 14 tested. The MTS assay was carried out as described by the manufacturer with formazan 15 production detected at 500 nm (reference wavelength: 690 nm) with a Spectracount 16 spectrophotometer (Packard, Warrenville, IL). 17 The assay routinely used to test the effect of the selected chemicals on the proliferation of 2 stable reporter cells was the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 3 bromide (MTT) colorimetric assay, which also permits to measure cellular metabolic 4 activity. H4IIE DR-CALUX ® , HepG2-Luc and MMV-Luc cells were seeded in 96-well 5 plates at a density of respectively 94000, 31000 and 62500 cells/cm 2 , in phenol red-free 6 DMEM supplemented with 10% heat-inactivated FBS for both H4IIE and HepG2-Luc 7 cell lines and with 10% charcoal-dextran-treated FBS, aiming at eliminating potential 8 hormones that could interfere during the assay, for the MMV-Luc cell line. MMV-Luc 9 cells were put in contact with the charcoal-dextran-treated FBS-enriched culture medium 10 24h before seeding. After overnight incubation, cells were exposed during 24h to serial 11 dilutions of OTA, DON, IMA or BEN, to culture medium alone, or to culture medium 12 containing 0.4% (v/v) of the vehicle for all tested concentrations (ethanol for OTA and 13 IMA, water for DON and DMSO for BEN). Final concentrations used for the three cell 14 lines were as follows. OTA: 4, 40, 400, 4000 and 40000 ng/ml. DON: 3.2, 16, 80, 400, 15 and 2000 ng/ml. BEN: 64, 320, 1600, 8000, and 40000 ng/ml. For IMA, two different 16 ranges of concentration were used: 312, 625, 1250, 2500, 5000, 10000, 20000, and 40000 17 ng/ml with both the H4IIE DR-CALUX ® and HepG2-Luc cell lines and 16, 125, 250, 18 500, and 4000 ng/ml with the MMV-Luc cell line. After medium removal, cells were 19 washed with 100 µl phosphate buffered saline (PBS) (Oxoïd, Hampshire, UK) and 20 incubated at 37°C with 100 µl yellow MTT reagent (0.5 mg/ml) (Sigma-Aldrich) during 21 3h. The colored insoluble formazan reagent produced by metabolically active 22 mitochondria was made soluble with DMSO and detected at 540 nm (reference 23 wavelength: 620 nm) with a Multiskan Ascent spectrophotometer (Thermo Fisher 24 Scientific Inc.) after plate agitation during 15 min. 25 26

Bacterial stress gene profiling assay 27

The entire procedure for the bacterial stress gene profiling assay is described in Dardenne 28 et al. (2007). The different E. coli strains were exposed for 90 min in the exponential 29 growth phase to a ½ serial dilution of OTA (1.6 -100 µg/ml), DON (0.031 -2 µg/ml), 30 IMA (0.4 -13 µg/ml) or BEN (0.78 -50 µg/ml) or to vehicle alone, corresponding to 1% 31 Activity of the promoter is calculated taking into account the growth inhibition of the 5 used strain. The results are presented as fold inductions at a given dose i relative to the 6 control values and were calculated through a set of formulas (Formula 3 and 4). 7

Activity i = 0,19 × OD 420nm PE -OD 420nm SE (OD 600nm PD × 90min) + ((OD 600nm PE -OD 600nm SE ) × 90min 2 )           8
Formula 3: Activity at a given dose i. PE, Post exposure; SE, start exposure (=post 9 dose); PD, pre-dose [START_REF] Dardenne | Dose-response relationships 1 and statistical performance of a battery of bacterial gene profiling assays[END_REF]. 10

Modulation of intestinal cytochrome P-450 biotransformation enzyme activity 13 7-Ethoxyresorufin-O-deethylase (EROD) activity assay 14

The measurement of the EROD activity allowed us to assess the effects of the tested 15 compounds on the catalytic activity of CYP1A1 in enterocytes. Caco-2 cells were 16 inoculated at 3.10 4 cells/cm 2 and cultivated until 16 days post-confluence (time of 17 maximal inducibility by B[a]P in our experimental conditions) in 48-well plates with 18 culture medium changes 3 times per week. Cells were then incubated for 6 or 24h with 19 OTA, DON, IMA or BEN diluted in FBS-free culture medium at a final concentration of 20 respectively 1 ng/ml, 250 ng/ml, 2500 ng/ml and 250 ng/ml. Control vehicles 21 corresponding to 0.0002% (v/v) DMSO for OTA, 0.05% (v/v) ethanol for DON, and 22 0.01% (v/v) DMSO for IMA and BEN were also tested. Positive controls consisted in 23 B[a]P (250 ng/ml) or TCDD (0.3 ng/ml) and negative control consisted in FBS-free 24 culture medium alone (untreated cells). Cells were rinsed with Hank's Balanced Salt 25 Solution (HBSS) (Lonza) and incubated for 30 minutes at 37°C with 300 µl of 7-26 ethoxyresorufin (5 µM) (Sigma-Aldrich) in phenol red-free DMEM (Lonza), in the 27 absence of contaminants or vehicles. Fluorescence due to the conversion of the substrate 28 to resorufin was measured, in the supernatants, using a Fluoroskan Ascent FL fluorimeter 1 (Thermo Fisher Scientific Inc.) with excitation and emission wavelengths of 530 and 585 2 nm, respectively. The EROD activity was normalized to the cell protein content 3 determined with the bicinchoninic acid protein (BCA) assay kit (Sigma-Aldrich) after 4 cell lysis with 1% (w/v) sodium deoxycholate (DOC) in water, at pH 11.3. 5

CYP3A4 activity assay 6

The CYP3A4 activity assay was used to estimate the inhibitory effect of the tested 7 compounds on the catalytic activity of CYP3A4 in enterocytes. Caco-2 cells were seeded 8 and DMSO for BEN). In assays to measure AhR antagonistic activities, the cells were 2 exposed during 24h simultaneously to a constant concentration of the AhR inducer 3 TCDD (0.1 or 3 ng/ml, for H4IIE DR-CALUX ® and HepG2-Luc, respectively) and to 4 increasing concentrations of OTA, DON, IMA or BEN (the same range as for the 5 agonistic assay). The inducer concentration was chosen to obtain a maximal luciferase 6 activity. Solvent concentration was 0.8% (v/v) in the culture medium for all tested 7 concentrations. In both assays (agonistic and antagonistic activities), the medium was 8 removed after incubation with the target analytes and the cells were lysed with 50 µl of a 9 solution containing 25 mM Tris, 2 mM 1,4-dithio-DL-thréitol (DTT), 2 mM trans-1,2-10 Diaminocyclohexane-N,N,N′,N′-tetraacetic acid monohydrate (DCTA), 10% (v/v) 11 glycerol and 1% (v/v) Triton X-100 (all from Sigma-Aldrich). An ORION II 12 luminometer equipped of an automatic injector (Berthold Detection System, Pforzheim, 13 DE) was used to add 100 µl of a solution containing both luciferin (0.15 mg/ml) 14 (Synchem OHG, Felsberg/Altenburg, DE) and ATP (0.3 mg/l) (Roche Diagnostics), and 15 to directly measure the emission of light resulting from the luciferase activity and 16 reported as Relative Light Units (RLUs). 17 18 In assays to measure AhR agonistic activities, when a dose-response curve was obtained, 19 the median effective concentration (EC 50 ) was calculated from a non linear equation 20 (Formula 5). The relative potency (REP) of a chemical was calculated as the ratio 21 between the EC 50 of the reference ligand and that of the chemical. Antagonistic activity 22 was expressed as a relative response, i.e. in percentage of the maximal cell response to 23 TCDD, corresponding to 100%. 24

2 1 0 1 A A x A y         + = 25

Human estrogen receptor (hER) agonistic/antagonistic activities 1

In order to test agonistic/antagonistic hER activities of dietary contaminants, MMV-Luc 2 cells were used. These cells were put in contact with the charcoal-dextran-treated FBS-3 enriched culture medium 24h before seeding, as explained previously for the cytotoxicity 4 assays. MMV-Luc cells were then seeded in 96-well plates at a density of 22000 5 cells/cm 2 and incubated overnight. In assays to measure hER agonistic activities, MMV-6 Luc cells were exposed during 24h to serial dilutions of OTA, DON, IMA or BEN, at the 7 same final concentrations as for the cytotoxicity assay, or to the culture medium 8 containing 0.4% (v/v) of the vehicle for all tested concentrations (ethanol for OTA and 9 IMA, water for DON and DMSO for BEN). In assays to measure hER antagonistic 10 activities, the cells were exposed during 24h simultaneously to a constant concentration 11 of the hER inducer E2 (1.4 ng/ml) (Sigma-Aldrich) and to increasing concentrations of 12 OTA, DON, IMA or BEN (the same range as for the agonistic assay), or to 60.7 ng/ml 13 (100 nM) of fulvestrant (Sigma-Aldrich) as a positive antagonistic control (Osborne et 14 al., 2004). The inducer concentration was chosen to obtain a maximal luciferase activity, 15 which was set at 100%. Solvent concentration was 0.8% (v/v) in the culture medium for 16 all tested concentrations. 17

The emission of light resulting from the luciferase activity was measured and data were 18 analyzed and expressed as previously described for the AhR assay. which means 1 ng/ml (2.46 nM) for OTA, 250 ng/ml (840 nM) for DON, 2500 ng/ml 10 (8400 nM) for IMA and 250 ng/ml (860 nM) for BEN. Details of calculation of the 11 TMDI and of realistic intestinal concentrations are shown in Table 1 for OTA and DON 12 and in Table 2 for IMA and BEN. For the assays with H4IIE DR-CALUX ® , HepG2-Luc 13 and MMV-Luc cells, as well as with E. coli strains, a range of concentrations including 14 the calculated maximal realistic intestinal concentration was used. intestinal concentrations considered for this study (see above) (not shown). However, 12 significant reductions of cell viability of approximately 20% or 30% occurred 13 respectively in HepG2-Luc cells at 400 ng OTA/ml and in H4IIE DR-CALUX ® cells at 14 40 µg OTA/ml, while the MMV-Luc cell line was not significantly affected, whatever the 15 OTA concentration used. A significant reduction of cell viability was observed after 16 exposure to concentrations of DON of 400 ng/ml and 2 µg/ml in the three reporter cell 17 lines. This decrease was approximately of 34%, 20% and 28% at 400 ng/ml and of 52%, 18

18% and 40% at 2 µg/ml in, respectively, H4IIE DR-CALUX ® , HepG2-Luc and MMV-19 Luc cells. Significant effects of IMA on cell viability were recorded at 20 µg/ml for 20 H4IIE DR-CALUX ® cells (±24% of decrease of viability), and at 40 µg/ml for HepG2-21 Luc cells (±40% of decrease of viability). IMA was not cytotoxic in the MMV-Luc cell 22 line, in the range of concentrations tested. BEN reduced cell viability only at the highest 23 tested dose (40 µg/ml) (± 35% in H4IIE DR-CALUX ® and HepG2-Luc cells and ±78% in 24 MMV-Luc cells). 25 26

Bacterial stress gene profiling assay 27

Prokaryotic gene expression profiles were obtained following 90 min exposure of the 28 different transgenic E. coli strains used to different concentrations of OTA (1.6 -100 29 µg/ml), DON (0.031 -2 µg/ml), IMA (0.4 -13 µg/ml) or BEN (0.78 -50 µg/ml). When 30 the lowest concentration of the tested range was higher than the calculated realistic 31 intestinal concentration and if no significant effect was observed at this lowest 1 concentration, effects at lower concentrations were not investigated. 2

In the case of OTA, significant inductions of genes related to membrane (MicF) and 3 DNA (UmuDC, Ada and SfiA) damages were obtained, but only starting from 3.13 µg/ml, 4 which represents more than 10000 times the calculated realistic intestinal concentration. 5

At concentrations of 12.5, 25 and 50 µg/ml clear dose-responses were observed for 6 different stress genes related to oxidative damage (KatG, Zwf, Soi28 and Nfo), membrane 7 damage and general cell stress (MicF, OsmY, UspA and ClpB) and DNA damage 8 (UmuDC, Ada, DinD and SfiA) (not shown). In the same way, no significant inductions of 9 stress genes were found after exposure of the transgenic E. coli strains to BEN at the 10 lowest tested concentration (0.78 µg/ml), representing more than 3 times the calculated 11 realistic intestinal concentration. At higher concentration (12.5 µg/ml), significant 12 inductions were revealed for endpoints related to membrane damage (MicF) and general 13 cell lesions induced by protein perturbation (ClpB) (not shown). Regarding DON, only 14 one marker for DNA damage (DinD) was induced upon exposure to the calculated 15 realistic intestinal concentration of 250 ng/ml (Figure 2A). Furthermore, at a 16 concentration ten times higher, significant induction for RecA, which is part of the SOS 17 response, was found. E. coli strains were also sensitive to IMA. At 2500 ng/ml, the 18 realistic intestinal concentration, statistically significant inductions were found for 19 markers related to protein perturbation, membrane integrity and osmotic stress (MicF and 20

ClpB) and oxidative stress (Zwf, Soi28 and SfiA) (Figure 2B). 21 22

Modulation of intestinal cytochrome P-450 biotransformation enzyme activity 23 Induction of CYP1A1 activity 24

The effect of OTA, DON, IMA and BEN at calculated realistic intestinal concentration 25 (see above) on CYP1A1 activity in Caco-2 cells after 6 and 24h exposure is illustrated in 26

Human estrogen receptor (hER) agonistic/antagonistic activities 1

Exposure of MMV-Luc cells to the reference ligand E2 during 24h resulted in a dose-2 dependent relationship (Figure 5), with an EC 50 of 0.003 ng/ml. Fulvestrant (ICI 182, 3 780) is a high affinity ER antagonist [START_REF] Osborne | Fulvestrant: an oestrogen receptor 9 antagonist with a novel mechanism of action[END_REF]. As a control, the antagonistic 4 activity was assayed in cells simultaneously exposed to E2 (1.4 ng/ml) and fulvestrant 5 (60.7 ng/ml). A 93% inhibition of the E2-related induction was observed, confirming the 6 antagonistic activity of fulvestrant in MMV-Luc cells, while no cytotoxic activity was 7 evidenced using the MTT assay (not shown). 8 9 OTA, DON and IMA showed no hER agonistic activity in MMV-Luc cells exposed 10 during 24h to the range of concentrations. On the contrary, we obtained a dose-response 11 relationship when cells were exposed to BEN with an EC 50 of 475 ng/ml (Figure 5). The 12 REP of BEN expressed as the ratio between the EC 50 of E2 and BEN was therefore 6.3 13 10 -6 . At the reference concentration considered in this paper, which is the realistic 14 intestinal concentration (250 ng/ml), the in vitro estrogenic activity of BEN was 15 equivalent to the effect produced by the EC 50 (0.003 ng/ml) of E2. 16 In antagonistic assays, MMV-Luc cells were exposed during 24h simultaneously to E2 17 and a range of concentrations of OTA, DON, IMA or BEN. In these conditions, none of 18 the tested contaminants were able to decrease the relative response induced by E2 at non 19 cyototoxic concentrations . 20 In all cases, vehicle controls had no effect on hER activation. of dietary contaminants occurring in the gastro-intestinal tract by using a dilution 1 hypothesis of the ingested daily intake in the intestinal lumen. The deterministic approach 2 followed allowed us to easily mimic intestinal chronic exposure with in vitro systems to a 3 broad range of contaminants, including those for which no ADI/TDI has been established 4 and no databases necessary in a probabilistic approach are available. The theoretical 5 maximum daily intake (TMDI) has been obtained by multiplication of European maximal 6 authorized level of contaminants in foodstuffs (contamination data) with average adult 7 daily consumption data of these foodstuffs from GEMS/FOOD Regional Diets, which are 8 based on national Food Balance Sheets. Data from GEMS/FOOD Regional diets greatly 9 underestimate short-term high percentile intakes. They are used in chronic exposure 10 assessment by the Joint Meeting on Pesticide Residues (JMPR) and the Joint FAO/ WHO 11 Expert Committee on Food Additives (JECFA), according to internationally accepted 12 methodologies (WHO, 1997), in order to assess long-term risks at the international level. 13 This method permits thus a valid comparison of TMDI with the 'Acceptable Daily 14 Intake' (ADI, for pesticides) or 'Tolerable Daily Intake' (TDI, for other contaminants), 15 which are based on long-term food consumption habits. 16 17 This very simple deterministic method, called 'by point estimates', has the particularity 18 not to be associated with any measurement of confidence and to slightly over-estimate 19 actual exposure, since it assumes that all individuals consume the same quantities of 20 specified foodstuff(s) and that the contaminant is always present in the foodstuff(s) at a 21 high level [START_REF] Kroes | Assessment of intake from the 8 diet[END_REF]. On the other hand, as compared to the probabilistic 22 approach, which is based on distributions rather than on single-point values, it requests 23 less extensive databases and offers more easily interpretable results [START_REF] Claeys | 38 Exposure assessment of the Belgian population to pesticide residues through fruit and 39 vegetable consumption[END_REF]. 24 It is therefore commonly used as a first step in exposure assessment and most appropriate 25 for screening purposes [START_REF] Verger | Méthodes classiques d'évaluation du risque[END_REF], in accordance with our study. The probabilistic 26 method is indeed very useful when the first deterministic estimation is at or above 27 ADI/TDI, since it allows to refine the assessment and to better evaluate if the chemical 28 exposure is a public health issue. Whatever the approach used, it should be kept in mind 29 that the quantity of contaminant residues effectively occurring in food can be lower than 30 in raw materials after processing and/or storage [START_REF] Kroes | Assessment of intake from the 8 diet[END_REF]. 31 In the present study, the main sources of ingestion of OTA, DON, IMA and BEN for 1 which European M(R)Ls in foodstuffs exist, have been taken into account in the 2 calculation of TMDI. For OTA, European MLs in cereal-based products, contributing to 3 50-70% of the dietary intake [START_REF] Petzinger | Ochratoxin A from a toxicological perspective[END_REF], dried vine fruit, coffee and 4 wine have been considered, while for DON, European MLs in cereal-based products were 5 the only ones to be used. The exposure assessment has resulted in a daily intake for OTA 6 and DON of respectively 13.05 ng/kg bw/day, representing 76% of TDI (considering that 7 TWI is equally spread over the week) and 2.37 µg/kg bw/day, or 237.7% of TDI. The fact 8 that TMDI exceeds TDI may be explained by the crude over-estimation induced by the 9 method. It is a preliminary indication that a potential risk should be taken into account 10 and that a more precise exposure assessment should be performed, for example with the 11 probabilistic method. By comparison, the European Scientific Cooperation (SCOOP) 12 tasks on mycotoxins give estimated daily intakes of 0.1-3.5 ng/kg bw/day for OTA and 13 0.078-0.48 µg/kg bw/day for DON, for participating member states displaying the lowest 14 and highest mean exposure (EC, 2002; EC, 2003). For OTA, other studies report dietary 15 exposure of 2-3 ng/kg bw/day for the average adult consumer (EFSA, 2006) or 0.7-4. 6 16 ng/kg bw/day for average consumers and 6-8 ng/kg bw/day for high consumers (SCF, 17 1998). In 2002, JECFA published a report where a probabilistic approach with French 18 data resulted in an intake of 92 ng OTA/kg bw/week (or 13.14 ng/kg bw/day) for 19 consumers of cereals at the 95 th percentile. Regarding DON, an estimation of the dietary 20 intake ranged from 0.77 µg/kg bw/day with the African diet to 2.4 µg/kg bw/day with the 21 Middle Eastern diet (WHO, 2001). 22 23 The oral exposure of human to IMA and BEN through consumption of contaminated 24 fruits and vegetables has been assessed in the present study to respectively 17.47 µg/kg 25 bw/day and 3.34 µg/kg bw/day and represents respectively 58.2% and 3.3% of their ADI. [START_REF] Van De Walle | Influence of 27 deoxynivalenol on NF-κB activation and IL-8 secretion in human intestinal Caco-2 cells[END_REF] In a study based on a Belgian pesticide monitoring program, the authors calculated an 27 exposure for the 97.5 th percentile consumer reaching ±25% and ±2% of ADI for IMA and 28 BEN, respectively [START_REF] Claeys | 38 Exposure assessment of the Belgian population to pesticide residues through fruit and 39 vegetable consumption[END_REF]. In USA, the exposure to IMA was estimated to 29 0.05 µg/kg bw/day, in the worst case, by considering residue data in products prepared as 30 for consumption [START_REF] Dougherty | Dietary exposures to food contaminants across the United States[END_REF]. In the case of IMA, an overestimation of the 31 showed a very slight but significant increase of LDH release (less than 2.5%) from 10 nM 6

(2.96 ng/ml), while 100 µM of DON (29 µg/ml) was necessary to strongly increase LDH 7 release (642% of control value), induce cellular apoptosis and diminish protein content 8 [START_REF] Maresca | The mycotoxin 3 deoxynivalenol affects nutrient absorption in human intestinal epithelial cells[END_REF]. Intestinal necrosis is indeed a major toxic effect of DON. In vivo, 9 the ingestion of contaminated food with high doses of DON may create lesions of the 10 mucosa in the gastrointestinal tract, resulting in extensive hemorrhage, vomiting and 11 gastroenteritis symptoms [START_REF] Pestka | Deoxynivalenol: toxicology and potential 23 effects on humans[END_REF]. The cytotoxicity of DON has 12 often been attributed to the inhibition of protein synthesis, by impairment of the peptidyl-13 transferase on the 60S ribosomal subunit. As a translational inhibitor binding to 14 ribosomes, DON rapidly activates the double-stranded RNA-activated protein kinase R 15 al., 2008). This overall process is called 'ribotoxic stress response'. The impact of DON 19 on cell viability was furthermore explained by pointing mitochondria and lysosomes as 20 targets, by showing an inhibition of protein and DNA synthesis, as well as through lipid 21 peroxidation [START_REF] Kouadio | Comparative study of cytotoxicity and oxidative stress induced by 4 deoxynivalenol, zearalenone or fumonisin B1 in human intestinal cell line Caco-2[END_REF][START_REF] Kouadio | Effects 45 of combinations of Fusarium mycotoxins on the inhibition of macromolecular synthesis, 46 malondialdehyde levels, DNA methylation and fragmentation, and viability in Caco-2 1 cells[END_REF]. Interestingly enough, we 22 observed that the E. coli strain transfected with the promoter DinD, responsive to DNA 23 damage, was induced at calculated realistic intestinal concentration. Furthermore, at a 24 concentration ten times higher, significant inductions for RecA, part of the SOS response, 25 were found. These results are in accordance with the genotoxic potential attributed to 26 DON [START_REF] Rotter | Toxicology of deoxynivalenol 3 (vomitoxin)[END_REF][START_REF] Bony | Genotoxicity assessment of 17 deoxynivalenol in the Caco-2 cell line model using the Comet assay[END_REF]. In addition to information about its mode of 27 action, this assay suggests that the toxicity of DON towards bacteria at realistic exposure 28 may imbalance gastro-intestinal populations, which may, on one hand, impair the 29 metabolism and bioavailability of food compounds and xenobiotics, and, on the other 30 hand, modify the systemic effects of the gut microbiota. 31 At realistic intestinal concentration, no cytotoxicity of OTA has been highlighted in 1 intestinal cells. Sensitivity of intestinal cells to OTA toxicity has yet been shown in vitro 2 but at higher concentrations than those used in the present work [START_REF] Berger | Interaction of ochratoxin A with human intestinal Caco-2 cells: possible 12 implication of a multidrug resistance-associated protein (MRP2)[END_REF]3 Creppy et al., 2004). The kidney is generally considered as the first and major organ 4 affected by OTA. Nevertheless, the IC 50 calculated in intestinal cells are often lower than 5 in other studies using renal cells, suggesting that the kidney has no specific sensitivity 6 towards OTA-induced cytotoxicity in comparison with cells of other origin (Creppy et 7 al., 2004;[START_REF] Kamp | 34 Ochratoxin A: induction of (oxidative) DNA damage, cytotoxicity and apoptosis in 35 mammalian cell lines and primary cells[END_REF], even if nephropathy is the main in vivo reported effect. The the liver is the primary target for IMA in experimental animals (EFSA, 2007) and a time-23 dependent cell death, accompanied by the depletion of intracellular ATP, GSH and 24 protein thiols, was observed in isolated rat hepatocytes by [START_REF] Nakagawa | Cytotoxic effects of postharvest fungicides, 34 ortho-phenylphenol, thiabendazole and imazalil, on isolated rat hepathocytes[END_REF]. 25 These authors have suggested that cytotoxicity caused by IMA might be related to lipid 26 peroxidation, due to an oxidative stress, as well as to the depletion of ATP via 27 impairment of the mitochondrial function. In the present study, we have shown in the 28 prokaryotic gene expression profile of IMA that significant inductions of genes related to 29 cellular and osmotic stress, as well as to oxidative stress, occur at realistic gastro-30 intestinal exposure. These results are in accordance with the hypothesis that IMA 31 Soliman, 1997) has shown that a 7-day treatment of rats significantly increased lipid 13 peroxidation and depleted liver GSH levels, suggesting that the hepatotoxicity induced by 14 BEN or its metabolites is due to an oxidative stress of cellular membranes. In the present 15 work, even though no response was recorded in the bacterial gene expression assay at 16 realistic concentration, significant inductions were observed at higher concentrations for 17 membrane damage-related endpoints, supporting the idea that lipid peroxidation may be 18 one of the modes of action of BEN. 19 20 Once absorbed from the lumen into intestinal cells, chemicals may undergo first-pass 21 metabolism. CYPs are major actors of these biotransformations. The study of the effects 22 of dietary chemicals on CYP expression and/or activity is of primary importance in the 23 comprehension of mechanisms of adverse and beneficial interactions in cells (Sergent et 24 al., 2008). Since CYPs have often broad substrate specificities, their induction by one 25 chemical may result in enhanced metabolism of other chemicals or drugs, thereby, 26 altering their bioavailability, pharmaco-or toxicokinetic properties as well as pharmaco-27 or toxicodynamic effects [START_REF] Ma | Induction of CYP1A1. The AhR/DRE Paradigm: transcription, Receptor 42 Regulation, and Expanding Biological Roles[END_REF]. 28 29 As in a previous publication from our group [START_REF] Sergent | 26 CYP1A1 induction and CYP3A4 inhibition by the fungicide imazalil in the human 27 intestinal Caco-2 cells-Comparison with other conazole pesticides[END_REF], we have again 30 highlighted that IMA, which is commonly recovered in consumed fruits and vegetables, 31 It is well known that CYP1A1 is implicated in the conversion of pro-carcinogens from 1 the PAH family to reactive cytotoxic metabolites, such as carcinogenic DNA-binding 2 species [START_REF] Schwarz | Differential 14 metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 15 variants[END_REF][START_REF] Shimada | Metabolic 42 activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes 43 P450 1A1 and P450 1B1 allelic variants and other human cytochromes P450 in 44 Salmonella typhimurium NM2009[END_REF]). An induction of CYP1A1 activity by 3 another compound, such as IMA in the present case, might further provoke either pro-4 carcinogen activation or detoxification by hydroxylation. Toxicity vs protection resulting 5 from CYP1A1 induction appears to depend on the cellular context, such as carcinoma 6 cells vs intact animal [START_REF] Uno | Oral exposure to benzo[a]pyrene in the mouse: detoxication by 24 inducible cytochrome P450 is more important than metabolic activation[END_REF], to tissue-or cell type-specificities as well as to 7 the involvement of phase II metabolism [START_REF] Nebert | Role of aryl hydrocarbon 37 receptor-mediated induction of the CYP1 enzymes in environmental toxicity and cancer[END_REF]. This comment emphasizes 8 the extreme care that has to be taken in the extrapolation of in vitro results to the in vivo 9 situation. Furthermore, it is difficult to conclude about consequences of CYP1A1 10 induction since other effects may arise in parallel, such as inhibition of TCDD-induced 11 CYP1A1 activity by IMA in Caco-2 cells, suggesting cytoprotective effects towards pro-12 carcinogen activation [START_REF] Sergent | 26 CYP1A1 induction and CYP3A4 inhibition by the fungicide imazalil in the human 27 intestinal Caco-2 cells-Comparison with other conazole pesticides[END_REF]. 13 14 In addition to xenobiotic metabolism, the CYP1A1 isoform is also involved in the 15 transformation of endobiotic compounds, such as atRA, the active metabolite of retinol 16 (vitamin A) [START_REF] Lampen | Metabolism of vitamin A and its 13 active metabolite all-trans-retinoic acid in small intestinal enterocytes[END_REF] and E2, which is known to act both as a promoter of 17 cell proliferation and as a pro-carcinogen [START_REF] Tsuchiya | Cytochrome P450-mediated 21 metabolism of estrogens and its regulation in human[END_REF][START_REF] Parl | Estrogen metabolism and 21 breast cancer[END_REF]. The 18 induction of CYP1A1 activity may thus lead in the disruption of atRA biological 19 activities, as well as in the resistance at the target site when atRA is used clinically. It 20 may also increase the metabolism of E2 to weak estrogenic [START_REF] Oh | Antiestrogenic activities of Ginkgo biloba extracts[END_REF], but 21 potentially toxic metabolites [START_REF] Tsuchiya | Cytochrome P450-mediated 21 metabolism of estrogens and its regulation in human[END_REF][START_REF] Parl | Estrogen metabolism and 21 breast cancer[END_REF]. Therefore, 22 intestinal CYP1A1 inducers, such as IMA, may influence the atRA and E2 small 23 intestinal metabolism and action. 24 25 Results from the present work confirm previously published material (Sergent et al., 26 2009) that shows a strong inhibition of CYP3A4 activity by a realistic concentration of 27 IMA in 1,25-vitD 3 -induced Caco-2 cells, but at lower concentration than ketoconazole, a 28 known CYP3A4 inhibitor in this cell line [START_REF] Gibbs | 6 Persistent inhibition of CYP3A4 by ketoconazole in modified Caco-2 cells[END_REF][START_REF] Hou | Possible inhibitory mechanism of Curcuma drugs on CYP3A4 27 in 1α,25 dihydroxyvitamin D3 treated Caco-2 cells[END_REF]. The 29 inhibition of the CYP3A4 activity but not gene transcription by IMA has been previously 30 observed in Caco-2 cells by [START_REF] Sergent | 26 CYP1A1 induction and CYP3A4 inhibition by the fungicide imazalil in the human 27 intestinal Caco-2 cells-Comparison with other conazole pesticides[END_REF]. On the other hand, the induction of 31 CYP3A4 activity and expression, as well as the activation of the human pregnane X 1 receptor (hPXR), by IMA have been previously demonstrated [START_REF] Muto | Induction and inhibition 31 of cytochrome P450 isoforms by imazalil, a food contaminant, in mouse small intestine 32 and liver[END_REF]2 Lemaire et al., 2006b). A broad range of drugs, carcinogens, but also endogenous steroids 3 (testosterone, progesterone, androstenedione, and cortisol) or E2 are substrates of 4 CYP3A4 [START_REF] Guengerich | Cytochrome P-450 3A4: Regulation and Role in Drug 20 Metabolism[END_REF][START_REF] Goodwin | Regulation of CYP3A gene 12 transcription by the pregnane X receptor[END_REF] and thus good candidates for 5 interactions at the intestinal level. Since more than the half of the existing drugs is 6 metabolized by CYP3A4, interactions with drugs constitute the main issue when foreign 7 compounds modulate CYP3A4 activity. For example, an increase of bioavailability and 8 plasma concentration of felodipine or of the pro-drug terfenadine, due to the diminution 9 of oxidation by CYP3A4 inhibitors such as grapefruit juice or ketoconazole, has been 10 associated with detrimental effects. On the other hand, it has been demonstrated that the 11 CYP3A4 inhibitor ketoconazole increases oral bioavailability of concurrently 12 administered cyclosporine and reduces the dosage required [START_REF] Guengerich | Role of cytochrome P450 enzymes in drug-drug interactions[END_REF]Dresser et 13 al., 2000). Therefore, the co-occurrence at the intestinal level of IMA and a CYP3A4-14 substrate drug may alter its efficiency, either adversely or positively, by pre-systemic 15 metabolism. In addition, the inhibition of CYP3A4 activity by IMA may have an impact 16 on the metabolism of food contaminants, since CYP3A4 activates some PAH 17 dihydrodiols [START_REF] Shimada | Metabolic 42 activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes 43 P450 1A1 and P450 1B1 allelic variants and other human cytochromes P450 in 44 Salmonella typhimurium NM2009[END_REF], as well as pro-carcinogens, such as aflatoxin B1 18 (AFB1) [START_REF] Shimada | Evidence for cytochrome P-450NF, the 38 nifedipine oxidase, being the principal enzyme involved in the bioactivation of aflatoxins 39 in human liver[END_REF][START_REF] Guengerich | Activation of procarcinogens by human 22 cytochrome P450 enzymes[END_REF]), but at 19 relatively high substrate concentration [START_REF] Gallagher | 45 Role of human microsomal and human complementary DNA-expressed cytochromes 46 P4501A2 and P4503A4 in the bioactivation of aflatoxin B1[END_REF]. 20 21

The ability of BEN to display hER agonistic activity, even at realistic gastro-intestinal 22 concentration, has been demonstrated in the present paper. The activation of the 23 transcription of the firefly luciferase reporter gene probably occurs after the binding of 24 the dimer complex 'ligand (BEN) -receptor (ER)' to the ERE elements present in the 25 vitellogenin promoter of the MMV-Luc cell line. An agonistic effect of BEN towards 26 ERα has indeed been reported previously in reporter HeLa cells (Lemaire et al., 2006a). 27 Furthermore, BEN was previously shown to bind the recombinant hERα in a binding 28 assay [START_REF] Scippo | Recombinant human estrogen, androgen and progesterone 18 receptors for detection of potential endocrine disruptors[END_REF]. The estrogenicity of BEN and its degradation product 29 carbendazim was also shown to act via the induction of the aromatase CYP19 activity, 30 catalyzing the conversion of androgens to estrogens, in a human ovarian granulose-like 31 [START_REF] Morinaga | A benzimidazole fungicide, benomyl, and its metabolite, carbendazim, induce 26 aromatase activity in a human ovarian granulose-like tumor cell line (KGN)[END_REF]. Therefore, it is likely that BEN acts as an 1 estrogenic compound by affecting both the action of estrogens, through binding to ERα 2 and activation of ERE downstream genes, and the synthesis of estrogens, by increasing 3 estrogens level. We therefore suspect that BEN acts as an endocrine disruptor, which may 4 lead to estrogen-mediated pathologies such as reproductive disorders. 5 6 The overall toolbox approach proposed in this paper and including four types of in vitro 7 assays revealed to be interesting in assessing chemical risks at the intestinal level upon 8 dietary consumption. In a first assay, genetically modified E. coli strains responding to 9 different types of stress permitted to assess toxicity on bacteria present in the human gut, 10 potentially leading to an imbalance in the populations and a modification of their activity 11 and in turn impacting the digestive function, the absorption and more generally some of 12 the various effects of the microbiota on the human health. The second in vitro assay 13 performed with intestinal Caco-2 cells allowed to give a quick insight into the modulation 14 of the activity of CYP1A1 and CYP3A4 in small intestinal cells. The third and fourth in 15 vitro assays focused on the AhR and ER, respectively. Whereas the AhR is undoubtedly 16 present in human enterocytes [START_REF] Fallone | Retinoids repress Ah 38 receptor CYP1A1 induction pathway through the SMRT corepressor[END_REF], controversy remains regarding the 17 presence of the ER. Nevertheless, ER has been identified and localized in the mouse 18 small intestine [START_REF] Kawano | 41 Identification and localization of estrogen receptor αand β-positive cells in adult male 42 and female mouse intestine at various estrogen levels[END_REF] and in the human colon [START_REF] Singh | Expression of oestrogen receptor and oestrogen-inducible 2 genes pS2 and ERD5 in large bowel mucosa and cancer[END_REF]. 19 Furthermore, its expression has been associated with a lower incidence of small intestinal 20 tumorigenesis in mice [START_REF] Giroux | Estrogen 9 receptor β deficiency enhances small intestinal tumorigenesis in ApcMin/+ mice[END_REF] and estrogen exposure in women results in a 21 reduction in colon cancer risk [START_REF] Grodstein | Postmenopausal hormone 15 therapy and the risk of colorectal cancer: a review and meta-analysis[END_REF] In the present work, we have proposed a package of in vitro experimental tools, which 5 responded to chemical stresses and proved to be efficient in assessing some relevant 6 dietary toxicological effects, even at realistic contaminant exposure in humans, as shown 7 by the comparison of the results obtained with four well-known contaminants with those 8 from the literature. Due to its high contribution in first-pass metabolism, as well as in the 9 bioavailability of dietary compounds, the intestinal compartment was the major focus of 10 the present study. Therefore, a method of easy evaluation of realistic concentrations of 11 dietary contaminants occurring in the human gastro-intestinal tract was proposed. This 12 method can be applied to a large range of dietary chemicals for which limited data exist 13 and is relevant in the risk assessment process. 14 15 This overall approach is not time-consuming and may be applied to high throughtput 16 screening of high numbers of samples. It allows to take a first 'fingerprint' of tested 17 dietary contaminants at realistic exposure, which may be useful for further risk 18 assessment studies and for limiting in vivo testing. The proposed experimental toolbox, 19 which gathers diverse yet existing test systems, offers an additional dimension in 20 comparison with the in vitro assays taken separately. In the scope of emerging risk 21 detection, this tool is very promising due to the diversity and complementarities of 22 endpoints measured and the biological relevance of the concentrations selected for the 23 experimental studies. 24 25

F

30

 30

2 3

 2 -activating substances, attention should be paid to molecules able to act on the 1 AhR/ER pathways.Considered together, the four in vitro experimental tools described above constitute a 4 package of complementary tests that may be considered as an efficient toolbox allowing 5 taking a first 'fingerprint' of potential toxicants. The aim of the present work was to 6 assess the pertinence of this toolbox by applying it to four well-known dietary 7 contaminants displaying distinct toxicological effects and showing human health concern: 8 two mycotoxins, namely ochratoxin A (OTA) and deoxynivalenol (DON), and two 9 fungicides, imazalil (IMA) and benomyl (BEN). The chemical structures of these 10 compounds are shown in Figure1. 1112

  limits (M(R)Ls) for both mycotoxins (Commission Regulation (EC) N°1881/2006 of 19 24 December 2006) and pesticides (Commission Regulation (EC) N°396/2005 of 23 25 February 2005, amended by Commission Regulation (EC) N°839/2008 of 31 July 2008 26 for BEN and Commission Regulation (EU) N°750/2010 of 7 July 2010 for IMA) in the 27

1000 12 - 15 Formula 2 :

 12152 TMDI: theoretical maximum daily intake [ng/person/day] 13 -V: volume of gastro-intestinal fluid [L/meal] (V = 1L) 14 -n: number of daily meals [meals/person/day] (n = 1) Maximal realistic intestinal concentration (C) 16 Thanks to this method, we obtained a maximal realistic intestinal concentration (C) 17 representative of chronic exposure. The concentration or concentration range of OTA, 18 DON, IMA or BEN used in cellular assays were chosen to be close to or to include the 19 calculated realistic intestinal concentration of the relevant contaminant. 20 21 Chemical contaminants 22 BEN (95%), DON (> 97%), IMA (> 97%), OTA (> 98%) and B[a]P were purchased 23 from Sigma-Aldrich (St Louis, MO). TCDD (99.9%) was supplied from BCP 24 Instruments, Wellington Laboratories Inc. (Guelph, CA)cells (ATCC, Rockville, MD), derived from a human colon adenocarcinoma, 29 were used between passages 26 and 34 and routinely grown in Dulbecco's Modified 30

  strains of E. coli, a common Proteobacterium from the human gut microbiota, used in 20 the present study (KatG, Zwf, Soi28, Nfo, MicF, OsmY, UspA, ClpB, MerR, RecA, 21 UmuDC, Ada, SfiA and DinD), except SfiA, are based on an E. coli K-12 derivative SF1 22 containing the mutations lac4169 deleting the entire lac operon, and rpsL. All the 23 different LacZ fusions are present as single chromosomal inserts. The strains, described 24 in Dardenne et al. (2007), were selected from the work of Orser et al. (1995) and respond 25 to different types of stress: DNA damage, oxidative stress, protein denaturation, 26 membrane damage, osmotic stress, general cellular stress and heavy metal presence. The 27

31

 31

3 Caco

 3 in the different eukaryotic cell lines. Colorimetric assays were 2 performed and cell morphology was routinely observed by phase contrast microscopy. 2H-tetrazolium (MTS) assay purchased as a kit (Celltiter 96 ® AQueous One 6 Solution Cell Proliferation Assay, Promega, Madison, WI) was used. It measures cellular 7

18 As

 18 another indicator of cytotoxicity, the release of cytosolic lactate dehydrogenase19 (LDH) by damaged and necrosed cells into the extracellular medium was measured in the 20 same conditions as for the EROD assay (see below). Caco-2 cells were seeded in 48-well 21 plates at a density of 3.10 4 cells/cm 2 and were cultivated until 16-day post-confluence 22 with culture medium changes three times per week. Cells were then incubated for 6 or 23 24h with OTA, DON, IMA, BEN or vehicles diluted in FBS-free culture medium at the 24 same final concentration as for the MTS assay. The negative control consisted in FBS-25 free culture medium alone and the positive control, corresponding to 100% of cell 26 mortality, consisted in cells treated with 1% (v/v) Triton  X-100 (Sigma-Aldrich) in 27 FBS-free culture medium. The activity of LDH in the medium was determined with the 28 Cytotoxicity detection kit (Roche diagnostics, Mannheim, DE) according to the 29 manufacturer's instructions with formazan production detected at 500 nm (reference 30 wavelength: 690 nm) with a Spectracount spectrophotometer (Packard). 31

  v) methanol for OTA and DON and 1% (v/v) DMSO for IMA and BEN, for all tested 1 concentrations. Afterwards, the cells were lysed and the β-galactosidase activity was 2 measured. 3 4

11 Formula 4 :

 114 Fold Induction at a given dose i (Dardenne et al., 2007). 12

at 4 . 23 24

 423 10 4 cells/cm 2 in 12-well plates pre-coated with type I collagen (Sigma-Aldrich) and 9 grown to confluence. As described by Schmiedlin-Ren et al.(1997), Caco-2 cells were 10 then cultivated during 2 weeks in 5% FBS-enriched DMEM and in the presence of 0.5 11 µM of 1,α25-(OH) 2 -vitamin D 3 (1,25-vitD 3 ) (Sigma-Aldrich), in order to express 12 functional CYP3A4 activity, with culture medium changes three times per week. The last 13 day, the cells were rinsed with PBS and incubated for 3h at 37°C with 500 µM 14 testosterone, in the absence of other compound (negative control) or in the presence of 15 ketoconazole (positive control), OTA, DON, IMA or BEN diluted in Hanks' balanced 16 salt solution (HBSS) at pH 7.4 at a final concentration of respectively 26.6 µg/ml (50 17 µM), 1 ng/ml, 250 ng/ml, 2500 ng/ml and 250 ng/ml. Supernatants were then collected 18 for 6β-(OH)-testosterone quantification through HPLC, as previously described in 19[START_REF] Sergent | 26 CYP1A1 induction and CYP3A4 inhibition by the fungicide imazalil in the human 27 intestinal Caco-2 cells-Comparison with other conazole pesticides[END_REF]. The limits of detection (LOD) and quantification (LOQ) were 100 20 and 300 nM, respectively. The CYP3A4 activity was normalized to the cell protein 21 content determined with the BCA assay kit (Sigma-Aldrich) after cell lysis with 1% 22 (w/v) DOC in water, pH 11.3.Human and rat aryl hydrocarbon receptor (AhR) agonistic/antagonistic activities 25The cell-based assays to screen for agonistic/antagonistic AhR activities of chemicals 26 were carried out as previously described (Van derHeiden et al., 2009). Briefly, 94000 27 cells/cm 2 were seeded in 96-well culture plates and incubated overnight. In assays to 28 measure AhR agonistic activities, H4IIE DR-CALUX ® or HepG2-Luc cells were exposed 29 during 6 or 24h to serial dilutions of OTA, DON, IMA or BEN, at the same final 30 concentrations as for the cytotoxicity assay, or to culture medium containing 0.4% (v/v) 31 for all tested concentrations (ethanol for OTA and IMA, water for DON 1

Formula 5 :

 5 Non linear equation of a dose-response curve allowing determining 26EC 50 . y is cell response, expressed in RLUs or in relative response (%); x is the analyte 27 concentration; A 0 is the maximal response expressed in RLUs or in relative response (%); 28 A 1 is EC 50 and A 2 is the slope of the linear part of the curve.29

Food1

  were expressed in means ± S.E.M. of three independent experiments performed 22 in triplicate (n=9). Statistical analysis was conducted with the SPSS 18.0 computer 23 program (IBM, Somers, NY), using one-way analysis of variance (ANOVA) coupled 24 with post-hoc test of Scheffe or Tukey. When comparison was made with a reference 25 group, post-hoc test of Dunnett was used. When homogeneity of variance was not 26 assumed, the correction of Welch was applied and coupled with the post-hoc test of 27 Dunnett T3. Homogeneity of variance was verified with the Levene statistic. A log 28 transformation was performed to achieve normality when necessary. Significant effects 29 were determined in comparison with respective vehicle conditions. 30 31 Page 17 of 53 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Determination of realistic intestinal concentrations 2 European dietary consumption data from GEMS/FOOD Regional Diets (WHO, 2003) 3 and European M(R)Ls of contaminants legally authorized in the main consumed 4 foodstuffs have allowed us to calculate TMDIs for OTA, DON, IMA and BEN, in a 5 deterministic way. A maximal realistic intestinal concentration of each of these dietary 6 contaminants has been calculated from the dilution hypothesis of TMDI in the gastro-7 intestinal fluid. Maximal realistic intestinal concentrations for experimental use with 8 intestinal cells have been chosen in the same range of magnitude as calculated ones, 9

15 16 17 CytotoxicityFood 3 4

 15173 Cytotoxicity of the contaminants has been assessed in the various cell lines by the means 18of different colorimetric assays evaluating the metabolic activity (considered as a good 19 approximation of the cell number) during the proliferation phase (MTS and MTT) or the 20 formation of cell necrosis (LDH release in culture medium) in 16-day post-confluent 21 cells.22 23 In intestinal Caco-2 cells, cytotoxicity of OTA, DON, IMA and BEN was assessed at the 24 estimated realistic intestinal concentration (see above). After 48h exposure in these 25 conditions, the MTS assay revealed a significant inhibition of cell proliferation (p<0.005) 26 by DON of approximately 40% in respect to untreated cells (not shown). No significant 27 effect was observed for the other contaminants in comparison with respective vehicle 28 controls (not shown). On the other hand, after 6 or 24h exposure of 16-day post-confluent 29 Caco-2 cells to OTA, DON, IMA or BEN at realistic intestinal concentration,LDH 30 activity in culture media of treated cells was not higher than in control untreated cells (not 31Page 18 of 53 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk In all cases, vehicle controls had no effect on cell proliferation or LDH release in 1 culture medium (<1% of positive control). No visual alterations were observed under 2 phase contrast microscope.Cell viability was also assessed by the mean of the MTT assay, with the stable reporter 5 rat and human hepatoma cell lines (H4IIE DR-CALUX ® and HepG2-Luc) and the stable 6 reporter human breast cancer cell line (MMV-Luc) exposed during 24h to a range of 7 concentration of OTA, DON, IMA or BEN, as described in materials and methods. The 8 percentage of reduction of cell viability was determined in respect to untreated cells. In 9 all cases, vehicle controls had not effect on cell viability. Results indicate that none of the 10 contaminants were cytotoxic for the tested cell lines at the reference calculated realistic 11

Figure 3 . 20 The 12 13 31 Page

 3201231 Figure 3. While OTA, DON and BEN had no effect, IMA revealed to be a significant 27 time-dependent CYP1A1 inducer (p<0.005), with a stronger induction after 24 than 6h 28 exposure. Vehicle controls had no inductive effect on CYP1A1 activity in comparison 29 with untreated cells (not shown). 30 Inhibition of CYP3A4 activity 31

21 22 23 InFood

 2123 Discussion the present paper, different cellular responses to four selected dietary chemical 24 contaminants (OTA, DON, IMA and BEN) were assessed at realistic human exposure. A 25 comparison of the results obtained with those from the literature has been performed in 26 order to validate and propose this methodology as a suitable toolbox to take a first in vitro 27 'fingerprint' of less studied dietary toxicants for risk assessment studies. 28 Since in risk assessment studies hazard characterization of dietary chemicals should 29 ideally be performed at exposure levels representative of 'normal consumers', we have 30 first proposed a methodology allowing the estimation of maximal realistic concentrations 31 Page 22 of 53 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

2 3

 2 our methodology may be caused by the fact that residues are mainly 1 located in the peel of citrus fruits, often removed before consumption.The dilution model of calculated TMDI in the gastro-intestinal fluid used in the present 4 work to generate realistic intestinal concentrations does not take into account inter-5 individual variations, which can obviously influence the amount of contaminants released 6 from the food matrix in the gastrointestinal tract during the digestion process and 7 accessible for intestinal absorption (i.e. gastric and intestinal lumen volume, rate of saliva 8 secretion, rate of pancreatic or biliary secretions, composition of these secretions). In 9 addition, the approach chosen in the present study does not at all allow estimating the 10 concentrations of the contaminants in the body fluids. The concentration of the 11 contaminant in the blood depends on individual-specific factors including the proportion 12 of contaminant absorbed by intestinal cells and released in the bloodstream, the first-pass 13 metabolism in intestinal and liver cells, the excretion back towards the lumen, the half-14 life and the degree of serum binding (mainly albumin), which can reach more than 99% 15 in the case of OTA[START_REF] Ringot | Toxicokinetics and 45 toxicodynamics of ochratoxin A, un update[END_REF]. The literature gives a few data for the presence 16 of contaminants in biological fluids. Depending on the study, the occurrence of OTA in 17 human blood ranged from 0.2 to 0.88 ng/ml (Studer-Rohr et al., 2000), from 0.17 to 0.56 18 ng/ml (EFSA, 2006) or from 0.18 to 1.19 ng/ml (EC, 2002), concentrations very close to 19 the calculated realistic intestinal concentration of the present study (1 ng/ml). In humans, 20 the concentration of DON in blood is poorly documented, while in urines, some authors 21 found mean levels of 37 ng/ml and 12 ng/ml, respectively in high and low-risk areas in 22China[START_REF] Meky | Development of a urinary biomarker of human exposure to 20 deoxynivalenol[END_REF]. To our knowledge, concentrations of IMA and BEN in human 23 body fluids have never been measured. 2425 As a prerequisite to in vitro assays, the cytotoxicity of OTA, DON, IMA and BEN was 26 assessed and further experiments were performed at non-cytotoxic concentrations. DON 27 revealed to be toxic in almost all cell types, even at realistic intestinal concentration (250 28 ng/ml) with proliferating Caco-2 cells. The inhibition of cell proliferation by DON in 29 intestinal cell lines was already shown in similar conditions of exposure (Maresca et al., 30 2002; Kouadio et al., 2005; Bony et al., 2006; Sergent et al., 2006; Van De Walle et al., 31 whereas in the HepG2 cell line, an IC 50 of 8.4 µg/ml has been calculated (Cetin 1 and Bullerman, 2005), suggesting that these cells are less sensitive to DON than Caco-2 2 cells. In the present study, a realistic exposure of 16-day post-confluent Caco-2 cells to 3 DON has not resulted in a marked increase of LDH release, a marker of cell necrosis. On 4 the contrary, cytotoxicity tests performed with DON in the intestinal cell line HT-29-D4, 5

(

  PKR) and the downstream mitogen-activated protein kinase (MAPK) cascades, which 16 drive apoptosis as well as activation of transcription factors implicated in the 17 inflammatory pathway (Zhou et al., 2003; Pestka and Smolinski, 2005; Van De Walle et 18

8 unchanged

 8 levels of LDH activity between control and OTA-treated intestinal cells, 9 confirmed by Berger et al. (2003), suggest that OTA does not induce cell necrosis in the 10 intestine at realistic intestinal concentration. Kamp et al. (2005) observed OTA-11 dependent oxidative DNA damage, suggesting that oxidative stress plays an important 12 role in OTA-derived toxicity/carcinogenicity in the kidney, whereas another hypothesis 13 for OTA carcinogenicity suggests covalent DNA adduct formation (for a review, see 14 Pfohl-Leszkowicz and Manderville (2007)). In the present study, bacterial stress genes 15 related to membrane, DNA and oxidative damage are significantly induced at high 16 concentration. The inductions of SfiA and Nfo more specifically suggest oxidative DNA 17 damage (Dardenne et al., 2008). 18 19 IMA revealed no cytotoxicity in eukaryotic cell lines in a realistic range of 20 concentrations. Little information is available regarding toxic effects of IMA. Different 21 studies have established oral LD 50 in rat ranging from 227 to 664 mg/kg bw. It seems that 22

4 5

 4 stress, as suggested by[START_REF] Nakagawa | Cytotoxic effects of postharvest fungicides, 34 ortho-phenylphenol, thiabendazole and imazalil, on isolated rat hepathocytes[END_REF].As a 1 consequence of the toxicity of a realistic dose of IMA on gastro-intestinal bacteria, an 2 imbalance of bacterial populations and/or a change in metabolism in the lumen may 3 occur. The fungicide BEN commonly induces hepatotoxicity (Igbedioh and Akinyele, 1992; 6 McCarroll et al., 2002). However, in the present study, none of the tested cell lines 7 showed reduced viability in a biological realistic range of concentrations. In a study 8 performed with the HepG2 cell line, Dierickx (1999) observed a reduced cell viability at 9 a non-physiological concentration of BEN, similar to the concentration showing reduced 10 viability of HepG2-Luc cells in the present work. He attributed the observed effects to the 11 butylcarbamoyl group of BEN. Interestingly enough, an in vivo study (Banks and 12

  at realistic exposure in intestinal Caco-2 cells. However, we 1 have not shown an AhR-mediated agonistic activity of IMA with the reporter gene assay.2 Our results are in accordance with those of a screening study on 200 pesticides with 3 mouse hepatoma (Hepa1c1c7) cells stably transfected to obtain dioxin-responsive 4 luciferase reporter cells[START_REF] Takeuchi | In vitro 13 screening for aryl hydrocarbon receptor agonistic activity in 200 pesticides using a highly 14 sensitive reporter cell line, DR-EcoScreen cells, and in vivo mouse liver cytochrome 15 P450-1A induction by propanil, diuron and linuron[END_REF]. This suggests that the classical AhR 5 pathway does not mediate the induction of CYP1A1 activity by IMA. An alternative 6 CYP1A1 regulation pathway, involving PPAR-α, another ligand-activated transcription 7 factor, and two PPAR response elements (PPRE) sites located within the CYP1A1 8 promoter gene, has been proposed in Caco-2 cells (Sérée et al., 2004; Fallone et al., 9 2005). Since it was previously observed that IMA is able to induce PPAR-α-mediated 10 transcriptional activity in reporter cells (Takeuchi et al., 2006), the PPAR-α-mediated 11 CYP1A1 regulation pathway might be considered as potentially responsible for the 12 observed induction of CYP1A1 activity by IMA in Caco-2 cells. The induction of 13 CYP1A1 activity by IMA has been previously demonstrated in the small intestine and 14 liver of rats as well as in Caco-2 cells (Muto et al., 1997; Sergent et al., 2009). However, 15 Sergent et al. (2009) have suggested that a post-transcriptional pathway, acting on the 16 protein rather than on the gene expression, is involved in the increase of CYP1A1 activity 17 following exposure of Caco-2 cells to IMA. 18 19 OTA, DON and BEN had no inductive effect on CYP1A1 activity at realistic intestinal 20 concentration. In accordance with these results, no AhR-mediated agonistic activity was 21 observed in the present study after exposure of reporter cells to different concentrations 22 of these contaminants. In the same trend, Takeuchi et al. (2008), who screened 200 23 pesticides with mouse hepatoma (Hepa1c1c7) cells stably transfected to obtain dioxin-24 responsive luciferase reporter cells, did not observe any AhR agonistic activity after 25 exposure to BEN. However, induction of CYP1A1 activity and expression by BEN and 26 its derivative carbendazim have been demonstrated in HepG2 cells and rabbit 27hepatocytes, but at concentrations much higher than those used in the present study(Rey- 28 Grobellet et al., 1996;[START_REF] Dierickx | CYP1/2 activation and glutathione-dependent cytotoxicity of four 11 pesticides in Hep G2 and Fa32 cells[END_REF]

3 4

 34 . In addition, recently, the decrease 22 of permeability of the intestinal barrier by ER agonists, including E2, has been 23 demonstrated both in rodents and in intestinal cell lines, such as Caco-2 (Braniste et al., 24 2009; Looijer-van Langen et al., 2011). AhR-or ER-mediated (ant)agonism by dietary 25 chemical compounds reaching the intestinal barrier at high concentration might therefore 26 have important consequences for human health. 27The proposed toolbox is a first inititative to associate different complementary, friendly-28 to-use and relevant tools. It may be further improved in the future with additional 29 approaches such as the inclusion of an in vitro simulated digestion of dietary chemicals 30 studies on intestinal cells, of inhibitory assays on CYP1A1 or 1 through the use of intestinal luciferase reporter cells responding to AhR and ER ligands. 2Conclusions

Figure 1 :

 1 Figure 1: Chemical structure of food contaminants investigated in the present study: ochratoxin A (OTA), deoxynivalenol (DON), imazalil (IMA) and benomyl (BEN).

Figure 2 :

 2 Figure 2: Prokaryotic gene expression profile after exposure of E. Coli strains to a concentration range of (A) DON (0.031 -2 µ µ µ µg/ml) and (B) IMA (0.4 -13 µ µ µ µg/ml). Values are means of three independent experiments performed in triplicates and are expressed as fold inductions, calculated with formulas as described in materials and methods. An arrow indicates the calculated realistic intestinal concentration. The different genes are responsive to: katG: oxidative stress, micF: membrane integrity and osmotic stress, osmY: osmotic stress, uspA: growth arrest, recA: SOS response, zwf: oxidative stress, clpB: protein perturbation, umuDC: radiation and/or chemically induced DNA damage, merR: heavy metals, ada: DNA damage (mainly methyl adducts), dinD: DNA damage, soi28: superoxide radical generating agents, nfo: single-and doublestranded DNA breaks and oxidative DNA damage, SfiA: SOS response to DNA damage.

Figure 3 :

 3 Figure 3: Modulation of CYP1A1 activity by OTA, DON, IMA and BEN in Caco-2 cells. Sixteen-day post-confluent cells seeded in 48-well plates (3.10 4 cells/cm 2 ) were exposed to OTA (1 ng/ml), DON (250 ng/ml), IMA (2500 ng/ml) or BEN (250 ng/ml) for 6 or 24h. Positive controls consisted in cells treated with B[a]P (250 ng/ml) or TCDD (0.3 ng/ml) and negative control consisted in untreated cells. CYP1A1 activity was assessed through the measurement of the fluorescence produced by the conversion of 5 µM 7-ethoxyresorufin to resorufin. Results are expressed in pmol resorufin produced/min/mg protein. Values are means ± S.E.M. of three independent experiments performed in triplicates (n=9). *** P < 0.005 as compared to respective vehicle controls.

Figure 4 : 2 cells. 43 HPLC

 4243 Figure 4: Modulation of CYP3A4 activity by OTA, DON, IMA and BEN in Caco-2 cells. Cells were seeded in 12-well plates (4.10 4 cells/cm 2 ) and treated during 14 daysfrom confluence with 0.5 µM 1,25-vitD 3 , in order to induce CYP3A4 activity. Cells were then exposed for 3h simultaneously to testosterone (500 µM) and to OTA (1 ng/ml), DON (250 ng/ml), IMA (2500 ng/ml), BEN (250 ng/ml) or ketoconazole (positive control) (26.6 µg/ml). The CYP3A4 activity of the cells was then assessed through the

Figure 5 :

 5 Figure 5: Dose-response curves of E2 and BEN in MMV-Luc cells. Exposure time was 24h. Results are expressed as means of the relative response (%) ± S.E.M. (100% with highest tested concentration of E2), of two independent experiments performed in triplicates (n=6), in function of the ligand concentration (ng/ml).

Figure 1 :Figure 2 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 122345 Figure 1: Chemical structure of food contaminants investigated in the present study: ochratoxin A (OTA), deoxynivalenol (DON), imazalil (IMA) and benomyl (BEN). 171x142mm (300 x 300 DPI)

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	1	WHO (2001). Evaluation of certain mycotoxins in food: fifty-sixth report of the Joint
	2	FAO/WHO Expert Committee on Food Additives. Geneva, World Health Organization:
	3	1-62.
	4	WHO (2003). GEMS/Food regional diets. Geneva, World health Organization, Food
	5	Safety Department: 1-27.
	6	Willemsen, P., Scippo, M.-L., Kausel, G., Figueroa, J., Maghuin-Rogister, G., Martial, J.
	7	A. and Muller, M. (2004). Use of reporter cell lines for detection of endocrine-disrupter
	8	activity. Analytical and Bioanalytical Chemistry 378: 655-663.
	9	Zhang, Q. Y., Dunbar, D., Ostrowska, A., Zeisloft, S., Yang, J. and Kaminsky, L. S.
	10 11 12 13	o F o F o F o F o F o F o F o F o (1999). Characterization of human small intestinal cytochromes P-450. Drug Metabolism and Disposition 27(7): 804-809. Zhou, H. R., Lau, A. S. and Pestka, J. J. (2003). Role of double-stranded RNA-activated protein kinase R (PKR) in deoxynivalenol-induced ribotoxic stress response. F o
	14	r r r r r r r r r Toxicological Sciences 74(2): 335-344. r
	15 16	P P P P P P P P P P
		e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r
		R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w
		O n O n O n O n O n O n O n O n O n O n
		l y l y l y l y l y l y l y l y l y l y
		41 42

Table 1 : Theoretical maximum daily intake (TMDI) and realistic intestinal concentration (C) of OTA and DON, estimated by a deterministic approach.

 1

	European maximal legal limits (MLs) in the main consumed foodstuffs specified in the
	Commission Regulation (EC) N°1881/2006 of 19 December 2006 and human European
	consumption data from GEMS/FOOD Regional Diets (WHO, 2003) (Q Ei ) were used
	(TWI, tolerable weekly intake; TDI, tolerable daily intake; bw, body weight).

ng/ml) 250 Page 45 of 53 http

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Table 2 : Theoretical maximum daily intake (TMDI) and realistic intestinal concentration (C) of IMA and BEN estimated by a

 2 Set by JMPR (http://www.inchem.org/pages/jmpr.html)2 Sum of benomyl and carbendazim, expressed as carbendazim3 Assuming a 70 kg body weight adult

	deterministic approach. European maximal limits of residues (MRLs) in food specified in the Commission Regulation (EC)
	N°396/2005 of 23 February 2005 (amended by Commission Regulation (EC) N°839/2008 of 31 July 2008 for BEN and Commission
	Regulation (EU) N o 750/2010 of 7 July 2010 for IMA) and human European consumption data from GEMS/FOOD Regional Diets
	(WHO, 2003) (Q Ei ) were used (ADI, acceptable daily intake; bw, body weight).
		Consumption data	IMA		BEN
				ADI 1 : 30 µg/kg bw/day	ADI 1 : 100 µg/kg bw/day
		Q Ei	GEMS	MRLs	TMDI	MRLs 2	TMDI
		(g/person/day)	code	(mg/kg)	(µg/person/day)	(mg/kg)	(µg/person/day)
	Cereals
	Barley	19.8	GC 640	0.05	0.99	2	39.6
	Maize	8.8	GC 645	0.05	0.44	0.01	0.088
	Oats	2	GC 647	0.05	0.1	2	4
	Rice	11.8	GC 649	0.05	0.59	0.01	0.118
	Rye	1.5	GC 650	0.05	0.075	0.1	0.15
	Wheat	178	GC 654	0.05	8.9	0.1	17.8
	Fruits
	Citrus fruits	44.6	FC 1	5	223	0.5	22.3
	Pome fruits	51.3	FP 9	2	102.6	0.2	10.26
	Stone fruits	23.3	FS 12	0.05	1.165
	Apricots	3.5	FS 240			0.2	0.7
	Cherries	3	FS 13			0.5	1.5
	Peaches	12.5	FS 247			0.2	2.5
	Plums	3.8	FS 14			0.5	1.9
	Small fruits and berries	23.5	FB 18	0.05	1.175
	Table grapes	13.8	FB 269			0.3	4.14
	Strawberries	5.3	FB 275			0.1	0.53
	Blueberries	0.5	FB 20			0.1	0.05
	Cranberries	0.3	FB 265			0.1	0.03
	Currants	0.3	FB 21			0.1	0.03
	Gooseberries	0.5	FB 268			0.1	0.05

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Acknowledgments

Public Service of Health, Food Chain, 27 Safety and Environment (contract RT 05/07 EMRISK).