Muhammad Imran

Frédéric Chalmel

Odile Sergent

Bertrand Evrard

Hélène Le Mentec

Antoine Legrand

Aurélien Dupont

Maëlle Bescher

Simon Bucher

Bernard Fromenty

Laurence Huc

Lydie Sparfel

Dominique Lagadic-Gossmann

Normand Podechard
email: normandpodechard@univ-rennes1.fr

Transcriptomic analysis in zebrafish larvae identifies iron-dependent mitochondrial dysfunction as a possible key event of NAFLD progression induced by benzo[a]pyrene/ethanol co-exposure

Keywords: NAFLD, iron homeostasis, mitochondrial dysfunction, AhR, B[a]P, ethanol, zebrafish Aryl hydrocarbon receptor, AhR-KO: AhR knock out, AMEN: Annotation, Mapping, Oxygen consumption rate, OXPHOS: Oxidative phosphorylation, PAH: Polycyclic aryl hydrocarbon, PBS: Phosphate-buffered saline, PMT: Photomultiplier tube, ROS: Reactive oxygen species, SEM: Standard error of the mean, T2DM: Type 2 Diabetes mellitus, TAFLD: Toxicant-Associated Fatty Liver Diseases, TASH: Toxicant-Associated Steatohepatitis, TCDD: 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin, TEM: Transmission electronic microscopy

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Transcriptomic analysis in zebrafish larvae identifies iron-dependent mitochondrial dysfunction as a possible key event of NAFLD progression induced by benzo[a]pyrene/ethanol co-exposure

Introduction

Non-alcoholic fatty liver disease (NAFLD) is now well recognized as a growing worldwide epidemic, responsible for an increasing number of chronic liver diseases and consecutive mortality [START_REF] Younossi | Non-alcoholic fatty liver disease -A global public health perspective[END_REF]. NAFLD covers a large panel of liver diseases, starting from liver steatosis to its pathological progression into non-alcoholic steatohepatitis (NASH), with possible evolution towards severe and irreversible complications such as cirrhosis and/or hepatocellular carcinoma [START_REF] Fazel | Epidemiology and natural history of nonalcoholic fatty liver disease[END_REF]. The global prevalence of NAFLD is around 25% of general population all over the world, while for NASH, it reaches between 3 to 5% [START_REF] Younossi | Non-alcoholic fatty liver disease -A global public health perspective[END_REF]). It should be noted that some subpopulations are particularly affected by NAFLD, notably patients exhibiting metabolic diseases such as type 2 Diabetes mellitus (T2DM) and obesity, in which NAFLD prevalence could rise to 60 or 90% respectively [START_REF] Younossi | Non-alcoholic fatty liver disease -A global public health perspective[END_REF].

Beyond principal etiological factors of NAFLD, i.e. metabolic diseases like obesity and T2DM, there are several others like dietary habits (including moderate/heavy alcohol consumption), genetic polymorphisms, gender, epigenetic factors as well as environmental factors [START_REF] Younossi | Non-alcoholic fatty liver disease -A global public health perspective[END_REF]. In this concern, the role of these latter factors has gained interest during last years, leading to the concept of TAFLD and TASH (Toxicant-Associated Fatty Liver Diseases and Toxicant-Associated Steatohepatitis) as proposed by Cave and co-workers (Joshi- [START_REF] Joshi-Barve | Alcoholic, Nonalcoholic, and Toxicant-Associated Steatohepatitis: Mechanistic Similarities and Differences[END_REF][START_REF] Wahlang | Mechanisms of Environmental Contributions to Fatty Liver Disease[END_REF]. In line with this, several pollutants, including ligands of aryl hydrocarbon receptor (AhR), have thus been shown to induce steatosis or favor its pathological progression [START_REF] Wahlang | Mechanisms of Environmental Contributions to Fatty Liver Disease[END_REF].

In this context, another challenging concern is the impact of chemical mixtures on NAFLD particularly in high-risk populations such as people already presenting fatty liver. In ACCEPTED MANUSCRIPT / CLEAN COPY that way, we have previously demonstrated that co-exposure to benzo[a]pyrene (B[a]P), the reference molecule of the PAH (polycyclic aryl hydrocarbon) family, in combination with a well-known hepatotoxicant, ethanol, induces the transition of steatosis towards a steatohepatitis-like state both in vitro and in vivo (Bucher et al. 2018b;Bucher et al. 2018a;[START_REF] Imran | Membrane Remodeling as a Key Player of the Hepatotoxicity Induced by Co-Exposure to Benzo[a]pyrene and Ethanol of Obese Zebrafish Larvae[END_REF][START_REF] Tête | Mechanisms involved in the death of steatotic WIF-B9 hepatocytes co-exposed to benzo[a]pyrene and ethanol: a possible key role for xenobiotic metabolism and nitric oxide[END_REF]. B[a]P, a widespread environmental contaminant, is a potent carcinogen to human (classified in group 1 by IARC), and a strong AhR ligand [START_REF] Das | Inevitable dietary exposure of Benzo[a]pyrene: carcinogenic risk assessment an emerging issues and concerns[END_REF][START_REF] Hardonnière | Role for the ATPase inhibitory factor 1 in the environmental carcinogen-induced Warburg phenotype[END_REF]; International Agency for Research on Cancer (IARC) 2012). B[a]P is a pollutant formed, like other PAHs, during incomplete combustion of organic compounds. Human exposure to B[a]P, excluding smoking or occupational exposure, is mainly food-borne notably with barbecued/grilled/broiled/smoked meats, grain and cereals [START_REF] Das | Inevitable dietary exposure of Benzo[a]pyrene: carcinogenic risk assessment an emerging issues and concerns[END_REF]; International Agency for Research on Cancer (IARC) 2012). Most of the toxic effects of B[a]P depend on its bioactivation by cytochrome P450s (CYP), which mainly occurs in liver. This thus explains the adverse effects of this pollutant on this organ through several mechanisms including oxidative stress, genotoxicity, mitochondrial dysfunction, cell death [START_REF] Das | Inevitable dietary exposure of Benzo[a]pyrene: carcinogenic risk assessment an emerging issues and concerns[END_REF][START_REF] Hardonnière | The environmental carcinogen benzo[a]pyrene induces a Warburg-like metabolic reprogramming dependent on NHE1 and associated with cell survival[END_REF]; International Agency for Research on Cancer (IARC) 2012; [START_REF] Tekpli | Membrane remodeling, an early event in benzo[a]pyrene-induced apoptosis[END_REF][START_REF] Uno | Cytochrome P450 1A1 (CYP1A1) protects against nonalcoholic fatty liver disease caused by Western diet containing benzo[a]pyrene in mice[END_REF]. B[a]P, as other toxicants, has been implicated in NAFLD development and progression [START_REF] Wahlang | Mechanisms of Environmental Contributions to Fatty Liver Disease[END_REF]. Regarding the impact of B[a]P/ethanol co-exposure, frequently observed in population, on steatosis progression, we recently evidenced several mechanisms using in vitro models such as the human hepatic cell line, HepaRG (Bucher et al. 2018a;[START_REF] Tête | Mechanisms involved in the death of steatotic WIF-B9 hepatocytes co-exposed to benzo[a]pyrene and ethanol: a possible key role for xenobiotic metabolism and nitric oxide[END_REF]. However, the underlying mechanisms involved in the exacerbation of NAFLD upon such a co-exposure remain to be deciphered in vivo.

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To this aim, we used an in vivo model of zebrafish larvae to have an integrative model in which the complexity and variety of cell and organ interactions are present and relevant to human NAFLD pathogenesis [START_REF] Chu | New school in liver development: lessons from zebrafish[END_REF][START_REF] Goessling | Zebrafish: An Important Tool for Liver Disease Research[END_REF][START_REF] Schlegel | Studying non-alcoholic fatty liver disease with zebrafish: a confluence of optics, genetics, and physiology[END_REF].

In addition to technical advantages like small size and transparency, zebrafish larvae also present broad similarities with humans concerning liver functions and sensitivity toward xenobiotics and alcohol (metabolism, toxicity, cellular and transcriptomic responses) [START_REF] Driessen | Gene expression markers in the zebrafish embryo reflect a hepatotoxic response in animal models and humans[END_REF][START_REF] Driessen | Exploring the zebrafish embryo as an alternative model for the evaluation of liver toxicity by histopathology and expression profiling[END_REF][START_REF] Goessling | Zebrafish: An Important Tool for Liver Disease Research[END_REF][START_REF] Goldstone | Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish[END_REF].

Besides, the full machinery for B[a]P metabolism exists in zebrafish notably with the expression of ahr2, the ligand-activated ortholog of AhR [START_REF] Goodale | AHR2 mutant reveals functional diversity of aryl hydrocarbon receptors in zebrafish[END_REF]. For the present study, we used a recently established zebrafish larvae model of high fat diet (HFD)-induced steatosis in which we observed steatosis progression following a 7 days-co-exposure to low doses of ethanol and B[a]P, respectively 43 mM and 25 nM (Bucher et al. 2018b;[START_REF] Imran | Membrane Remodeling as a Key Player of the Hepatotoxicity Induced by Co-Exposure to Benzo[a]pyrene and Ethanol of Obese Zebrafish Larvae[END_REF]. In order to elucidate the mechanisms involved in the pathological progression of steatosis induced upon B[a]P/ethanol co-exposure in HFD-zebrafish larvae, a transcriptomic approach was performed in the present study. We identified and confirmed by RT-qPCR, disruptions of key processes, i.e. mitochondrial dysfunctions, alterations of heme and iron metabolism, AhR signaling and oxidative stress, which were then more deeply investigated.

Our main results strongly suggest the involvement of a mitochondrial overload of labile iron as a key event in the liver disease progression upon B[a]P/ethanol co-exposure. Furthermore, these effects were prevented by the common iron-chelating drug, deferoxamine.

Materials and Methods

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Details for all Materials and methods are available in supplementary material (File S1).

Results

Transcriptomic analysis identifies heme homeostasis and mitochondrial dysfunction as potential events of liver disease progression in B[a]P/ethanol co-exposed HFD zebrafish larvae

In order to decipher the cellular mechanisms involved in the progression of liver steatosis in HFD zebrafish larvae co-exposed to B[a]P and ethanol, we performed a transcriptomic analysis based on affymetrix microarray technology (GeneChip™ Zebrafish Gene 1.0 ST Array). Briefly, HFD zebrafish larvae were chronically exposed to 43 mM ethanol and/or to 25 nM B[a]P from 5 dpf until 12 dpf (doses chosen in rationale to human level exposure and already shown to induce progression of steatosis in our zebrafish model (Bucher et al. 2018b;[START_REF] Imran | Membrane Remodeling as a Key Player of the Hepatotoxicity Induced by Co-Exposure to Benzo[a]pyrene and Ethanol of Obese Zebrafish Larvae[END_REF]).

From transcriptomic analysis, 525 differentially expressed genes (DEGs) were found after coexposure compared to control. Those genes were further partitioned into 8 expression clusters (termed P1-P8) organized in two broad expression patterns corresponding to upregulated (315 DEGs, corresponding to P1-4) and down-regulated (210 DEGs, P5-8) genes (see supplementary Table S1 for all data) (Figure 1a andb). The functional analysis revealed that up-regulated genes were significantly associated with porphyrin metabolism and mitochondria (GO:0006787 porphyrin-containing compound catabolic process; GO:0031966 mitochondrial membrane; KEGG:00860 porphyrin and chlorophyll metabolism) (Figure 1c).

Interestingly, these terms were also found to be significantly enriched in the 4 up-regulated clusters (P1-4) thereby indicating that the observed alterations were not just due to one toxicant; rather both B[a]P and ethanol were effective, particularly when used in combination (Figure 1b). Regarding down-regulated genes, most of the terms appeared to be related to ACCEPTED MANUSCRIPT / CLEAN COPY immunity (for example: GO:0060333 IFNg-mediated signaling pathways; GO:0043312 neutrophil degranulation or GO:0030670 phagocytic vesicle membrane) (Figure 1b). To improve relevance of findings regarding human health, we performed an Ingenuity Pathways Analysis (IPA) following conversion of the 525 zebrafish DEGs into 259 human homologs (supplementary Table S2); several terms were then presented for canonical pathways, toxicity lists and toxicity functions (summary presented in supplementary Table S3; full results are provided in supplementary Table S4). In line with the functional analysis performed in AMEN, the selected terms highlight mitochondria dysfunction and alterations in heme homeostasis, involvement of AhR signaling and oxidative stress for canonical pathways and toxicity lists (supplementary Table S3). Finally, analysis of toxicity functions markedly outlined the impact of B[a]P/ethanol co-exposure on liver diseases (supplementary Table S3) in agreement with our previous work (Bucher et al. 2018b;[START_REF] Imran | Membrane Remodeling as a Key Player of the Hepatotoxicity Induced by Co-Exposure to Benzo[a]pyrene and Ethanol of Obese Zebrafish Larvae[END_REF][START_REF] Tête | Mechanisms involved in the death of steatotic WIF-B9 hepatocytes co-exposed to benzo[a]pyrene and ethanol: a possible key role for xenobiotic metabolism and nitric oxide[END_REF].

Validation and investigation by RT-qPCR of molecular dysregulations induced by coexposure

In order to validate the microarray results from our zebrafish model, we performed RT-qPCR assays on zebrafish samples (Figure 2a to 2c) (Note that for all genes studied by RT-qPCR and presented by abbreviation, full names could be found in the supplementary file S5 with primer information). One of the main processes identified through the transcriptomic analysis in in vivo steatosis progression upon B[a]P/ethanol co-exposure appeared to be mitochondrial dysfunction. In fact, this was in line with our recent work realized in vitro in human HepaRG cell line (Bucher et al. 2018a). In order to get further insight into mitochondrial dysfunction, especially in vivo, we decided to perform RT-qPCR on several target genes selected either from our transcriptomic screening or according to their role in mitochondrial function (Figure 2a).

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Most of the transcripts found to be dysregulated in our microarray (indicated by an arrow in Figure 2a to 2c), were validated by RT-qPCR with significant changes (22 transcripts out of the 25 found in microarray). Among mitochondria-related genes, the expression of several transporters of metabolites (abcg2a, slc25a25a, slc25a47a, slc25a48 and tspo) was induced, thus suggesting alterations of mitochondrial metabolism (Figure 2a). Interestingly, abcg2a and tspo are also known to be involved in heme homeostasis. Regarding genes related to electron transport chain, mitochondrial respiration and ATP production, several were found to be induced (sdha, sdhaf3, uqcc1 and uqcc3), thus further pointing to alterations of mitochondrial respiration capacity (Figure 2a). In addition, expression of known regulators of mitochondrial activity (hebp2, parla, sirt3) was modified upon B[a]P/ethanol co-exposure; it could be noted that hepb2 and parla have been implicated in cell death [START_REF] Ishihara | PARL paves the way to apoptosis[END_REF][START_REF] Szigeti | Induction of necrotic cell death and mitochondrial permeabilization by heme binding protein 2/SOUL[END_REF] in agreement with the deleterious effects of B[a]P/ethanol co-exposure previously reported in vitro (Bucher et al. 2018b;Bucher et al. 2018a;[START_REF] Tête | Mechanisms involved in the death of steatotic WIF-B9 hepatocytes co-exposed to benzo[a]pyrene and ethanol: a possible key role for xenobiotic metabolism and nitric oxide[END_REF].

Furthermore, another key process revealed by our microarray analysis was porphyrin metabolism, notably included in the more general term of heme metabolism (as suggested by changes of expression for tspo and abcg2a) (Figure 1c). In addition, IPA identified iron homeostasis as a new process affected by B[a]P/ethanol co-exposure, which is also closely linked to heme metabolism (supplementary Table S3). Therefore, we decided to evaluate the expression of several genes associated with heme and iron metabolism (Figure 2b). Most of the changes in gene expression, found by microarray analysis, were validated by fech,blvra,blvrb,tfa) (Figure 2b). In addition, co-exposure significantly altered the expression of several genes involved in heme synthesis (alas1, alas2), in iron transport (slc25a37 and slc25a28 alias mitochondrial iron transporters mitoferrin 1 and 2; slc40a1 alias ferroportin 1), and in iron storage (fth1a and fthl30, ferritin subuntits; tfa for transferrin a)

ACCEPTED MANUSCRIPT / CLEAN COPY (Figure 2b). Altogether, these results strongly support the idea of a perturbation in heme and iron homeostasis following B[a]P/ethanol co-exposure in our steatotic model of zebrafish larvae.

In addition, oxidative stress-related genes (markers of oxidative stress and/or genes involved in ROS/NOS production/elimination) were also investigated by RT-qPCR. Our data thus confirmed the changes observed in our microarray in the expression of catalase (cat) and peroxiredoxin 1 (prdx1); they also showed induction of nos2a, prdx6, sod2 and sod3b (Figure 2c), thus suggesting the implication of oxidative stress. Finally, potential activation of AhR signaling was explored by assessing the expression of several AhR-related genes (Figure 2c). Three genes, for which expression was increased in transcriptomic analysis, were validated by RT-qPCR (cyp1a, gstp1, nfe2le2a alias nrf2). In addition, nqo1 (NAD(P)H dehydrogenase quinone 1, an indirect target of AhR) was also found to be significantly induced by B[a]P/ethanol co-exposure as well as ahr2, the ortholog of human AhR (Figure 2c).

Finally, in order to evaluate the relevance of the results found in zebrafish toward human NAFLD progression upon similar co-exposure, we assessed expression of several genes related to the potential mechanisms involved (mitochondrial dysfunction, alterations of heme metabolism and iron homeostasis, and AhR signaling) in HepaRG cells, an in vitro human model of hepatocytes, supplemented with fatty acids (FA) in which B[a]P/ethanol co-exposure was demonstrated to induce steatosis progression toward a steatohepatitis-like state (Bucher et al. 2018b) (Figure 2d). As illustrated in Figure 2d, regarding most of the studied genes, the changes in mRNA expression observed in zebrafish larvae (as indicated by white arrows) were also reproduced in steatotic HepaRG cells co-exposed to B[a]P and ethanol, even though the amplitude of changes could be less. This is particularly observable for mRNA expression changes highly significant in zebrafish, i.e. abcg2a, tspo, uqcc3, abcb6a, fech, blvra, slc25a37, ACCEPTED MANUSCRIPT / CLEAN COPY slc25a28, nqo1. Indeed for 7 of them, significant changes were also found in HepaRG (except for ABCB6 and SLC25A28, close to significance) (Figure 2d). Therefore, these results further validated the HFD-fed zebrafish larvae as a suitable model to study human NAFLD progression, then prompting us to more thoroughly investigate the in vivo role of the observed alterations.

Assessment of mitochondrial dysfunction

Due to the well-recognized role of mitochondrial dysfunction in NAFLD [START_REF] Begriche | Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease[END_REF]) and as it was identified in our transcriptomic analysis, we decided to evaluate mitochondrial respiration in co-exposed steatotic zebrafish larvae (Figure 3a). To this aim, we used a protocol adapted from [START_REF] Raftery | A bioenergetics assay for studying the effects of environmental stressors on mitochondrial function in vivo in zebrafish larvae[END_REF] based on the Agilent Seahorse technology, which allows the measurement of oxygen consumption from living zebrafish larvae [START_REF] Raftery | A bioenergetics assay for studying the effects of environmental stressors on mitochondrial function in vivo in zebrafish larvae[END_REF]) (described in supplementary Materials and Methods). We observed that coexposure significantly inhibited both basal and maximal respiration without any effect on spare and non-mitochondrial respiration (Figure 3a; representative curves of OCR could be found in Figure S3). Then, in order to get further insight into mitochondrial dysfunction, we performed transmission electron microscopy (TEM) to study the ultrastructure of liver cells in co-exposed HFD zebrafish larvae (Figure 3b). Images obtained at low magnification (panel of pictures on the left) showed well-organized hepatocytes under control conditions and few biliary canaliculi. However, under B[a]P/ethanol co-exposure conditions, disorder of hepatocyte morphology was observed. Regarding more specifically the mitochondria in HFD control larvae, they appeared to represent a large part of hepatocyte surface, with a circular form and numerous cristae (central and right panels of pictures in Figure 3b) under control conditions. In contrast, after a 7-days-co-exposure to toxicants, only a few mitochondria per hepatocyte could be observed, with a smaller size and flatter form, and less observable cristae.

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Therefore, these ultrastructure observations were in accordance with the decreased oxygen consumption induced by B[a]P/ ethanol co-exposure.

Involvement of AhR in in vivo mitochondrial dysfunction and liver damages during steatosis progression induced by B[a]P/ethanol co-exposure

AhR and/or B[a]P have already been reported to be involved in mitochondrial dysfunction in diverse in vitro models (Bucher et al. 2018a;[START_REF] Hardonnière | Role for the ATPase inhibitory factor 1 in the environmental carcinogen-induced Warburg phenotype[END_REF][START_REF] Hwang | Mitochondrial-targeted aryl hydrocarbon receptor and the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cellular respiration and the mitochondrial proteome[END_REF].

Based upon the fact that our transcriptomic analysis outlined AhR signaling (see supplementary Table S3), we decided to investigate the role of this receptor in the mitochondrial dysfunction both at a transcriptomic level and at a functional level, i.e. mitochondrial oxygen consumption. To do so, we used the specific AhR antagonist CH223191

(1 µM) [START_REF] Wang | Benzo(a)pyrene facilitates dermatophagoides group 1 (Der f 1)-induced epithelial cytokine release through aryl hydrocarbon receptor in asthma[END_REF]. To ensure that this antagonist did inhibit AhR activation in our zebrafish model, we analyzed mRNA expression of known AhR target genes after CH223191 treatment of co-exposed larvae (Figure 4a). As expected, we observed an inhibition of several B[a]P/ethanol-induced genes, cyp1a, nqo1 and nfe2l2 (Figure 4a). It was not the case for gstp1 mRNA expression; rather CH223191 alone induced its expression to a similar level as upon toxicant co-exposure, with a further increase upon co-treatment with the three molecules (Figure 4a). Thus, it probably means that the observed effect on gstp1 expression would be a secondary response linked to its role in detoxification rather than being directly targeted by AhR. Looking at genes related to mitochondria (Figure 4b), we found that CH223191 was able to prevent co-exposure effects on abcg2a and partially on sdha (even if, for this last gene, there is no statistical difference between co-exposure conditions (BE vs CH+BE), there is no more effect of co-exposure in presence of CH223191 (CH vs CH+BE)). Regarding tspo and uqcc3, a significant induction was observed with the antagonist alone, but no further increase occurred, when larvae were co-exposed to B[a]P/ethanol (Figure 4b). Regarding heme and ACCEPTED MANUSCRIPT / CLEAN COPY iron-related genes, CH223191 reversed co-exposure effect on fech, slc25a28, slc40a1 and tfa but not blvra, fth1a, fthl30 and slc25a37 (Figure 4c). Taken together, these observations suggest that AhR activation might disturb mitochondrial activity partly through a transcriptional action. In order to further test the in vivo role of AhR in the steatosis progression upon co-exposure, the effects of CH223191 on the expression of mRNA markers of toxicity were analyzed by RT-qPCR (Figure 4d). We found that CH223191 inhibited the increase in mRNA expression of both casp3 and il6, thus suggesting reduction of cell death and inflammation, whereas it increased prdx1 expression. As gstp1, this latter increase might be associated with a secondary cell response against co-exposure-induced oxidative damages.

Still trying to be relevant of human NAFLD progression upon similar co-exposure, these alterations of gene expression were evaluated in AhR-knock-out HepaRG cells (Figure 4e). By this way, we found that most of the changes induced by co-exposure in the expression of genes related to mitochondria (ABCG2, UQCC3), iron homeostasis (FECH, SLC25A28, SLC40A1, TF) or, as expected, AhR activation (NFE2L2 alias NRF2 and NQO1), were prevented (Figure 4e). Finally, we investigated CH223191 effect on mitochondrial function and liver integrity (Figure 4f and g respectively). Using the Seahorse technology, we found that alterations in mitochondrial respiration induced in co-exposed HFD larvae (especially the decrease in basal respiration), were prevented by CH223191 (Figure 4f; representative curves of OCR could be found in Figure S3). We also noted that CH223191 alone could induce both maximal and nonmitochondrial respiration (Figure 4f). In addition, this AhR antagonist was able to reduce liver cell damages induced by toxicant co-exposure as shown by Hematoxylin-Eosin-Safranin (HES) staining (Figure 4g; representative pictures can be found in supplementary Figure S1). In total, these results indicate a crucial involvement of AhR in mitochondrial disruption and liver ACCEPTED MANUSCRIPT / CLEAN COPY injuries following B[a]P/ethanol co-exposure in agreement with in vivo progression of liver steatosis.

B[a]P/ethanol co-exposure leads to disruption of heme metabolism and to an oxidative stress involved in liver injury

As our transcriptomic data clearly pointed to heme metabolism as a possible target of B[a]P/ethanol co-exposure, alterations of this process were more thoroughly analyzed through biochemical assessment. Thus, levels of heme, hemin (the oxidized and free form of heme) and bilirubin (one of the major metabolic compounds of heme degradation) were determined in our model of HFD-fed zebrafish larvae. Co-exposure was found to increase the levels of both heme and hemin (Figure 5a and b, respectively), whereas bilirubin levels remained unchanged (Figure 5c). As increased cellular heme and hemin amounts have been reported to be toxic for cells mainly via oxidative stress [START_REF] Kumar | Free heme toxicity and its detoxification systems in human[END_REF], we decided to test whether B[a]P/ethanol co-exposure induced such a phenomenon under our experimental conditions, and if so, whether it was involved in the related liver damages using common antioxidants, quercetin and vitamin E [START_REF] Batiha | The Pharmacological Activity, Biochemical Properties, and Pharmacokinetics of the Major Natural Polyphenolic Flavonoid: Quercetin[END_REF][START_REF] Burton | Vitamin E as an in Vitro and in Vivo Antioxidant[END_REF]. Thus, lipid peroxidation was evaluated after co-exposure by fluorescent imaging using C11-bodipy 583/591 staining in liver of zebrafish larvae (Figure 5d; representative pictures can be found in supplementary Figure S4). As expected, an increase in lipid peroxidation was observed upon B[a]P/ethanol co-exposure, such an effect being prevented by quercetin (25 µM) or vitamin E (100 µM) co-treatment (Figure 5d). In order to test the involvement of oxidative stress in liver disease progression, liver damages were estimated by counting the damaged cells after histological HES staining (Figure 5e; representative pictures can be found in supplementary Figure S1). As previously reported (Bucher et al. 2018b), we found that coexposure increased the proportion of damaged cells in liver, and quercetin as vitamin E, at ACCEPTED MANUSCRIPT / CLEAN COPY least partly, prevented this effect. These data therefore showed the involvement of oxidative stress in the pathological progression of steatosis upon B[a]P/ethanol co-exposure of HFD-fed zebrafish larvae.

Iron is a crucial player in the liver mitochondrial dysfunction and toxicity induced by B[a]P/ethanol co-exposure of HFD zebrafish larva

Our present results have highlighted the role of (i) mitochondrial dysfunction, (ii) dysregulation of heme homeostasis, and (iii) oxidative stress in co-exposure-induced in vivo steatosis progression. In addition, the transcriptomic analysis suggested changes in iron homeostasis, notably in mitochondria. In this context, it appeared necessary to further explore the involvement of iron homeostasis in mitochondrial dysfunction and liver toxicity under our experimental conditions. First, the level of labile iron in mitochondria (mostly represented by ferrous iron Fe 2+ (Lv and Shang 2018)) was evaluated. To do so, alive HFD-fed zebrafish larva, co-exposed or not, were loaded with the fluorescent mitochondrial Fe 2+ -sensitive probe, Mito-FerroGreen, followed by imaging of the liver by confocal microscopy (Figure 6a). We observed that co-exposure markedly increased the level of labile iron in mitochondria, as shown by a stronger fluorescence intensity in the liver of treated animals compared to untreated counterparts (Figure 6a, zebrafish liver is delineated by a white dot-line). In addition, quantitative analysis of liver fluorescent intensities, detected in several larvae, clearly showed the significant increase of the mitochondrial Fe 2+ pool in liver upon B[a]P/ethanol co-exposure (Figure 6b).

In order to determine how this increase of iron might affect mitochondria and thus could lead to toxicity upon co-exposure in our zebrafish model, we decided to test the potential protective effect of the iron chelator, deferoxamine (100 µM) (Figure 6c-e). We firstly evaluated the content of liver mitochondrial Fe 2+ using Mito-FerroGreen fluorescence ACCEPTED MANUSCRIPT / CLEAN COPY imaging. As expected, a co-treatment of larvae with deferoxamine totally prevented the increase of mitochondrial Fe 2+ content (Figure 6c). Then, the role of iron in the toxicity was evaluated by RT-qPCR assessment of genes related to mitochondria (Figure 6d), to heme and iron homeostasis (Figure 6e), and to cellular stress and toxicity (Figure 6f). Interestingly, deferoxamine inhibited some of the changes induced by co-exposure related to mitochondria (abcg2a, uqcc3) (Figure 6d), or to iron and heme homeostasis (fech, slc25a28, slc40a1, tfa) (Figure 6e). In addition, regarding the expression of the gene markers of toxicity (i.e. prdx1 for oxidative stress, casp3a for cell death and il6 for inflammation), deferoxamine inhibited all the changes induced by co-exposure (Figure 6f). It could be noted that deferoxamine did not prevent the effects of co-exposure on the mRNA expression of cyp1a, nbfe2l2 and nqo1, thus confirming that the protective effect of deferoxamine was linked to iron chelation rather than an inhibition of AhR signaling (Figure 6g). Finally, the role of mitochondrial iron overload in liver toxicity was confirmed by the observation of a protective effect of deferoxamine upon liver cell damages induced by B[a]P/ethanol co-exposure (Figure 6h ; representative pictures can be found in supplementary Figure S1). Taken together, these results are in favor of the involvement of an iron overload in the mitochondrial dysfunction induced by B[a]P/ethanol co-exposure, and hence related toxicity.

Finally, in order to better understand the involvement of AhR signaling in the deleterious effect of B[a]P/ethanol co-exposure on the liver, we assessed the impact of AhR inhibition on liver mitochondrial iron accumulation and oxidative stress (Figure 7 and supplementary Figure S2). Interestingly, we observed that AhR inhibition was fully protective against liver mitochondrial iron accumulation (Figure 7a) and against lipid peroxidation (Figure 7b).

Therefore, these results show that AhR activation is a key event on the pathological ACCEPTED MANUSCRIPT / CLEAN COPY progression of steatosis to steatohepatitis induced by co-exposure in our zebrafish larva model.

Discussion

With the rise of NAFLD prevalence over the last decades, this liver disease has become a major health issue, due to its pathological complications like NASH, cirrhosis and cancer.

Besides the well-known etiological factors of NAFLD (i.e. genetic factors, nutrition, obesity, diabetes), environmental toxicants have been put forward by an increasing number of studies; this has led to the emergence of the concept of TAFLD and TASH (Joshi- [START_REF] Joshi-Barve | Alcoholic, Nonalcoholic, and Toxicant-Associated Steatohepatitis: Mechanistic Similarities and Differences[END_REF][START_REF] Wahlang | Mechanisms of Environmental Contributions to Fatty Liver Disease[END_REF][START_REF] Wahlang | Toxicant-associated steatohepatitis[END_REF]). Recently we have found, both in vitro and in vivo, that co-exposure to B[a]P and ethanol, at doses not toxic in the absence of steatosis, could favor the NAFLD progression (Bucher et al. 2018b;Bucher et al. 2018a;[START_REF] Imran | Membrane Remodeling as a Key Player of the Hepatotoxicity Induced by Co-Exposure to Benzo[a]pyrene and Ethanol of Obese Zebrafish Larvae[END_REF][START_REF] Tête | Mechanisms involved in the death of steatotic WIF-B9 hepatocytes co-exposed to benzo[a]pyrene and ethanol: a possible key role for xenobiotic metabolism and nitric oxide[END_REF]. In this context, several mechanisms have been identified in vitro including alterations of B[a]P and ethanol metabolism (Bucher et al. 2018b;[START_REF] Tête | Mechanisms involved in the death of steatotic WIF-B9 hepatocytes co-exposed to benzo[a]pyrene and ethanol: a possible key role for xenobiotic metabolism and nitric oxide[END_REF]), NOS production [START_REF] Tête | Mechanisms involved in the death of steatotic WIF-B9 hepatocytes co-exposed to benzo[a]pyrene and ethanol: a possible key role for xenobiotic metabolism and nitric oxide[END_REF], and mitochondrial dysfunction (Bucher et al. 2018a). However, the underlying mechanisms remained to be deciphered in vivo. To this aim, we used our recently developed model of HFD fed zebrafish larva, proven to be reliable to human models, in which B[a]P/ethanol co-exposure was involved in NAFLD steatosis progression toward a steatohepatitis-like state.

Thus, we performed a non-targeted approach by transcriptomic analysis in this model.

From this, we focused on the 525 genes presenting significant changes in expression after B[a]P/ethanol co-exposure in HFD larvae. Thus, following clustering of these genes considering their expression found upon exposure to either toxicant alone or together, we performed a functional analysis based on GO enrichment (Figure 1c). Most of the annotation regarding down-regulated genes was linked to immunity. Such a result was not surprising, even in the ACCEPTED MANUSCRIPT / CLEAN COPY context of liver inflammation and NASH previously observed in our in vivo B[a]P/ethanol coexposure model (Bucher et al. 2018b;[START_REF] Imran | Membrane Remodeling as a Key Player of the Hepatotoxicity Induced by Co-Exposure to Benzo[a]pyrene and Ethanol of Obese Zebrafish Larvae[END_REF]. Indeed each toxicant is known to induce immunosuppressive effects [START_REF] Cella | Aryl hydrocarbon receptor: Linking environment to immunity[END_REF][START_REF] Liamin | Genome-Wide Transcriptional and Functional Analysis of Human T Lymphocytes Treated with Benzo[α]pyrene[END_REF][START_REF] Szabo | Alcohol's Effect on Host Defense[END_REF]. Interestingly, most of the immunosuppressive effects previously reported refer to host defense, which is coherent with the biological process term returned by functional analysis (Figure 1b; IFNg-mediated signaling pathways, neutrophil degranulation or phagocytic vesicle for example). Taken together, our results support the idea that, in the presence of NAFLD, in vivo exposure to toxicants might also favor infections, and doing so, potentially further aggravate liver diseases.

Concerning up-regulated genes, the functional analysis revealed a significant association with terms related to porphyrin/heme/iron metabolism, mitochondrial dysfunction, NAFLD/liver toxicity and AhR signaling (Figure 1 and supplementary Table S3). Some of these observations were in line with our recent work on the mechanisms involved in the in vitro impact of B[a]P/ethanol co-exposure on the progression of steatosis in the human hepatocarcinoma HepaRG cell line; indeed, we found a role for an AhR-dependent mitochondrial dysfunction (Bucher et al. 2018a). Based on this, we decided to further investigate these processes in our in vivo zebrafish model, with special emphasis on AhR signaling and mitochondrial iron/heme homeostasis.

Mitochondrial dysfunction is well-known to be involved in liver diseases, notably NAFLD [START_REF] Grattagliano | Targeting mitochondria to oppose the progression of nonalcoholic fatty liver disease[END_REF]. From transcriptomic and RT-qPCR analysis, several alterations related to mitochondria were presently identified, with an up-regulation in the expression of several genes involved in respiratory complexe formation (sdha, sdhaf3 in complex II, upcc1 and uqcc3 in complex III), and a down-regulation of sdhc, part of complex III (Figure 2a), thus suggesting disruption of mitochondrial respiration. Note that a similar change ACCEPTED MANUSCRIPT / CLEAN COPY in UQCC3 expression was also found in human HepaRG cells under these conditions (Figure 2d). Disruption of mitochondrial respiration in B[a]P/ethanol co-exposed HFD zebrafish larvae was confirmed by the decrease in basal mitochondrial respiration (Figure 3a) prevented by AhR inhibition (Figure 4f), like in co-exposed steatotic HepaRG cells (Bucher et al. 2018a). Such functional alterations were related to changes in the ultrastructure of mitochondria (smaller and flattened, less cristae) (Figure 3b) that might be associated with more mitochondrial fission and ultimately with promotion of NAFLD, as previously reported [START_REF] Li | Mitochondria-Mediated Pathogenesis and Therapeutics for Non-Alcoholic Fatty Liver Disease[END_REF].

Considering all these results, one might then hypothesize that the decrease in mitochondria respiration detected under our conditions could be associated with a decrease of the respiratory chain ability to produce ATP and/or a decrease of fatty acid oxidation; both potentially depending on and promoting oxidative stress in a vicious cycle [START_REF] Begriche | Mitochondrial Dysfunction Induced by Xenobiotics: Involvement in Steatosis and Steatohepatitis[END_REF][START_REF] Li | Mitochondria-Mediated Pathogenesis and Therapeutics for Non-Alcoholic Fatty Liver Disease[END_REF]. With respect to respiratory chain activity and related ATP production (i.e. parameters related to oxidative phosphorylation [OXPHOS]), it is worth emphasizing that B[a]P and ethanol, used alone or in combination, have already been reported to decrease these parameters in steatotic human hepatocytes and other models [START_REF] Begriche | Mitochondrial Dysfunction Induced by Xenobiotics: Involvement in Steatosis and Steatohepatitis[END_REF]Bucher et al. 2018b;Bucher et al. 2018a;[START_REF] Das | Inevitable dietary exposure of Benzo[a]pyrene: carcinogenic risk assessment an emerging issues and concerns[END_REF][START_REF] Hardonnière | The environmental carcinogen benzo[a]pyrene induces a Warburg-like metabolic reprogramming dependent on NHE1 and associated with cell survival[END_REF][START_REF] Tête | Mechanisms involved in the death of steatotic WIF-B9 hepatocytes co-exposed to benzo[a]pyrene and ethanol: a possible key role for xenobiotic metabolism and nitric oxide[END_REF]. Such an alteration of OXPHOS is commonly associated with an increase of ROS production and progression of NAFLD toward NASH particularly upon exposure to xenobiotic [START_REF] Wahlang | Mechanisms of Environmental Contributions to Fatty Liver Disease[END_REF]. In line with this, a lipid peroxidation in liver was detected under our experimental conditions and was found to be involved in co-exposure toxicity (Figure 5d,e).

Concerning AhR regulation and mitochondrial functions, it's interesting to note that inhibition of AhR by the antagonist CH223191 had for some genes similar effect than coexposure (BE) ( ). There is no clear explanation yet but such effects might rely on previously described ACCEPTED MANUSCRIPT / CLEAN COPY AhR mitochondrial pool and its consequences on mitochondrial function [START_REF] Tappenden | The aryl hydrocarbon receptor interacts with ATP5α1, a subunit of the ATP synthase complex, and modulates mitochondrial function[END_REF]. In this context, compensatory mechanisms might be hypothesized.

Another altered process revealed by transcriptomic analysis was heme metabolism.

Interestingly, several studies have shown that heme accumulation can favor oxidative stress and NAFLD progression, and have suggested that elevation of heme catabolism (through increase in heme oxygenase 1 (HO1), for example) could be a therapeutic perspective of NAFLD [START_REF] Severson | Genetic factors that affect nonalcoholic fatty liver disease: A systematic clinical review[END_REF]). In the context of steatosis progression toward a steatohepatitislike state induced by B[a]P/ethanol co-exposure, we have found for the first time that there is an accumulation of both heme and hemin while there is no change in the heme catabolic process, as visualized by the absence of increase in bilirubin level (Figure 5), despite a significant increase in blvra (biliverdin reductase) gene expression (Figure 2b). Previous studies have reported that the strong AhR ligand, TCDD, could alter heme homeostasis and promote NASH in mice (Fader et al. 2017). This observation, along with our present results, would thus put heme homeostasis as a potential central hub in response to cellular chemical stress during NAFLD. As the expression of several genes of heme biosynthesis was found to be elevated in zebrafish larvae (Figure 2b) or in HepaRG cells (Figure 2d), one could suggest that heme and hemin elevations would depend on a transcriptional regulation rather than a down-regulation of the catabolic pathway. In line with this, note that no change in hmox1a mRNA expression was detected under our conditions (Figure 2b); furthermore, the bilirubin level remained unchanged (Figure 5c). A common consequence of heme accumulation and particularly of hemin accumulation, is an increase of oxidative stress responsible for cell death [START_REF] Kumar | Free heme toxicity and its detoxification systems in human[END_REF]. Thus, the presently observed biochemical effects would fit well with the increase in lipid peroxidation and its role in hepatotoxicity (Figure 5). However, several studies have also highlighted that heme and hemin are potential inducers of cellular ACCEPTED MANUSCRIPT / CLEAN COPY antioxidant systems and could then act to protect cells against oxidative stress [START_REF] Donegan | Handling heme: The mechanisms underlying the movement of heme within and between cells[END_REF][START_REF] Luan | Hemin Improves Insulin Sensitivity and Lipid Metabolism in Cultured Hepatocytes and Mice Fed a High-Fat Diet[END_REF]. In this context, the elucidation of the precise role of heme and hemin in our in vivo model of pathological progression of steatosis will require further experiments.

Even if the exact role of heme homeostasis remains to be determined in our model, it is well recognized that heme metabolism is closely linked to iron homeostasis, known to cause oxidative stress through Fenton reaction, and also implicated in NAFLD [START_REF] Britton | Iron and non-alcoholic fatty liver disease[END_REF][START_REF] Corradini | Iron and steatohepatitis[END_REF]. As a mitochondrial dysfunction was presently detected, the content of labile iron contained in mitochondria was evaluated. Despite the fact that an increased deposition of iron has previously been detected in NAFLD [START_REF] Corradini | Iron and steatohepatitis[END_REF], to our knowledge, this is the first time that an increase (2-fold) in mitochondrial Fe 2+ pool is observed in the context of in vivo pathological progression of steatosis upon exposure to toxicants (Figure 6a andb). Besides an elevation of mitochondrial iron in liver, we cannot yet exclude that iron content could also be elevated in other cell compartments, notably in ferritin complex, or in blood (bound to transferrin or hemoglobin). This elevation of mitochondrial iron was actually in agreement with the up-regulation of numerous iron-related genes like ferritin (fth1a, fthl30) and mitoferrin 1 and 2 (slc25a37, slc25a28). Interestingly, most of the gene regulations regarding iron homeostasis observed under our experimental conditions were prevented in vivo by CH223191 or in vitro in human AhR-KO HepaRG cells (Figure 4c and4e). Thus, this strongly suggested a role for AhR in the increase in iron content. This hypothesis was confirmed by the protective effect of the AhR inhibitor CH223191 against mitochondrial iron accumulation and lipid peroxidation (Figure 7). In order to estimate the role of this mitochondrial iron accumulation in our context, we used a recognized iron chelator, namely deferoxamine (Figure 6). Not only this chelator prevented the alterations in ACCEPTED MANUSCRIPT / CLEAN COPY mitochondrial iron pool (Figure 6c) and in mRNA expression detected for most of the genes linked to heme and iron homeostasis (Figure 6e), but it also prevented the induction of gene markers of cell death (casp3a), inflammation (il6), and oxidative stress (prdx1) (Figure 6f); finally, it also protected from liver injuries induced by B[a]P/ethanol co-exposure (Figure 6h).

It should be noted that AhR inhibition prevented, in our in vivo model, the decrease in mitochondrial respiration and gene transcriptomic changes related to cell death and inflammation and liver cell damages (Figure 4). Several in vitro and in vivo studies favor a role for iron in the liver alterations induced by environmental contaminants such as TCDD and polychlorinated biphenyls (PCBs) (Fader et al. 2017;[START_REF] Smith | Synergy of iron in the toxicity and carcinogenicity of polychlorinated biphenyls (PCBs) and related chemicals[END_REF]. Especially concerning B[a]P toxicity, this PAH as well as TCDD have been reported in vivo to modulate expression of hepcidin, to disturb heme homeostasis and to induce liver inflammation (Fader et al. 2017;Fader and Zacharewski 2017;[START_REF] Wang | Hepcidin gene expression induced in the developmental stages of fish upon exposure to Benzo[a]pyrene (BaP)[END_REF]). Nevertheless, a clear link between xenobiotic exposure, iron overload and liver toxicity has been, so far, only demonstrated in vivo for TCDD, but not for B[a]P (Fader and Zacharewski 2017). Nonetheless, B[a]P was shown in vitro to increase iron uptake in an hepatic cell line with consecutive increase of oxidative stress and cell death [START_REF] Gorria | Membrane fluidity changes are associated with benzo[a]pyrene-induced apoptosis in F258 cells: protection by exogenous cholesterol[END_REF]. Regarding co-exposure to B[a]P and ethanol, only one study, from our team, has previously demonstrated in vitro that this mixture induced an iron overload responsible for an exacerbation of oxidative stress, and hence of hepatocyte death [START_REF] Collin | Cooperative interaction of benzo[a]pyrene and ethanol on plasma membrane remodeling is responsible for enhanced oxidative stress and cell death in primary rat hepatocytes[END_REF]. Considering the mechanisms of iron accumulation, several possibilities could be proposed and will need to be further investigated in future. First, transcriptional regulation induced by co-exposure might favor iron uptake and retention, and limit iron export from liver. This hypothesis is coherent with the induction of genes such as ferritins (fth1a, fthl30) and mitoferrin (slc25a37, slc25a28) in our co-exposed HFD-fed zebrafish larvae model, and with increase of iron uptake in F258 hepatic cells following B[a]P exposure (Gorria et al. ACCEPTED MANUSCRIPT / CLEAN COPY 2006b). The second possibility refers to membrane remodeling, i.e change in membrane fluidity and/or modulation of lipid raft signaling. Indeed, such a membrane remodeling has been involved in B[a]P-induced iron elevation and apoptosis in rat hepatic epithelial F258 cells (Gorria et al. 2006a), and in labile iron increase, lysosomal membrane permeabilization, oxidative stress and cell death induced cooperatively by B[a]P and ethanol in hepatic WIF-B9 cells [START_REF] Collin | Cooperative interaction of benzo[a]pyrene and ethanol on plasma membrane remodeling is responsible for enhanced oxidative stress and cell death in primary rat hepatocytes[END_REF]. Regarding this latter point, it is worth noting that we previously demonstrated an implication of membrane remodeling in the pathological progression of steatosis in our model of HFD-fed zebrafish larvae upon B[a]P/ethanol co-exposure (Imran et al. 2018).

Altogether, our data strongly indicate that mitochondrial iron accumulation, dependent on AhR activation, would be largely responsible for the progression from steatosis to a steatohepatitis-like state following B[a]P/ethanol co-exposure. Iron accumulation, notably in mitochondria, is thus shown for the first time to our knowledge as a key event in toxicant-induced liver disease exacerbation in a context of NAFLD. Remarkably, these conclusions seem to be relevant of human heath as iron deposition in human liver has been correlated with oxidative stress and progression toward steatohepatitis or fibrosis [START_REF] Britton | Iron and non-alcoholic fatty liver disease[END_REF]). However, the role for xenobiotic exposure, particularly in mixture, in human liver iron overload and NAFLD is still not elucidated and need to be explored. In this context, one interesting concept should be investigated deeper : the ferroptosis. Ferroptosis is a recently described specific type of non-apoptotic cell death dependant on iron and lipid peroxidation [START_REF] Cao | Mechanisms of ferroptosis[END_REF][START_REF] Mou | Ferroptosis, a new form of cell death: opportunities and challenges in cancer[END_REF][START_REF] Qi | Ferroptosis Affects the Progression of Nonalcoholic Steatohepatitis via the Modulation of Lipid Peroxidation-Mediated Cell Death in Mice[END_REF][START_REF] Tsurusaki | Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis[END_REF]. Thus, ferroptosis is known to be suppressed by iron chelators as deferoxamine and with antioxidants [START_REF] Mou | Ferroptosis, a new form of cell death: opportunities and challenges in cancer[END_REF][START_REF] Tsurusaki | Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis[END_REF] which is very coherent with results observed in our zebrafish model. In addition, this type of cell death has been also associated with NASH progression (Qi ACCEPTED MANUSCRIPT / CLEAN COPY et al. 2020;[START_REF] Tsurusaki | Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis[END_REF]. Under our experimental conditions, it would thus be interesting to test such an involvement of ferroptosis notably by testing the effects of a specific inhibitor, like ferrostatin-1 [START_REF] Cao | Mechanisms of ferroptosis[END_REF][START_REF] Skouta | Ferrostatins inhibit oxidative lipid damage and cell death in diverse disease models[END_REF].

Conclusions

On the whole, this study, using a model of HFD-fed steatotic zebrafish larva, has shed new light on the mechanisms involved in the in vivo transition of liver steatosis toward steatohepatitis-like state induced by B[a]P/ethanol co-exposure. Indeed, this co-exposure activates AhR, then leading to transcriptomic alterations of heme and iron homeostasis, and also of mitochondrial functions. The consequences of such alterations were notably an elevation of heme and hemin content in zebrafish larvae, probably dependent on an increase in heme synthesis, since no change in bilirubin could be observed. The role of heme and hemin are still speculative but they most likely play a role in NAFLD progression through oxidative stress. Looking at mitochondrial disruptions, the main changes impacted morphology (smaller and flattened mitochondria), mitochondrial respiration, and labile iron (Fe 2+ ) content. We proposed that iron overload in mitochondria, possibly depending on AhR activation and likely acting via oxidative stress, would represent a key event in the steatosis progression induced by B[a]P/ethanol co-exposure of HFD zebrafish larvae as iron chelation has been found to be largely protective towards cell death and inflammation. are also very grateful to MRiC and H2P2 platforms (UMS BIOSIT, Rennes, France), notably Stéphanie Dutertre (MRic) for confocal microscopic imagery, Alain Fautrel and Pascal Belaud (H2P2) for their help on histological staining, and finally Agnes Burel (MRiC) for her expertise on electron microscopy. We also wish to thank Dr Olivier Loréal (UMR 1241, Inserm, Rennes) for the fruitful discussion regarding iron homeostasis. Muhammad Imran was the recipient of a fellowship from the Higher Education Commission, Pakistan. Simon Bucher was recipient of fellowships from the Région Bretagne (ARED) and from the Agence Nationale de la Recherche (ANR). We also wish to thank ANR and the Institut Thématique Multi-Organisme Cancer (ITMO Cancer) d'Aviesan for financial supports to our work (STEATOX project, "ANR-13-CESA-0009" and METAhCOL project, n°17CE040_00). For these experiments, some larvae were also co-treated with 1 µM CH223191 (CH), a specific AhR antagonist. In (e), HepaRG cells wild-type (WT) or knock-out for AhR (KO) (see reference (Bucher et al. 2018a)

Cellular toxicity & NAFLD progression

Highlights

• A transcriptomic approach was used on an in vivo model of zebrafish larvae to reveal mechanisms of NAFLD progression induced by B[a]P/ethanol co-exposure.

• B[a]P/ethanol co-exposure aggravates NAFLD through mitochondrial dysfunction.

• AhR activation induced by B[a]P/ethanol co-exposure is responsible of mitochondrial iron overload and oxidative stress involved in toxicant-induced NAFLD progression.

Graphical Abstract

  Fig 4a: gstp1; Fig 4b: tspo and uqcc3; Fig 4c: fech, blvra, fth1a, fthl30; Fig 4d:

  prdx1

Figure 1

 1 Figure legends:

Figure 2 Figure 3

 23 Figure 2 Validation of zebrafish transcriptomic screening and relevance in human HepaRG cells. (a, b, c) Zebrafish larvae were fed with HFD from 4 dpf and then treated with control vehicle (C) or exposed to 25nM B[a]P and 43 mM ethanol (BE) for seven days (from 5 to 12 dpf). mRNA samples were collected from pools of 10 to 20 larvae, and mRNA expression was evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) for different groups of function-related genes, i.e. mitochondria-related genes (a) (n≥10), heme & iron-related genes (b) (n≥5), oxidative stress & AhR signaling-related genes (c) (n≥7). (d) HepaRG cells were supplemented with fatty acids for 2 days (150 µM stearic acid and 150 µM oleic acid) and co-treated (BE) or not (C) with B[a]P (2.5 μM) and ethanol (25mM) for 2 weeks (see reference (Bucher et al. 2018b) for further details) (n≥3). mRNA expression was evaluated by RT-qPCR for different groups of function-related genes. Data are expressed relative to mRNA levels found in control condition (C), set at 0 (log 2 change). Values are the mean ± SEM. *, **, *** statistically different from HFD control with respectively p<0.05; p<0.01 and p<0.001. Trends of change in gene expression found in zebrafish either in microarray (a, b, c)or byRT-qPCR (d) are indicated by white arrows when consistent with microarray or RT-qPCR observations or in grey arrows when not (by an up arrow for up-regulated genes and down arrow for down regulated genes)

Figure 4

 4 Figure 4 Involvement of AhR activation in mitochondrial dysfunction and liver toxicity induced by B[a]P/ethanol co-exposure. Zebrafish larvae were fed with HFD from 4 dpf and then treated at 5 dpf with control vehicle (C) or exposed to B[a]P and ethanol (BE) (25 nM B[a]P + 43 mM ethanol for 7 days in (a-d, g) or 1 µM B[a]P + 173 mM ethanol for 1 day in (f).

Figure 5 Figure 6

 56 Figure 5 Disruption of heme metabolism and involvement of oxidative stress in liver injury induced by B[a]P/ethanol co-exposure. Zebrafish larvae were fed with HFD from 4 dpf and then treated with control vehicle (C) or exposed to 25 nM B[a]P and 43 mM ethanol (BE) for 7 days ( from 5 to 12 dpf). Some larvae were also co-treated with 25 µM quercetin (Que) or 100 µM vitamin E (Vit E). Levels of heme (a) (n≥18), hemin (b) (n≥7) and bilirubin (c) (n≥11) were evaluated from homogenates of pools of whole larva. (d) Lipid peroxidation, marker of oxidative stress, was assessed in zebrafish larvae by quantification of fluorescence intensities in liver following staining of larvae with C11-Bodipy 581/591 (BC11) (n≥6). (e) Counting of liver damaged cells was performed on histological sections of zebrafish stained by HES from at least 3 larvae per condition. Values are the mean ± SEM. *, **, *** indicate a statistically significant effect of BE co-exposure vs control counterpart in presence or not of antioxidant (BE vs C; Que+BE vs Que; Vit E+BE vs Vit E) with p<0.05, p<0.01 and p<0.001 respectively; § indicates a statistically significant effect of quercetin or vitamin E vs counterpart without antioxidant (Que vs C; Que+BE vs BE; Vit E vs C; Vit E+BE vs BE) with p<0.05 (d, e); # indicates a statistically significant interaction between co-exposure and antioxidant (d, e) with p<0.05

Figure 7 :

 7 Figure 7: Involvement of AhR in liver mitochondrial iron accumulation and lipid peroxidation induced by B[a]P/ethanol co-exposure of steatotic zebrafish larva. Zebrafish larvae were fed with HFD from 4 dpf and then treated with control vehicle (C) or exposed to 25 nM B[a]P and 43 mM ethanol (BE) for 7 days ( from 5 to 12 dpf). Some larvae were also co-treated with a protective agent: 1 µM CH223191 (CH). (a) The relative mitochondrial iron content was assessed by quantification of green fluorescence intensities in the liver using confocal images. (b) Lipid peroxidation, a marker of oxidative stress, was assessed in zebrafish larvae by quantification of fluorescence intensities in the liver following staining with C11-Bodipy 581/591 (BC11). Values are the mean ± SEM (n≥7). *, **, *** indicate a statistically significant effect of BE co-exposure vs control counterpart in presence or not of CH (BE vs C and CH+BE vs CH) with p<0.05, p<0.01 and p<0.001 respectively; § indicates a statistically significant effect of CH vs counterpart without CH (CH vs C; CH+BE vs BE) with p<0.05; # indicates a statistically significant interaction between co-exposure and CH treatment with p<0.05.

Acknowledgements

We first wish to thank INRA-LPGP (Institut National de la Recherche Agronomique, Laboratoire de Physiologie et Génomique des Poissons, Rennes, France) for providing zebrafish eggs. We ACCEPTED MANUSCRIPT / CLEAN COPY

Funding: This work was supported by the Agence Nationale de la Recherche (ANR) and the Institut Thématique Multi-Organisme Cancer (ITMO Cancer) d'Aviesan (STEATOX project, "ANR-13-CESA-0009" and METAhCOL project, n°17CE040_00).

Author contribution: Conception and design of the study (NP, MI, DLG, OS); Acquisition of data (MI, FC, BE, HLM, AD, MB, SB, AL); Analysis and interpretation of data (MI, NP, DLG, FC, LS, LH); Drafting the article or revising it critically for important intellectual content (MI, NP, DLG, OS, LS, BF, LH).

Data Availability: All transcriptomic data are available in our supplemental files. Consent for publication: All authors approve of this submission for publication.

Declarations

ACCEPTED MANUSCRIPT / CLEAN COPY

Competing interests: The authors declare no competing interests.

Supplementary material captions:

-Supplementary file S1: Details for all Materials and methods.

-Supplementary Figure S1: Illustrations of histological sections of zebrafish larvae to evaluate liver damages upon B[a]P/ethanol co-exposure of steatotic zebrafish larva with or without co-treatment with protective agents.

-Supplementary Figure S2: Illustrations of fluorescent staining of zebrafish larvae to evaluate liver mitochondrial iron accumulation and lipid peroxidation upon B[a]P/ethanol co-exposure of steatotic zebrafish larva with or without co-treatment with AhR antagonist, CH223191.

-Supplementary Figure S3: Effect of B[a]P/ethanol co-exposure on oxygen consumption rale in zebrafish larva and impact of the AhR antagonist, CH223191 (OCR curves).

-Supplementary Figure S4: Illustrations of fluorescent staining of zebrafish larvae to evaluate lipid peroxidation upon B[a]P/ethanol co-exposure of steatotic zebrafish larvae with or without co-treatment with an antioxidant, quercetin or vitamin E.

-Supplementary Table S1: Excel file containing fold change data of the 525 DEGs.

-Supplementary Table S2: Excel file containing the table of conversion of the 525 zebrafish DEGs into 259 human homologs with associated retained fold change.

-Supplementary Table S3: Excel file containing the table of several terms resulted from the Ingenuity Pathways Analysis (IPA) of the 259 human homologs of DEGs.

-Supplementary Table S4: Excel file containing tables of full results from the Ingenuity Pathways Analysis (IPA) of the 259 human homologs of DEGs.

-Supplementary Table S5: Excel file containing tables of sequences of the tested primers used in RT-qPCR.

-Supplementary Table S6: Excel file containing all compiled data and sample size information.

ACCEPTED MANUSCRIPT / CLEAN COPY