Danielle Naville

Diane Rebourcet

Marie-Agnès Chauvin

Nathalie Vega

Audrey Jalabert

Michèle Vigier

Emmanuelle Loizon

Martine Bégeot

Dr Brigitte Le Magueresse-Battistoni

Naville Danielle

Rebourcet Diane

Vega Nathalie

Jalabert Audrey

Vigier Michèle

Loizon Emmanuelle

Bégeot Martine

Le Magueresse-Battistoni Brigitte

2 3

Keywords: Dioxin, Leydig cell, in vitro, AhR, chemokine

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Polycyclic aromatic hydrocarbons (PAHs), as well as halogenated aromatic hydrocarbons (HAHs) such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) and related compounds, are highly persistent environmental pollutants. Dioxins are by-products from incineration processes and pesticide production, and thus they are not intentionally produced.

They are considered endocrine disruptors [START_REF] Diamanti-Kandarakis | Endocrine-disrupting chemicals: an Endocrine Society scientific statement[END_REF][START_REF] Hotchkiss | Fifteen years after "Wingspread"-environmental endocrine disrupters and human and wildlife health: where we are today and where we need to go[END_REF][START_REF] Lundqvist | The effects of PCBs and dioxins on child health[END_REF], i.e. chemical substances that interfere with the endocrine systems of humans and wildlife. As such, they are of high concern for human health because endocrine disrupters are suspected to be responsible for apparent changes seen over recent decades, including congenital malformations, cancer and declining sperm counts [START_REF] Skakkebaek | Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects[END_REF][START_REF] Toppari | Male reproductive health and environmental xenoestrogens[END_REF]. Dioxins are not genotoxic but cause a broad spectrum of adverse effects including hepatotoxicity, immune system suppression, developmental toxicity, and skin defects. TCDD mediates its toxicity by binding to the aryl hydrocarbon receptor (AhR) and subsequent alteration of the expression of target genes which exhibit dioxin response elements in their promoter moiety including the cytochrome CYP1A1 [START_REF] Barouki | The aryl hydrocarbon receptor, more than a xenobiotic-interacting protein[END_REF][START_REF] Mimura | Functional role of AhR in the expression of toxic effects by TCDD[END_REF]. Apart from phase I and phase II enzymes of the detoxification machinery, microarray studies allowed identifying chemokines as potential new target genes in liver [START_REF] Boutros | Dioxin-dependent and dioxin-independent gene batteries: comparison of liver and kidney in AHR-null mice[END_REF][START_REF] Boutros | Transcriptomic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in liver: comparison of rat and mouse[END_REF] but also in testis [START_REF] Rebourcet | The effects of an in utero exposure to 2,3,7,8-tetrachloro-dibenzo-p-dioxin on male reproductive function: identification of Ccl5 as a potential marker[END_REF], suggesting that TCDD exposure may induce inflammatory responses in addition to detoxifying processes. Evidences also accumulated showing that inflammatory cytokines including the tumor necrosis factor  (TNF) were downstream targets of AhR signalling pathways [START_REF] Haarmann-Stemmann | Growth factors, cytokines and their receptors as downstream targets of arylhydrocarbon receptor (AhR) signaling pathways[END_REF]).

In the adult testis, Leydig cells, which are fully differentiated cells, produce the male primary steroid hormone, testosterone, in response to their trophic Luteinizing Hormone, LH. In addition to being under hormonal control, Leydig cells are under a local control exerted by autocrine and/or paracrine factors such as cytokines and chemokines [START_REF] Benahmed | Role of tumor necrosis factor in the male gonad[END_REF][START_REF] Guazzone | Cytokines and chemokines in testicular inflammation: A brief review[END_REF]. Leydig cells are presumed to be somewhat resistant to various types of chemotoxicants due to the insufficiency of CYP1A1 expression and activity as demonstrated elsewhere [START_REF] Chung | Cellular defense mechanisms against benzo[a]pyrene in testicular Leydig cells: implications of p53, aryl-hydrocarbon receptor, and cytochrome P450 1A1 status[END_REF]. The xenobiotic metabolizing enzyme CYP1B1 can also be induced by treatment with AhR agonists [START_REF] Shimada | Metabolic activation of polycyclic aromatic hydrocarbons to carcinogens by cytochromes P450 1A1 and 1B1[END_REF] and in contrast to CYP1A1, CYP1B1 is a predominantly extra-hepatic CYP enzyme.

For example, it is highly expressed in adrenals and gonads [START_REF] Leung | Developmental expression and endocrine regulation of CYP1B1 in rat testis[END_REF]. However, its regulation by PAHs in steroidogenic tissues is controversial [START_REF] Deb | CYP1B1 expression in rat testis and Leydig cells is not inducible by aryl hydrocarbon receptor agonists[END_REF][START_REF] Mandal | 7,12-Dimethylbenz[]anthracene inhibition of steroid production in MA-10 mouse Leydig tumor cells is not directly linked to induction of CYP1B1[END_REF]).

In the present study, we were interested in delving further into the adverse effects caused by TCDD on testis physiology focusing on Leydig cells using a model of primary cultures. We demonstrated that Leydig cells expressed AhR, its repressor AhRR and Cyp1B1 genes, and that they are under TCDD regulation. The chemokines Cxcl4 and Ccl5, previously identified in vivo as target genes for TCDD [START_REF] Rebourcet | The effects of an in utero exposure to 2,3,7,8-tetrachloro-dibenzo-p-dioxin on male reproductive function: identification of Ccl5 as a potential marker[END_REF] were directly (Cxcl4) or indirectly (Ccl5) regulated by TCDD. However, TCDD did not alter steroidogenic outcome of Leydig cells. Collectively, our data support the hypothesis that Leydig cells and thus the testis is not inert with respect to toxic insult. It also suggested that TCDD impacted the expression of target genes in Leydig cells under the complex network of hormones and cytokines.

Materials and Methods

Leydig cell preparation

Swiss male CD-1 mice aged of 8 weeks were purchased from Harlan Laboratories France (Gannat, France). Animals were killed by CO 2 asphyxia. Testes were removed and immediately used for cell preparations. All procedures were performed with the approval of the Regional Committee of Ethics for Animal Experiments.

Leydig cells were isolated and cultured in HAM's F12-DMEM (PAA Laboratories, Les Mureaux, France) at 32°C in an humidified atmosphere of 5% CO 2 as previously described [START_REF] Carreau | Stimulation of adult rat Leydig cell aromatase activity by a Sertoli cell factor[END_REF][START_REF] Mazaud Guittot | A comprehensive survey of the laminins and collagens type IV expressed in mouse Leydig cells and their regulation by LH/hCG[END_REF]. Briefly, testes were decapsulated and digested with 0.25 mg/ml collagenase at 32°C for 10 min. The digestion procedure was stopped by dilution with fresh medium. Interstitial cells were collected in the supernatant and the pellet containing aggregates of Sertoli and germ cells was discarded. Interstitial cells were purified on a discontinuous Percoll density gradient (layers of 21,26,34,40 and 60% Percoll). The gradient was centrifuged at 800 g for 30 min. The interface between 40 and 60% was collected and washed with medium to remove the Percoll. The presence of 3- hydroxysteroid dehydrogenase (3beta-HSD) activity was revealed by a histochemical technique described in details elsewhere [START_REF] Bilinska | Paracrine effect of seminiferous tubule factors on rat Leydig cell testosterone production: role of cytoskeleton[END_REF] using the anti-3beta-HSD antibody provided by Dr I. Mason (Reproductive and Developmental Sciences Division, Edinburgh, UK). It was used to determine the purity of Leydig cells (95%). Contamination by intestitial macrophages did not exceed 3%. Cells were resuspended in fresh culture medium supplemented with 2% Fetal Calf Serum (FCS). They were plated in 12-well plates (400,000 cells/well). After 24 h, culture media were replaced with serum-free culture media.

2 Leydig cell treatment

Two days after plating, culture media were renewed and 0.5 ml of fresh serum-free culture media was added per well in the presence or not of different products listed below for the time periods indicated in the text. These products included human chorionic gonadotropin (hCG; 100 ng/ml, Organon Schering-Plough, France) to mimic the effect of LH, dibutyryl cyclic AMP (dbcAMP; 0.2 mM, Sigma-Aldrich, France), recombinant Tumor Necrosis Factor alpha (TNF; 20 ng/ml; Peprotech France), Interleukin 1 alpha (IL1; 20 ng/ml, Peprotech France), Lipopolysaccharides (LPS; 10 microg/ml; Sigma-Aldrich, France). Cytokines were used at a 20 ng/ml concentration to maximally stimulate Leydig cells [START_REF] Benahmed | Role of tumor necrosis factor in the male gonad[END_REF]Hong et al. 2004). For 2,3,7,8-tetrachlorodibenzo-p-dioxin (ref ED-901-C; LGC Promochem, Molscheim France), stock solutions were prepared in dimethylsulfoxide (DMSO) and appropriately diluted in culture medium. DMSO volume content per well was 0.04 microl in a final volume of 0.5ml per well. Cell viability was determined by the MTS

[3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] reduction method
according to manufacturer's instructions (Promega, Charbonnières, France). Treatments did not alter cell viability which was over 95%. Doses of TCDD higher than 25 nM resulted in loss of cell viability and were not used in the present study.

Testosterone Measurement by Enzyme Immunoassay

Testosterone levels in the culture media were measured directly with a testosterone immunoassay kit purchased from Cayman Chemical Company (Interchim, Montlucon France). The sensitivity of the assay was 6 pg/ml.

RNA extraction, Reverse Transcription and Quantitative PCR (RT-qPCR)

Total RNA was extracted from cultured Leydig cells using TRI Reagent (Applied Bioystems, Courtaboeuf, France). RNA integrity was determined with the Agilent 2100 Bioanalyzer and RNA 6000 Nano Kit (Agilent Technologies, Massy, France). Briefly, Firststrand cDNAs were synthesized from 1 µg of total RNA in the presence of 100 U of Superscript II (Invitrogen, Eragny, France) and a mixture of random hexamers and oligo(dT) primers (Promega). Real-time PCR assays were performed in duplicates for each sample using a Rotor-Gene TM 6000 (Corbett Research, Mortlake, Australia), as described [START_REF] Rebourcet | The effects of an in utero exposure to 2,3,7,8-tetrachloro-dibenzo-p-dioxin on male reproductive function: identification of Ccl5 as a potential marker[END_REF]. The list of the primers used (Invitrogen, Eragny, France) is available on Table 1.

Briefly, PCR was performed in the presence of 0.4 µM of specific sense and antisense primers and 10 µl Absolute QPCR SYBR Green ROX mix (Thermo Fisher Scientific, Courtaboeuf, France), in a total volume of 20 µl. After the initial denaturation step of 15 min at 95°C, the reaction conditions were 40 cycles of 95°C for 15 sec, 55 to 62°C (depending on the primers, Table 1) for 10 sec, and 72°C for 20 sec. Melting curve analyses were performed immediately following the final PCR cycle to verify the specificity of the PCR product by checking its Tm.

Rpl19 (ribosomal protein L19) gene was chosen for normalizing target genes in the testis. It was consistently and reproducibly expressed in all samples, and it did not vary following treatments including TCDD treatment. Relative quantification was made by the standard curve method for both target and housekeeping gene (endogenous control) in each sample. A series of dilutions of calibrator sample (external standard) was included in each experiment in order to generate an external standard curve. Then the concentration of the target in each sample was divided by the concentration of the housekeeping gene in each sample, to correct for sample heterogeneity and variability of detection.

Statistical analysis

All experiments have been performed at least three times, with independent preparations of cells. Data were expressed as means ± SEM and the comparisons between treatments were made by one-way analysis of variance (ANOVA) followed by the post hoc Fisher PLSD test for multiple comparisons. A p value of less than 0.05 was considered significant. All statistical analyses were done with the aid of Statview 5.0 software package (SAS Institute Inc. Cary, NC 27513).

Results

Steroidogenic outcome of Leydig cells treated with TCDD

Addition of TCDD (25 nM) to Leydig cells did not alter basal and dbcAMP-induced testosterone production after 6 h of treatment (Fig. 1a). RT-qPCR experiments were also developed to survey expression levels of genes that mediate the first steps in steroidogenesis including the Steroidogenic acute regulatory protein (StAR) and Cyp11a1 in Leydig cells treated with TCDD, and stimulated or not with hCG or dbcAMP for 6 h. Within these conditions, TCDD had no impact on StAR (Fig. 1b) or Cyp11a1 (data not shown) gene expression levels.

Gene expression of AhR, the repressor AhRR and Cyp1b1, and their regulation by TCDD and hCG or dbcAMP

TCDD mainly exerts its action through binding to AhR. This step is followed by a rapid activation of target genes including the repressor AhRR, Cyp1A1 and Cyp1B1 [START_REF] Abel | An introduction to the molecular basics of aryl hydrocarbon receptor biology[END_REF][START_REF] Barouki | The aryl hydrocarbon receptor, more than a xenobiotic-interacting protein[END_REF]). Cyp1A1 is not expressed at all in the testis [START_REF] Chung | Cellular defense mechanisms against benzo[a]pyrene in testicular Leydig cells: implications of p53, aryl-hydrocarbon receptor, and cytochrome P450 1A1 status[END_REF][START_REF] Rebourcet | The effects of an in utero exposure to 2,3,7,8-tetrachloro-dibenzo-p-dioxin on male reproductive function: identification of Ccl5 as a potential marker[END_REF] nor was it induced following TCDD challenging in Leydig cells (data not shown). We found that Leydig cells expressed AhR, the repressor AhRR and Cyp1b1. TCDD added for 6 h regulated significantly (p<0.05) the level of mRNAs encoding AhR (a 40% decrease), AhRR (a 7 fold-increase) and Cyp1b1 (a 2.5 fold-increase) (Fig. 2). Addition of hCG 100 ng/ml or dbcAMP 0.2 mM had no effect on AhR and Cyp1b1 gene expression levels (Fig. 2). However, hCG significantly halved the TCDDinduced increase in Ahrr gene expression levels after 6 h of treatment (Fig. 2b). This effect was also observed after 24 h of treatment with a 4-fold decrease in hCG and TCDD co-treated cells versus TCDD-treated cells (data not shown). Addition of dbcAMP also opposed TCDD enhancement of AhRR gene expression levels after 6 h of treatment, indicating the involvement of Protein Kinase A (Fig. 2b).

Expression of the chemokines Ccl5 and Cxcl4 in primary culture of Leydig cells and their regulation by TCDD

We previously demonstrated that Ccl5 and Cxcl4 were 2 chemokines whose expression levels were altered in rat testis exposed to TCDD at gestational age 15. In addition, CCL5 immuno-reactivity was mostly confined to Leydig cells [START_REF] Rebourcet | The effects of an in utero exposure to 2,3,7,8-tetrachloro-dibenzo-p-dioxin on male reproductive function: identification of Ccl5 as a potential marker[END_REF]. We therefore determined by RT-qPCR that the chemokines Ccl5 and Cxcl4 were expressed at the mRNA level in the mouse cultured Leydig cells. In addition, we found that Cxcl4 (Fig. 3a) but not Ccl5 (Fig. 3b) gene expression levels were significantly up-regulated by TCDD and dbcAMP. Combined treatment resulted in additive effects (Fig. 3a).

Local regulation of the TCDD targeted genes by TNF

We investigated potential interactions between TNF and TCDD in the model of primary culture of Leydig cells. Cells were treated for 24 h. We found that TNF stimulated significantly (p<0.05) AhR but not AhRR gene expression levels (Fig. 4). The negative effects of TCDD on AhR gene expression levels observed at 6 h of treatment (Fig. 2a) were not observed at 24 h of treatment. However, TCDD-treated cells did no longer respond to TNF by enhanced AhR mRNA levels (Fig. 4a). Gene expression levels of Cxcl4 and Ccl5 were also investigated. The addition of TNF, IL1 and LPS, described as stimulators of chemokines [START_REF] Graves | Chemokines, a family of chemotactic cytokines[END_REF][START_REF] Nomiyama | The evolution of mammalian chemokine genes[END_REF]) up-regulated Cxcl4 (Fig. 5a) and Ccl5 (Fig. 5b) gene expression levels in primary culture of Leydig cells. TNF was a potent stimulator for the two chemokines (4 and 4.4-fold increase for Cxcl4 and Ccl5 respectively, p<0.05) (Fig. 5). By contrast, IL1 stimulated more Cxcl4 (7-fold increase) than Ccl5 (1.8fold-increase) gene expression. LPS stimulated more Ccl5 (8-fold increase) than Cxcl4 (2.8fold increase) gene expression (Fig. 5). Extending data presented in Fig. 3, we observed that TCDD could significantly (p<0.05) oppose the TNF-induced increase in Ccl5 mRNA levels (Fig. 5b); with no effect on TNF-induced increase in Cxcl4 mRNA levels (Fig. 5a). A summary of all the observed effects is provided on Table 2.

Discussion

Understanding the detrimental effects caused by TCDD is limited using animal models, and primary cell culture models represent useful tools to decipher mechanistic toxicities. In the present study, we were interested in delving further into the adverse effects caused by TCDD on Leydig cell physiology focusing on AhR dependent genes and chemokines, recently identified as TCDD target genes.

We first demonstrated that TCDD did not impact the steroidogenic outcome of Leydig cells assessed through measurement of StAR and Cyp11a1 expression levels, and testosterone production in Leydig cells cultured in basal conditions and under stimulation by the trophic hormone or dbcAMP. These data are consistent with previous in vitro studies showing no direct effect of TCDD on steroidogenesis in MA-10 mouse Leydig tumor cells and adult rat Leydig cells [START_REF] Mandal | 7,12-Dimethylbenz[]anthracene inhibition of steroid production in MA-10 mouse Leydig tumor cells is not directly linked to induction of CYP1B1[END_REF]. These data are also consistent with our previous study indicating no change in testosterone levels in the testes of rats exposed in utero to low doses of TCDD [START_REF] Rebourcet | The effects of an in utero exposure to 2,3,7,8-tetrachloro-dibenzo-p-dioxin on male reproductive function: identification of Ccl5 as a potential marker[END_REF], whereas reduced testosterone levels were observed in adult rats treated with very high doses of TCDD [START_REF] Johnson | 2,3,7,8-Tetrachlorodibenzo-p-dioxin reduces the number, size, and organelle content of Leydig cells in adult rat testes[END_REF][START_REF] Kleeman | Inhibition of testicular steroidogenesis in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats: evidence that the key lesion occurs prior to or during pregnenolone formation[END_REF][START_REF] Moore | Androgenic deficiency in male rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin[END_REF].

Leydig cells recovered from fully mature animals are known to express AhR [START_REF] Schultz | Expression of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator messenger ribonucleic acids and proteins in rat and human testis[END_REF], but no reports have yet identified the TCDD regulation of AhR in Leydig cells nor determined if the repressor AhRR was present in Leydig cells and regulated by TCDD. We found that TCDD down-regulated transiently the expression of AhR gene, and enhanced the expression of AhRR gene, suggesting negative feedback to limit AhR signalling effect. Indeed, it has been previously shown that AhRR functions as a naturally occurring dominant-negative factor limiting AhR signalling pathway [START_REF] Abel | An introduction to the molecular basics of aryl hydrocarbon receptor biology[END_REF][START_REF] Mimura | Identification of a novel mechanism of regulation of Ah (dioxin) receptor function[END_REF]. Hence, AHRR is an important determinant of tissue specific responsiveness to TCDD, and strong evidences have been reported on an inverse relationship between AHRR expression and sensitivity to induction of xenobiotic-metabolizing enzymes caused by TCDD [START_REF] Korkalainen | Primary structure and inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) of aryl hydrocarbon receptor repressor in a TCDD-sensitive and a TCDD-resistant rat strain[END_REF]). The data presented herein i.e., high expression of AhRR and no detectable Cyp1a1 induction are in keeping with these observations. Interestingly, in conditions of increased gene expression levels of StAR following hCG or dbcAMP stimulation, genes encoding AhR and AhRR had unchanged expression levels. We next investigated the combined effects of TCDD with hCG/dbcAMP or TNF. Indeed, TNF may originate from the systemic circulation or from the neighbouring interstitial macrophages [START_REF] Guazzone | Cytokines and chemokines in testicular inflammation: A brief review[END_REF]. We found that addition of hCG which protects testis function against TCDD in adult rats [START_REF] Lai | Inhibition of CYP450scc expression in dioxin-exposed rat Leydig cells[END_REF], while not acting directly on AhR or AhRR gene expression levels, attenuated the TCDD-induced stimulation of AhRR. Unlike hCG, TNF did not reduce TCDD action on the AhRR gene expression levels, and enhanced AhR expression levels. Altogether, these data are in keeping with previous studies showing that gonadotrophins and TNF exert inverse effects on Leydig cells [START_REF] Benahmed | Role of tumor necrosis factor in the male gonad[END_REF][START_REF] Guazzone | Cytokines and chemokines in testicular inflammation: A brief review[END_REF]).

Testicular Cyp1B1 is localized predominantly in Leydig cells [START_REF] Ge | Gene expression in rat leydig cells during development from the progenitor to adult stage: a cluster analysis[END_REF]) and subject to developmental regulation in rat testis [START_REF] Leung | Developmental expression and endocrine regulation of CYP1B1 in rat testis[END_REF]. Herein, we found that TCDD enhanced Cyp1b1 expression levels in Leydig cells but hCG and dbcAMP had no effect. These data do not support previous studies [START_REF] Deb | Regulation of Cytochrome P450 1B1 Expression by Luteinizing Hormone in Mouse MA-10 and Rat R2C Leydig Cells: Role of Protein Kinase A[END_REF][START_REF] Deb | CYP1B1 expression in rat testis and Leydig cells is not inducible by aryl hydrocarbon receptor agonists[END_REF]. Such a discrepancy may result from the use of primary cultures of adult Leydig cells in our system versus Leydig cell lines in Deb and collaborators. In addition, adult Leydig cells are less responsive to gonadotropins with aging [START_REF] Midzak | Leydig cell aging and the mechanisms of reduced testosterone synthesis[END_REF]) and, Deb and collaborators [START_REF] Deb | CYP1B1 expression in rat testis and Leydig cells is not inducible by aryl hydrocarbon receptor agonists[END_REF]) also observed that in vivo treatment with TCDD increased testicular CYP1B1 protein levels in adult rats. The meaning of the enhancement of Cyp1b1 by TCDD is unclear. TCDD is not metabolized by CYP1B1. Besides, CYP1B1 is not required for mammalian development, reproductive vitality and physiological homeostasis as demonstrated by the absence of major phenotype in Cyp1b1-null mice [START_REF] Buters | Cytochrome P450 CYP1B1 determines susceptibility to 7, 12-dimethylbenz[a]anthracene-induced lymphomas[END_REF].

More studies are required to elucidate the role CYP1B1 may play in Leydig cell physiology.

It has been recently demonstrated that TCDD apart from the classical battery of genes of the detoxification machinery, may provoke inflammatory adverse effects [START_REF] Haarmann-Stemmann | Growth factors, cytokines and their receptors as downstream targets of arylhydrocarbon receptor (AhR) signaling pathways[END_REF]. For example, it was demonstrated that TCDD could enhance TNF secretion in a model of cultured adipocytes [START_REF] Nishiumi | 2,3,7,8tetrachlorodibenzo-p-dioxin impairs an insulin signaling pathway through the induction of tumor necrosis factor-alpha in adipocytes[END_REF] as well as in the serum of rats orally treated with TCDD [START_REF] Ciftci | Protective effect of curcumin on immune system and body weight gain on rats intoxicated with 2,3,7,8-Tetrachlorodibenzo-pdioxin (TCDD)[END_REF]. It was also shown that dioxin through binding to AhR could stimulate Ccl5 gene expression in an in vitro model of endometriosis [START_REF] Yu | Combination of estrogen and dioxin is involved in the pathogenesis of endometriosis by promoting chemokine secretion and invasion of endometrial stromal cells[END_REF][START_REF] Zhao | Dioxin stimulates RANTES expression in an in-vitro model of endometriosis[END_REF], Ccl1, a chemokine involved in cardiovascular diseases and inflammatory or allergic processes (N' Diaye et al. 2006) and Ccl2 in multiple organs of mice [START_REF] Vogel | Modulation of the chemokines KC and MCP-1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice[END_REF]. TCDD also inhibited Cxcl12 gene expression and its receptor Cxcr4 in MCF-7 breast cancer cells [START_REF] Hsu | A proposed mechanism for the protective effect of dioxin against breast cancer[END_REF]. Chemokines are a subset of small chemotactic cytokines produced by both immuno-competent cells and nonimmune cells. They are secondary pro-inflammatory mediators that are induced by cytokines such as interleukin-1 or TNF [START_REF] Graves | Chemokines, a family of chemotactic cytokines[END_REF][START_REF] Nomiyama | The evolution of mammalian chemokine genes[END_REF]. We here found that Leydig cells could express Ccl5 and Cxcl4, and that these chemokines responded to stimulation by LPS, IL1 and TNF. In addition, depending on the conditions (presence or not of TNF), these two chemokines were targeted by TCDD in primary cultures of adult mouse Leydig cells, consistently with our previous in vivo findings [START_REF] Rebourcet | The effects of an in utero exposure to 2,3,7,8-tetrachloro-dibenzo-p-dioxin on male reproductive function: identification of Ccl5 as a potential marker[END_REF].

Collectively, we demonstrated that TCDD can directly and indirectly impact in vitro adult Leydig cell function through alteration in the gene expression levels of chemokines as well as AhR signalling pathway, supporting the hypothesis that Leydig cells and thus the testis are not inert with respect to toxic insult. If considering that TCDD may stimulate TNF secretion, our data raise the hypothesis that the resulting AhR activation in testis following an in vivo TCDD insult might be the sum of a direct action by TCDD itself on Leydig cells and an indirect inflammatory response with elevated systemic TNF subsequently activating AhR signalling pathway in Leydig cells. More insight into the molecular mechanisms behind the influence of TCDD on Leydig cells will require determining the involvement of the TNF signalling transduction pathways, specifically the involvement of NF-B and mitogenactivated protein kinases (MAPKs). Indeed, and depending on cell types and culture conditions, it was shown that hCG could attenuate NF-B activation and cytokine expression [START_REF] Huber | Human chorionic gonadotrophin attenuates NF-kappaB activation and cytokine expression of endometriotic stromal cells[END_REF], and trigger transient MAPK kinase activation [START_REF] Brion | MAPK phosphatase-1 (MKP-1) expression is up-regulated by hCG/cAMP and modulates steroidogenesis in MA-10 Leydig cells[END_REF].

Therefore, integrated responses to TCDD would be highly dependent on the cell type, its local and hormonal environment.

  the expected PCR fragment in base pairs (bp) and the hybridation temperature (Topt) for annealing are reported.

Figure 1 :

 1 FIGURE LEGENDS:

Figure 2 :

 2 Figure 2: Relative expression of Ahr (a), Ahrr (b) and Cyp1b1 (c) in primary cultures of Leydig cells recovered from 8-week old mouse testes treated for 6 h with various factors and assessed by RT-qPCR analysis. Factors added included TCDD (25 nM), hCG (100 ng/ml), or dbcAMP (0.2 mM). Ahr (a), Ahrr (b) and Cyp1b1 (c) levels were normalized using Rpl19 and are expressed relatively to controls arbitrarily fixed to 1. Values are the mean ± SEM of n= 4 separate experiments. a, p<0.05 versus its respective control; b, p<0.05 versus TCDD-treated samples in (b). Boxes in black correspond to controls and boxes in grey indicate treatment with TCDD.

Figure 3 :

 3 Figure 3: Relative expression of Cxcl4 (a) and Ccl5 (b) in primary cultures of Leydig cells recovered from 8-week old mouse testes treated for 24 h with various factors and assessed by RT-qPCR analysis. Factors added included TCDD (25 nM), dbcAMP (0.2 mM) or the two. Cxcl4 (a) and Ccl5 (b) levels were normalized using Rpl19 and are expressed relatively to controls arbitrarily fixed to 1. Values are the mean ± SEM of n= 6 separate experiments. a, p<0.05 versus its respective control; b, p<0.05 versus dbcAMP and TCDD-treated cells. Boxes in black correspond to controls and boxes in grey indicate treatment with TCDD.

Figure 4 :

 4 Figure 4: Relative expression of Ahr (a) and Ahrr (b) in primary cultures of Leydig cells recovered from 8-week old mouse testes treated for 24 h with various factors and assessed by RT-qPCR analysis. Factors added included TCDD (25 nM), TNF (20 ng/ml) or the two. Ahr and Ahrr levels were normalized using Rpl19 and are expressed relatively to controls arbitrarily fixed to 1. Values are the mean ± SEM of n= 8 separate experiments. a, p<0.05

Table 2 : Summary of the effects described

 2

			Stimuli added in vitro
	Genes surveyed	TCDD	hCG/dbcAMP	TNF
	Star and
	Cyp11a1	no effect	strong up-regulation	not determined
		down-regulation at 6 h, not at 24 h;
	AhR	TCDD opposed TNF-effect on	no effect	up-regulation at 24 h
		AhR at 24 h
	AhRR	up-regulation at 6 & 24 h	no effect per se; can oppose	no effect
			TCDD-effect on AhRR at 6 & 24 h
	Cyp1b1	up-regulation at 6 h	no effect	not determined
	Cxcl4	up-regulation at 24 h	up-regulation at 24 h	up-regulation at 24 h
	Ccl5	no effect per se; can oppose	no effect	up-regulation at 24 h
		TNF-effect at 24 h

Acknowledgments :

We are grateful to Emilie Christin for technical assistance. We thanked Dr I Mason (Reproductive and Developmental Sciences Division, Edinburgh, UK) for providing the anti-3beta-HSD antibody. This work was supported by Inserm (Institut National de la Santé et de la Recherche Médicale), INRA (Institut National de la Recherche Agronomique), University of Lyon 1 and specific grants from AFSSET (EST-2006/1/33) and ANR (ANR-06-PNRA-006-01). DR was funded by ANR (ANR-06-PNRA-006-01) and Schering-Plough (FARO Organon).

BLM and DN designed the study. DN, MAC and BLM performed the cultures and treatment. NV, MV, EL performed the QPCR dosages. DR performed the immunostaining and cell viability tests. AJ and MV performed the western blotting experiments. BLM, DN and MB analyzed the data and wrote the paper. All authors read and approved the final manuscript.

versus its respective control; b, p<0.05 versus TCDD or TCDD and TNF-treated cells in (b). Boxes in black correspond to controls and boxes in grey indicate treatment with TCDD.


PhD Brigitte Le Magueresse-Battistoni

Hubert Vidal

Danielle Naville

Life-long consumption of low-dosed food pollutants on metabolic health

Abstract:

Obesity is a major public health problem because it is a risk factor for metabolic disorders including type 2 diabetes and cardiovascular disorders. Notably, food pollutants have been charged to contribute to the etiology of obesity and type 2 diabetes. Chemicals are either persistent or short-lived but in either case they have invaded our lives, and diet is the main route of exposure. However, the current threshold model used in risk assessment focused on single chemicals and does not take into account potential effects from mixtures containing pollutants at environmental doses, e.g. the real life exposure. Furthermore, recent data suggest that the deleterious metabolic impact of pollutants may be aggravated in a context of obesity.

These findings stress the importance of better delineating the metabolic consequences of lifelong consumption of food pollutants, notably when considering vulnerable populations including fetuses and young children as well as populations at risk for metabolic disorders such as overweight and obese people. With this in mind, we recently demonstrated that a mixture of food pollutants at doses lower than the accepted ineffective concentrations does indeed have metabolic and health consequences in chronically exposed obese mice. This proof-of-concept-study highlights the needs for breakthrough of ideas to strengthen public health action for a better protection.

Obesity is a major public health problem because it is a risk factor for metabolic disorders including type 2 diabetes and cardiovascular disorders. Its evolution is pandemic with more than 1.4 billion adults overweight in 2008, and the WHO predicts that by 2015 about 700 million people will be obese. Nowadays, an estimate of 347 million people worldwide has diabetes, and diabetes is projected to be the seventh leading cause of death in 2030 [START_REF] Naville | Obesity and Overweight[END_REF] . In addition, diabetes has a heavy cost on the economy and quality of life. For example, the total estimated cost of diagnosed diabetes in 2012 in the USA was $245 billion 2 . Type 2 diabetes results from the body's ineffective use of insulin, a hypoglycaemic hormone secreted by the pancreas that plays a major metabolic role in maintaining blood glucose levels in the normal range. The first step leading to diabetes is insulin resistance that develops in the metabolically active tissues (i.e., liver, skeletal muscle, and adipose tissues) and eventually leads to the classic triad of hyperinsulinemia, hyperglycemia and hypertriglyceridemia, which are the characteristic features of type 2 diabetes 3 .

Obesity is a multifactorial disease in which, besides genetic predisposition, excessive food intake combined with physical inactivity has generally been implicated. However, it was recently suggested that food pollutants could contribute to the etiology of obesity and type 2 diabetes [START_REF] Thayer | Role of environmental chemicals in diabetes and obesity: a National Toxicology Program workshop review[END_REF] . This hypothesis is based on several findings including the speed with which the incidence of obesity and its related metabolic disorders increases that excludes the only involvement of genetic factors and strengthens the environmental origin hypothesis. Furthermore, the production and usage of synthetic chemicals that have paralleled the incidence of diabetes [START_REF] Neel | The paradox of progress: environmental disruption of metabolism and the diabetes epidemic[END_REF] support the hypothesis that the exposure to chemicals may have damaged physiological mechanisms regulating glucose and lipid metabolism [START_REF] Pf | Chemical toxins: a hypothesis to explain the global obesity epidemic[END_REF] . This assumption is also based on epidemiological studies, which associated the prevalence of type 2 diabetes with elevated body burdens of chemicals 4 7 , and experimental studies in rodents, which established a causal relationship between exposure to chemicals and the development of metabolic disorders including obesity and insulin resistance [START_REF] Chamorro-Garcia | Transgenerational inheritance of increased fat depot size, stem cell reprogramming, and hepatic steatosis elicited by prenatal exposure to the obesogen tributyltin in mice[END_REF][START_REF] Ibrahim | Chronic consumption of farmed salmon containing persistent organic pollutants causes insulin resistance and obesity in mice[END_REF][START_REF] Ruzzin | Persistent organic pollutant exposure leads to insulin resistance syndrome[END_REF] .

Chemical pollutants end up in the plates

Based on their biodegradability, chemicals may be either highly persistent in the environment or non-persistent. Persistent Organic Pollutants (POPs) are chemicals created intentionally by industrial activities such as the polychlorobiphenyls (PCBs) made for their flame-retardant properties. Other POPs may be unwanted industrial by-products such as the dioxins which result from incomplete combustion during industrial processes. Production of POPs is limited (eg, dioxins) or was banned (eg, PCBs) in the 1970s because of accumulating evidences of their deleterious effects on wildlife [START_REF] Edwards | Environmental exposures and gene regulation in disease etiology[END_REF] . However, owing to their persistence, all environmental compartments remain contaminated. These highly lipophilic compounds accumulate higher up the food chain in a process called bioaccumulation, being present in virtually all categories of foods and especially in fatty foods [START_REF] Schafer | Persistent toxic chemicals in the US food supply[END_REF] . Other chemicals are shortlived compounds, but, because of their massive industrial production, particularly in the plastic industry, they are omnipresent in the environment. For example, bisphenol A (BPA) used in the manufacture of polycarbonate is constitutive of many food containers. Similarly, resins containing BPA are used in the lining of cans. Phthalates are another class of chemicals that is widely used in plastics to make them soft and flexible. BPA and phthalates can leach from food and beverage containers and packaging to cause contamination by migration, especially when heated 13 14 . Thus, diet is the main route of exposure to pollutants whether persistent or not. In addition, pollutants can transfer from the mother to the fetus through the placenta during pregnancy and through breastfeeding [START_REF] Heindel | Role of nutrition and environmental endocrine disrupting chemicals during the perinatal period on the aetiology of obesity[END_REF] .

Chemical pollutants, endocrine and metabolic disruption

Many chemicals are considered as Endocrine Disrupting Compounds (EDCs). Although the definition of an EDC is still a matter of debate, the WHO definition states that an endocrine disrupter is an exogenous substance or a mixture that alters function(s) of the endocrine system and consequently causes adverse effects in an intact organism, or its progeny, or (sub)populations [START_REF] Who | Global assessment of the state of the science of endocrine disruptors[END_REF] . There are multiple mechanisms through which EDCs may interfere with the endocrine system. In addition to acting as direct agonist or antagonist compounds through binding to nuclear receptors, EDCs may affect the synthesis, transport, metabolism and excretion of hormones, thus altering the plasma concentrations of natural hormones.

Originally, EDCs have been charged to contribute, among others, to declining sperm counts in the offspring of exposed pregnant mothers and increased incidence of male children born with genital malformations [START_REF] Jea | Male reproductive health and environmental xenoestrogens[END_REF] . It was next suggested that some EDCs may as well exhibit metabolic disrupting activities through interaction with nuclear receptors that were hormone receptors, xenosensors or liposensors such as the peroxisome proliferator receptors (PPARs) [START_REF] Casals-Casas | Endocrine disruptors: from endocrine to metabolic disruption[END_REF] . A subset was identified as obesogens because they promote adipocyte differentiation and fat storage [START_REF] Grun | Perturbed nuclear receptor signaling by environmental obesogens as emerging factors in the obesity crisis[END_REF] .

These nuclear receptors are expressed in the metabolically active tissues, both at central and peripheral levels, and they play crucial roles in integrating the complexities of homeostasis and development, including insulin secretion and sensitivity to this hormone which is instrumental in the regulation of glucose and lipid metabolisms and in adipogenesis [START_REF] Casals-Casas | Endocrine disruptors: from endocrine to metabolic disruption[END_REF] . For example, estrogens play a pivotal role in regulating energy homeostasis [START_REF]Estrogen and androgen receptors: regulators of fuel homeostasis and emerging targets for diabetes and obesity[END_REF] . Besides, inappropriate activation of androgen receptor (AR), thyroid hormone receptor (TR) and glucocorticoid receptor (GR) also leads to impaired metabolic homeostasis 18 21 22 23 .

Furthermore, the xenosensors activated by numerous steroids and EDCs are involved in metabolic disease states such as steatosis, obesity and insulin resistance in addition to their primarily role in xenobiotic detoxification [START_REF] Kretschmer | CAR and PXR: xenosensors of endocrine disrupters? Chemicobiological interactions[END_REF][START_REF] Merrill | Toxicological function of adipose tissue: focus on persistent organic pollutants[END_REF][START_REF] Spruiell | Role of pregnane X receptor in obesity and glucose homeostasis in male mice[END_REF] . As such, the final consequences of an exposure to chemicals will depend on the complexities of the interactions between the diverse members of the nuclear receptor family to convey normal metabolic regulations [START_REF] Casals-Casas | Endocrine disruptors: from endocrine to metabolic disruption[END_REF] . Also, chemicals might target different nuclear receptors depending on the metabolic tissues (as described above) and their integrative effects would rely on cross-talk with the other nuclear receptors, as shown with the obesogen tributyltin (TBT). TBT is a powerful biocide used as an "antifouling" agent in paints for ships' hulls, and now banned from use, which acts as an adipogenic agonist through interference both with estrogen signalling and the PPAR pathway [START_REF] Grun | Perturbed nuclear receptor signaling by environmental obesogens as emerging factors in the obesity crisis[END_REF] . On top of this, POPs like dioxins might also display inflammatory activities favouring the development of resistance to insulin in adipose tissues [START_REF] Merrill | Toxicological function of adipose tissue: focus on persistent organic pollutants[END_REF] . It is also important to keep in mind that these events depend on sex, the developmental age, and the food matrix (particularly the lipid content).

Chemical pollutants and the limits of the current threshold model used in risk assessment

Human health risk assessments focused on single chemicals by setting up Tolerable Daily Intake (TDI) reference doses that are defined by international agencies, such as the U.S.

Environmental Protection Agency (EPA) or the European Food Safety Agency (EFSA). TDI reference doses derived from the no-observed-adverse-effect levels (NOAELs) or the lowestobserved-adverse-effect levels (LOAELs) in animal studies. The supposedly most sensitive animal is being used and the most sensitive endpoint is being considered for the establishment of either the NOAEL or the LOAEL. Then this value is to be divided by an uncertainty factor ranging from 100 to 1000 to take into account interspecies as well as interindividual variation [START_REF] Dorne | Metabolism, variability and risk assessment[END_REF] . Thus, the TDI is an estimate of the amount of a given chemical in food or drinking water that can be ingested daily over a lifetime without a significant health risk. While these reference doses are protective from a toxicological point of view, they may not sufficiently protect the population from an endocrine and metabolic point of view, and it was recently proposed that the principles of endocrinology should be considered in the making of the regulatory decisions on EDCs [START_REF] Vandenberg | Regulatory decisions on endocrine disrupting chemicals should be based on the principles of endocrinology[END_REF] . Specifically, since EDCs mimic/antagonize hormones, chemicals defined as EDCs should behave as hormones in many features, that is, they should act at low doses, exhibit non-linear, and often non-monotonic dose-response curves and their effects should be life-stage dependent in the context of multi-hormonal exposure.

Consistently, adverse metabolic effects have been documented using doses near and even below the TDIs, especially for BPA 29 30 . Moreover, the population is exposed to a cocktail of pollutants [START_REF] Koch | Human body burdens of chemicals used in plastic manufacture[END_REF] , which resulting effect cannot be predicted on the basis of the effects of each individual pollutant, eluding possible agonist, antagonist or synergistic endocrine/metabolic interactions between chemicals in a context of multi-exposure. For dioxins and related POP congeners whose toxic effects are presumed to result from the binding to the xenosensor aryl hydrocarbon receptor (AhR), it is assumed that the combined effects of the congeners produce additive effects [START_REF] Van Den Berg | Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife[END_REF] . A concentration addition model was also proposed for mixtures of antiandrogenic compounds [START_REF] Hass | Adverse effects on sexual development in rat offspring after low dose exposure to a mixture of endocrine disrupting pesticides[END_REF] . These concepts are largely debated nowadays because the biological effects of one particular EDC are different depending on the presence of the other EDCs. Moreover, the effects would be different depending on the doses of the other EDCs and on related endogenous hormone levels making uncountable the number of situations [START_REF] Hertzberg | A four-step approach to evaluate mixtures for consistency with dose addition[END_REF][START_REF] Carvalho | Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they? Toxicological sciences : an official[END_REF][START_REF] Zoeller | Endocrine-disrupting chemicals and public health protection: a statement of principles from The Endocrine Society[END_REF][START_REF] Ruzzin | Public health concern behind the exposure to persistent organic pollutants and the risk of metabolic diseases[END_REF] . Thus, while the dose is a critical factor in toxicology, the vulnerability of the target as well as the possible complex non-monotonous relationship between dose and effect observed for some pollutants have to be taken into account with EDCs. An additional degree of complexity was further provided when interactions were evidenced between obesity and serum concentrations of pollutants on the prevalence of type 2 diabetes 37 . Interactions were also observed in animal experiments and it was found that the metabolic deleterious impact of BPA was aggravated in animals fed a high-fat diet [START_REF] Wei | Perinatal exposure to bisphenol A at reference dose predisposes offspring to metabolic syndrome in adult rats on a high-fat diet[END_REF] . Thus, exposure to pollutants and diet imbalance may be two sides of the same coin as suggested by Barouki et al [START_REF] Barouki | Developmental origins of noncommunicable disease: Implications for research and public health[END_REF] to pinpoint that adaptation to either stress is sharing many of the same characteristics as chemicals originally identified for their endocrine disrupting properties are also capable of metabolic disturbances [START_REF] Casals-Casas | Endocrine disruptors: from endocrine to metabolic disruption[END_REF] . Furthermore, recent reports indicated that EDCs could regulate patterns of gene expression through epigenetic modifications such as DNA methylation and histone modifications. The process does not directly modify the DNA sequence but it alters the methylated or acetylated environment of the DNA sequence, facilitating/repressing transcription factors' access to the promoters of the targeted genes and altering gene expression 40 41 . Thus, pollutants through epigenetic mechanisms could interfere with the metabolic programming trajectories set up during the maternal life, a highly vulnerable period of development, as described in case of nutritional stress with long lasting health effects affecting the entire life of the individuals. This is the DOHaD hypothesis for Developmental Origins of Health and Disease [START_REF] Barker | The developmental origins of insulin resistance[END_REF] . Long-lasting effects may also pass the generations, and individuals may suffer from exposure of their grandparents. For example, in utero exposure of the F1 generation to TBT induces hepatic steatosis and adipose tissue expansion in the nonexposed subsequent generations [START_REF] Chamorro-Garcia | Transgenerational inheritance of increased fat depot size, stem cell reprogramming, and hepatic steatosis elicited by prenatal exposure to the obesogen tributyltin in mice[END_REF] . Transgenerational obesogenic effects were also described in the F3 generation originated from F1 generation rats in utero exposed to either mixtures of plasticizers (BPA, phthalates), or the insecticide dichlorodiphenyltrichloroethane (DDT) [START_REF] Skinner | Environmental stress and epigenetic transgenerational inheritance[END_REF] .

Development of a proof-of-concept study

In this context of uncertainties related to the impact of pollutants on human health, notably during fetal and perinatal periods considered of very high vulnerability, it is essential to study the cocktail effect of low-dosed chemicals. While it is hardly possible to estimate the risk for all scenarios, relevant mixtures of well-characterized pollutants may be studied by taking into account parameters such as geographical locations, sex and diet habits. Having this in mind, we recently developed an experimental model, [START_REF] Naville | Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice[END_REF] which is original because of the lifelong exposure to pollutants, encompassing maternal life (preconception, gestation and lactation). In addition, mice were fed a high-fat high sucrose diet (HFSD) to mimic Western diet and reflect occidental diet habits. Thus, these mice are at high risk of insulin resistance and other metabolic disorders [START_REF] Gout | Metabolic and melanocortin gene expression alterations in male offspring of obese mice[END_REF] . The mixture comprised pollutants largely occurring in foods, especially fatty food, either persistent (PCB153; 2, 3, 7, 8-tetrachlorodibenzo-p-dioxine, TCDD) or not persistent (BPA; Di-2-EthylHexylPhtalate, DEHP) and activating a broad range of signalling pathways. They are classified as EDCs and are of high concern for human health because of their established links with metabolic diseases in epidemiological and experimental studies 18 37 . Specifically, TCDD mainly acts through binding the AhR and exerts estrogenic-like effects depending on the hormonal context [START_REF] Fea | Modulation of oestrogen receptor signalling by association with the activated dioxin receptor[END_REF] ; PCB153 is a non-dioxin-like PCB with estrogenic-like activities and is considered as a surrogate for the total PCB exposure [START_REF] Hagmar | Intra-individual variations and time trends 1991-2001 in human serum levels of PCB, DDE and hexachlorobenzene[END_REF] ; BPA is the archetypal example of an estrogenomimetic EDC interacting with several nuclear receptors to promote insulin secretion, obesity and hepatic insulin resistance [START_REF] Garcia-Arevalo | Exposure to bisphenol-A during pregnancy partially mimics the effects of a high-fat diet altering glucose homeostasis and gene expression in adult male mice[END_REF] ; DEHP is a well-known activator of the PPARs [START_REF] Casals-Casas | Endocrine disruptors: from endocrine to metabolic disruption[END_REF] . Pollutants in the mixture were low-dosed, in the range of the TDI defined by international agencies for humans, and thus at least 500 fold lower than the NOAEL for each chemical supposedly defined on the most sensitive species and on the most sensitive end-point. As a preliminary study in the setting of the experimental model, we used a mixture with doses in the range of the NOAEL for each pollutant, and a general toxicity related to litter size and pup survival was observed [START_REF] Naville | Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice[END_REF] . No such toxicity was described using the mixture in the TDI range. However, we observed significant sexdependent metabolic modifications in the absence of weight modification, underscoring the importance of studying both males and females. In males, although pollutants did not alter glucose and insulin metabolic tests or plasma cholesterol levels, we observed changes in cholesterol metabolism with a decrease in hepatic total cholesterol together with enhanced expression of genes encoding the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAR) which is the rate limiting-enzyme in cholesterol synthesis, and proteins involved in the bile salt pathway [START_REF] Naville | Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice[END_REF] . In females, there was deterioration of glucose tolerance, and we hypothesized that this effect was related to the twofold induction of estrogen sulfotransferase (EST/SULT1E1), impairing estrogen signalling in the liver. Indeed, through conjugation of a sulfonate group, EST/SULT1E1 inactivates estrogens preventing their binding to the highaffinity estrogen receptor, thus modulating estrogen bioavailability [START_REF]Estrogen sulfotransferase: intracrinology meets metabolic diseases[END_REF] . We interpreted these data as an indication that the cocktail of pollutants could have opposed the well-described protective effects of estrogens from metabolic disorders such as insulin resistance, in females.

More experiments will aid in defining whether these events represented an adverse or a compensatory adaptive response to the mixture of pollutants.

Conclusion

Collectively, our experimental model is a proof-of-concept that low doses of pollutants, in the range of doses considered ineffective for humans, are in fact not harmless when exposure is chronic and when pollutants are administered in combination in a context of obesity caused by a calorie-rich diet. Of course, there are limitations in these experiments. We did not assess individually each pollutant that made up the mixture; nor did we yet get data with other concentrations of the mixture. Indeed, the two higher doses tested which were in the range of the NOAEL doses for each pollutant and 10-times higher, resulted in general toxicity with a reduced litter size and lowered pup survival [START_REF] Naville | Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice[END_REF] . Another limitation is that these data were yielded in a rodent model. However, unless it is considered that the adverse effects seen in rodents are not expected to occur in humans (and the epidemiologic studies performed so far do not fit this hypothesis), the principle of precaution should guide the making of decisions, especially when considering women of childbearing age and young infants particularly exposed because of mouthing activities. Substitute replacement, while promising, should be carried out with caution because interference with the endocrine system was reported for several structural analogues of BPA [START_REF] Rosenmai | Are structural analogues to bisphenol a safe alternatives? Toxicological sciences : an official[END_REF] . In the meantime, it is estimated that removing BPA from food sources would allow sparing $ 2 billion annually in the US [START_REF] Trasande | Further limiting bisphenol a in food uses could provide health and economic benefits[END_REF] because of prevention of childhood obesity and of its complications over the life course.

Effective solutions rely on a breakthrough of ideas for better life as well as economic benefits.

They require huge support from governments and economic players to contribute to the improvement of public health.

Acknowledgments:

The authors are very grateful to the colleagues Claudie Pinteur, Nathalie Vega, Yoan Menade, Michèle Vigier, Alexandre Le Bourdais, Emmanuel Labaronne, Cyrille Debard, Céline Luquain-Costaz and Martine Bégeot, all from CarMeN laboratory Lyon.