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.

Non-alcoholic fatty liver disease (NAFLD) is a worldwide epidemic for which environmental contaminants are increasingly recognized as important etiological factors. Among them, the combination of benzo[a]pyrene (B[a]P), a potent environmental carcinogen, with ethanol, was shown to induce the transition of steatosis toward steatohepatitis. However, the underlying mechanisms involved remain to be deciphered. In this context, we used high-fat diet fed zebrafish model, in which we previously observed progression of steatosis to a steatohepatitis-like state following a 7-day-co-exposure to 43 mM ethanol and 25 nM B[a]P. Transcriptomic analysis highlighted the potent role of mitochondrial dysfunction, alterations in heme and iron homeostasis, involvement of aryl hydrocarbon receptor (AhR) signaling, and oxidative stress. Most of these mRNA dysregulations were validated by RT-qPCR. Moreover, similar changes were observed using a human in vitro hepatocyte model, HepaRG cells. The mitochondria structural and functional alterations were confirmed by transmission electronic microscopy and Seahorse technology, respectively. Involvement of AhR signaling was evidenced by using in vivo an AhR antagonist, CH223191, and in vitro in AhR-knock-out HepaRG cells. Furthermore, as co-exposure was found to increase the levels of both heme and hemin, we investigated if mitochondrial iron could induce oxidative stress. We found that mitochondrial labile iron content was raised in toxicant-exposed larvae. This increase was prevented by the iron chelator, deferoxamine, which also inhibited liver co-exposure toxicity. Overall, these results suggest that the increase in mitochondrial iron content induced by B[a]P/ethanol co-exposure causes mitochondrial dysfunction that contributes to the pathological progression of NAFLD.

Protective Action of Ostreococcus tauri and Phaeodactylum tricornutum Extracts towards Benzo[a]Pyrene-Induced Cytotoxicity in Endothelial Cells.

Marine microalgae are known to be a source of bioactive molecules of interest to human health, such as n-3 polyunsaturated fatty acids (n-3 PUFAs) and carotenoids. The fact that some of these natural compounds are known to exhibit anti-inflammatory, antioxidant, anti-proliferative, and apoptosis-inducing effects, demonstrates their potential use in preventing cancers and cardiovascular diseases (CVDs). Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), is an ubiquitous environmental pollutant known to contribute to the development or aggravation of human diseases, such as cancer, CVDs, and immune dysfunction. Most of these deleterious effects are related to the activation of the polycyclic aromatic hydrocarbon receptor (AhR). In this context, two ethanolic microalgal extracts with concentrations of 0.1 to 5 µg/mL are tested, Ostreoccoccus tauri (OT) and Phaeodactylum tricornutum (PT), in order to evaluate and compare their potential effects towards B[a]P-induced toxicity in endothelial HMEC-1 cells. Our results indicate that the OT extract can influence the toxicity of B[a]P. Indeed, apoptosis and the production of extracellular vesicles were decreased, likely through the reduction of the expression of CYP1A1, a B[a]P bioactivation enzyme. Furthermore, the B[a]P-induced expression of the inflammatory cytokines IL-8 and IL1-ß was reduced. The PT extract only inhibited the expression of the B[a]P-induced cytokine IL-8 expression. The OT extract therefore seems to be a good candidate for counteracting the B[a]P toxicity.

Environmental carcinogenesis and pH homeostasis: Not only a matter of dysregulated metabolism.

According to the World Health Organization, around 20% of all cancers would be due to environmental factors. Among these factors, several chemicals are indeed well recognized carcinogens. The widespread contaminant benzo[a]pyrene (B[a]P), an often used model carcinogen of the polycyclic aromatic hydrocarbons' family, has been suggested to target most, if not all, cancer hallmarks described by Hanahan and Weinberg. It is classified as a group I carcinogen by the International Agency for Research on Cancer; however, the precise intracellular mechanisms underlying its carcinogenic properties remain yet to be thoroughly defined. Recently, the pH homeostasis, a well known regulator of carcinogenic processes, was suggested to be a key actor in both cell death and Warburg-like metabolic reprogramming induced upon B[a]P exposure. The present review will highlight those data with the aim of favoring research on the role of H+ dynamics in environmental carcinogenesis.

Zebrafish larva as a reliable model for in vivo assessment of membrane remodeling involvement in the hepatotoxicity of chemical agents.

The easy-to-use in vivo model, zebrafish larva, is being increasingly used to screen chemical-induced hepatotoxicity, with a good predictivity for various mechanisms of liver injury. However, nothing is known about its applicability in exploring the mechanism called membrane remodeling, depicted as changes in membrane fluidity or lipid raft properties. The aim of this study was, therefore, to substantiate the zebrafish larva as a suitable in vivo model in this context. Ethanol was chosen as a prototype toxicant because it is largely described, both in hepatocyte cultures and in rodents, as capable of inducing a membrane remodeling leading to hepatocyte death and liver injury. The zebrafish larva model was demonstrated to be fully relevant as membrane remodeling was maintained even after a 1-week exposure without any adaptation as usually reported in rodents and hepatocyte cultures. It was also proven to exhibit a high sensitivity as it discriminated various levels of cytotoxicity depending on the extent of changes in membrane remodeling. In this context, its sensitivity appeared higher than that of WIF-B9 hepatic cells, which is suited for analyzing this kind of hepatotoxicity. Finally, the protection afforded by a membrane stabilizer, ursodeoxycholic acid (UDCA), or by a lipid raft disrupter, pravastatin, definitely validated zebrafish larva as a reliable model to quickly assess membrane remodeling involvement in chemical-induced hepatotoxicity. In conclusion, this model, compatible with a high throughput screening, might be adapted to seek hepatotoxicants via membrane remodeling, and also drugs targeting membrane features to propose new preventive or therapeutic strategies in chemical-induced liver diseases. Copyright © 2016 John Wiley & Sons, Ltd.

Alkyl galactofuranosides strongly interact with Leishmania donovani membrane and provide antileishmanial activity.

We investigated the in vitro effects of four alkyl-galactofuranoside derivatives, i.e., octyl-ß-D-galactofuranoside (compound 1), 6-amino-ß-D-galactofuranoside (compound 2), 6-N-acetamido-ß-D-galactofuranoside (compound 3), and 6-azido-ß-D-galactofuranoside (compound 4), on Leishmania donovani. Their mechanism of action was explored using electron paramagnetic resonance spectroscopy (EPR) and nuclear magnetic resonance (NMR), and ultrastructural alterations were analyzed by transmission electron microscopy (TEM). Compound 1 showed the most promising effects by inhibiting promastigote growth at a 50% inhibitory concentration (IC50) of 8.96±2.5 µM. All compounds exhibit low toxicity toward human macrophages. Compound 1 had a higher selectivity index than the molecule used for comparison, i.e., miltefosine (159.7 versus 37.9, respectively). EPR showed that compound 1 significantly reduced membrane fluidity compared to control promastigotes and to compound 3. The furanose ring was shown to support this effect, since the isomer galactopyranose had no effect on parasite membrane fluidity or growth. NMR showed a direct interaction of all compounds (greatest with compound 1, followed by compounds 2, 3, and 4, in descending order) with the promastigote membrane and with octyl-galactopyranose and octanol, providing evidence that the n-octyl chain was primarily involved in anchoring with the parasite membrane, followed by the putative crucial role of the furanose ring in the antileishmanial activity. A morphological analysis of compound 1-treated promastigotes by TEM revealed profound alterations in the parasite membrane and organelles, but this was not the case with compound 3. Quantification of annexin V binding by flow cytometry confirmed that compound 1 induced apoptosis in >90% of promastigotes. The effect of compound 1 was also assessed on intramacrophagic amastigotes and showed a reduction in amastigote growth associated with an increase of reactive oxygen species (ROS) production, thus validating its promising effect.

Deciphering the Role of PIG1 and DHN-Melanin in Scedosporium apiospermum Conidia.

Scedosporium apiospermum is a saprophytic filamentous fungus involved in human infections, of which the virulence factors that contribute to pathogenesis are still poorly characterized. In particular, little is known about the specific role of dihydroxynaphtalene (DHN)-melanin, located on the external layer of the conidia cell wall. We previously identified a transcription factor, PIG1, which may be involved in DHN-melanin biosynthesis. To elucidate the role of PIG1 and DHN-melanin in S. apiospermum, a CRISPR-Cas9-mediated PIG1 deletion was carried out from two parental strains to evaluate its impact on melanin biosynthesis, conidia cell-wall assembly, and resistance to stress, including the ability to survive macrophage engulfment. ΔPIG1 mutants did not produce melanin and showed a disorganized and thinner cell wall, resulting in a lower survival rate when exposed to oxidizing conditions, or high temperature. The absence of melanin increased the exposure of antigenic patterns on the conidia surface. PIG1 regulates the melanization of S. apiospermum conidia, and is involved in the survival to environmental injuries and to the host immune response, that might participate in virulence. Moreover, a transcriptomic analysis was performed to explain the observed aberrant septate conidia morphology and found differentially expressed genes, underlining the pleiotropic function of PIG1.

NHE-1 relocation outside cholesterol-rich membrane microdomains is associated with its benzo[a]pyrene-related apoptotic function.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are ubiquitous toxic environmental pollutants capable of inducing cell death. Intracellular pH plays a key role in the regulation of cell survival and death. Our previous works have demonstrated that intracellular alkalinization mediated by Na(+)/H(+) exchanger 1 (NHE-1) is a critical event involved in B[a]P-induced apoptosis. The aim of this study was to further elucidate the mechanisms of NHE-1 activation upon B[a]P exposure. METHODS: We tested the effects of plasma membrane cholesterol enrichment or depletion on B[a]P-induced NHE-1 activation related to apoptosis. We isolated cholesterol-rich plasma membrane microdomains to assess NHE-1 submembrane location and immunoprecipitated NHE-1 from the different sub-membrane fractions obtained to examine NHE-1 protein interactions during B[a]P-induced apoptosis. RESULTS: We found that NHE-1 is preferentially located in cholesterol-rich microdomains and that B[a]P activates NHE-1 via its relocation and binding of calmodulin outside these specialized plasma membrane microstructures; these events are necessary for the execution of the apoptosis-related intracellular alkalinization. CONCLUSION: Plasma membrane location of NHE-1 affects its protein interactions and apoptotic function.

Membrane remodeling, an early event in benzo[a]pyrene-induced apoptosis.

Benzo[alpha]pyrene (B[alpha]P) often serves as a model for mutagenic and carcinogenic polycyclic aromatic hydrocarbons (PAHs). Our previous work suggested a role of membrane fluidity in B[alpha]P-induced apoptotic process. In this study, we report that B[alpha]P modifies the composition of cholesterol-rich microdomains (lipid rafts) in rat liver F258 epithelial cells. The cellular distribution of the ganglioside-GM1 was markedly changed following B[alpha]P exposure. B[alpha]P also modified fatty acid composition and decreased the cholesterol content of cholesterol-rich microdomains. B[alpha]P-induced depletion of cholesterol in lipid rafts was linked to a reduced expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). Aryl hydrocarbon receptor (AhR) and B[alpha]P-related H(2)O(2) formation were involved in the reduced expression of HMG-CoA reductase and in the remodeling of membrane microdomains. The B[alpha]P-induced membrane remodeling resulted in an intracellular alkalinization observed during the early phase of apoptosis. In conclusion, B[alpha]P altered the composition of plasma membrane microstructures through AhR and H(2)O(2) dependent-regulation of lipid biosynthesis. In F258 cells, the B[alpha]P-induced membrane remodeling was identified as an early apoptotic event leading to an intracellular alkalinization.

MEHP/ethanol co-exposure favors the death of steatotic hepatocytes, possibly through CYP4A and ADH involvement.

Liver steatosis has been associated with various etiological factors (obesity, alcohol, environmental contaminants). How those factors work together to induce steatosis progression is still scarcely evaluated. Here, we tested whether phthalates could potentiate death of steatotic hepatocytes when combined with ethanol. Pre-steatotic WIF-B9 hepatocytes were co-exposed to mono (2-ethylhexyl) (MEHP, 500 nM; main metabolite of di (2-ethylhexyl) phthalate or DEHP) and ethanol (5 mM) for 5 days. An increased apoptotic death was detected, involving a DNA damage response. Using 4-Methypyrazole to inhibit ethanol metabolism, and CH-223191 to antagonize the AhR receptor, we found that an AhR-dependent increase in alcohol dehydrogenase (ADH) activity was essential for cell death upon MEHP/ethanol co-exposure. Toxicity was also prevented by HET0016 to inhibit the cytochrome P450 4A (CYP4A). Using the antioxidant thiourea, a role for oxidative stress was uncovered, notably triggering DNA damage. Finally, co-exposing the in vivo steatosis model of high fat diet (HFD)-zebrafish larvae to DEHP (2.56 nM)/ethanol (43 mM), induced the pathological progression of liver steatosis alongside an increased Cyp4t8 (human CYP4A homolog) mRNA expression. Altogether, these results further emphasized the deleterious impact of co-exposures to ethanol/environmental pollutant towards steatosis pathological progression, and unraveled a key role for ADH and CYP4A in such effects.

Extracellular vesicles released by polycyclic aromatic hydrocarbons-treated hepatocytes trigger oxidative stress in recipient hepatocytes by delivering iron.

A growing body of evidences indicate the major role of extracellular vesicles (EVs) as players of cell communication in the pathogenesis of liver diseases. EVs are membrane-enclosed vesicles released by cells into the extracellular environment. Oxidative stress is also a key component of liver disease pathogenesis, but no role for hepatocyte-derived EVs has yet been described in the development of this process. Recently, some polycyclic aromatic hydrocarbons (PAHs), widespread environmental contaminants, were demonstrated to induce EV release from hepatocytes. They are also well-known to trigger oxidative stress leading to cell death. Therefore, the aim of this work was to investigate the involvement of EVs derived from PAHs-treated hepatocytes (PAH-EVs) in possible oxidative damages of healthy recipient hepatocytes, using both WIF-B9 and primary rat hepatocytes. We first showed that the release of EVs from PAHs -treated hepatocytes depended on oxidative stress. PAH-EVs were enriched in proteins related to oxidative stress such as NADPH oxidase and ferritin. They were also demonstrated to contain more iron. PAH-EVs could then induce oxidative stress in recipient hepatocytes, thereby leading to apoptosis. Mitochondria and lysosomes of recipient hepatocytes exhibited significant structural alterations. All those damages were dependent on internalization of EVs that reached lysosomes with their cargoes. Lysosomes thus appeared as critical organelles for EVs to induce apoptosis. In addition, pro-oxidant components of PAH-EVs, e.g. NADPH oxidase and iron, were revealed to be necessary for this cell death.

Ethanol induces oxidative stress in primary rat hepatocytes through the early involvement of lipid raft clustering.

UNLABELLED: The role of the hepatocyte plasma membrane structure in the development of oxidative stress during alcoholic liver diseases is not yet fully understood. Previously, we have established the pivotal role of membrane fluidity in ethanol-induced oxidative stress, but no study has so far tested the involvement of lipid rafts. In this study, methyl-beta-cyclodextrin or cholesterol oxidase, which were found to disrupt lipid rafts in hepatocytes, inhibited both reactive oxygen species production and lipid peroxidation, and this suggested a role for these microstructures in oxidative stress. By immunostaining of lipid raft components, a raft clustering was detected in ethanol-treated hepatocytes. In addition, we found that rafts were modified by formation of malondialdehyde adducts and disulfide bridges. Interestingly, pretreatment of cells by 4-methyl-pyrazole (to inhibit ethanol metabolism) and various antioxidants prevented the ethanol-induced raft aggregation. In addition, treatment of hepatocytes by a stabilizing agent (ursodeoxycholic acid) or a fluidizing compound [2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate] led to inhibition or enhancement of raft clustering, respectively, which pointed to a relationship between membrane fluidity and lipid rafts during ethanol-induced oxidative stress. We finally investigated the involvement of phospholipase C in raft-induced oxidative stress upon ethanol exposure. Phospholipase C was shown to be translocated into rafts and to participate in oxidative stress by controlling hepatocyte iron content. CONCLUSION: Membrane structure, depicted as membrane fluidity and lipid rafts, plays a key role in ethanol-induced oxidative stress of the liver, and its modulation may be of therapeutic relevance.

Cisplatin-induced apoptosis involves membrane fluidification via inhibition of NHE1 in human colon cancer cells.

We have previously shown that cisplatin triggers an early acid sphingomyelinase (aSMase)-dependent ceramide generation concomitantly with an increase in membrane fluidity and induces apoptosis in HT29 cells. The present study further explores the role and origin of membrane fluidification in cisplatin-induced apoptosis. The rapid increase in membrane fluidity following cisplatin treatment was inhibited by membrane-stabilizing agents such as cholesterol or monosialoganglioside-1. In HT29 cells, these compounds prevented the early aggregation of Fas death receptor and of membrane lipid rafts on cell surface and significantly inhibited cisplatin-induced apoptosis without altering drug intracellular uptake or cisplatin DNA adducts formation. Early after cisplatin treatment, Na+/H+ membrane exchanger-1 (NHE1) was inhibited leading to intracellular acidification, aSMase was activated, and ceramide was detected at the cell membrane. Treatment of HT29 cells with Staphylococcus aureus sphingomyelinase increased membrane fluidity. Moreover, pretreatment with cariporide, a specific inhibitor of NHE1, inhibited cisplatin-induced intracellular acidification, aSMase activation, ceramide membrane generation, membrane fluidification, and apoptosis. Finally, NHE1-expressing PS120 cells were more sensitive to cisplatin than NHE1-deficient PS120 cells. Altogether, these findings suggest that the apoptotic pathway triggered by cisplatin involves a very early NHE1-dependent intracellular acidification leading to aSMase activation and increase in membrane fluidity. These events are independent of cisplatin-induced DNA adducts formation. The membrane exchanger NHE1 may be another potential target of cisplatin, increasing cell sensitivity to this compound.

Disturbances in H+ dynamics during environmental carcinogenesis.

Despite the improvement of diagnostic methods and anticancer therapeutics, the human population is still facing an increasing incidence of several types of cancers. According to the World Health Organization, this growing trend would be partly linked to our environment, with around 20% of cancers stemming from exposure to environmental contaminants, notably chemicals like polycyclic aromatic hydrocarbons (PAHs). PAHs are widespread pollutants in our environment resulting from incomplete combustion or pyrolysis of organic material, and thus produced by both natural and anthropic sources; notably benzo[a]pyrene (B[a]P), i.e. the prototypical molecule of this family, that can be detected in cigarette smoke, diesel exhaust particles, occupational-related fumes, and grilled food. This molecule is a well-recognized carcinogen belonging to group 1 carcinogens. Indeed, it can target the different steps of the carcinogenic process and all cancer hallmarks. Interestingly, H+ dynamics have been described as key parameters for the occurrence of several, if not all, of these hallmarks. However, information regarding the role of such parameters during environmental carcinogenesis is still very scarce. The present review will thus mainly give an overview of the impact of B[a]P on H+ dynamics in liver cells, and will show how such alterations might impact different aspects related to the finely-tuned balance between cell death and survival processes, thereby likely favoring environmental carcinogenesis. In total, the main objective of this review is to encourage further research in this poorly explored field of environmental molecular toxicology.

PAHs increase the production of extracellular vesicles both in vitro in endothelial cells and in vivo in urines from rats.

Environmental contaminants, to which humans are widely exposed, cause or worsen several diseases, like cardiovascular diseases and cancers. Among these molecules, polycyclic aromatic hydrocarbons (PAHs) stand out since they are ubiquitous pollutants found in ambient air and diet. Because of their toxic effects, public Health agencies promote development of research studies aiming at increasing the knowledge about PAHs and the discovery of biomarkers of exposure and/or effects. Extracellular vesicles (EVs), including small extracellular vesicles (S-EVs or exosomes) and large extracellular vesicles (L-EVs or microvesicles), are delivery systems for multimolecular messages related to the nature and status of the originating cells. Because they are produced by all cells and detected within body fluids, EV releases could act as cell responses and thereby serve as biomarkers. To test whether EVs can serve as biomarkers of PAHs exposure, we evaluate the effects of these pollutants on EV production using an in vitro approach (human endothelial cell line, HMEC-1) and an in vivo approach (urine samples from PAHs-exposed rats). Our study indicates that, i) PAH exposure increases in vitro the EV production by endothelial cells and in vivo the release of EVs in urine, and that the stimulating effects of PAHs concern both S-EVs and L-EVs; ii) PAH exposure and more particularly exposure to B[a]P, can influence the composition of exosomes produced by endothelial cells; iii) the aryl hydrocarbon receptor, a cytosolic receptor associated to most deleterious effects of PAHs, would be involved in the PAH effects on the release of S-EVs, but not L-EVs. These results suggest that EVs may have utility for monitoring exposure to PAHs, and more particularly to B[a]P, considered as reference PAH, and to detect the related early cellular response prior to end-organ damages.

Polycyclic Aromatic Hydrocarbons Can Trigger Hepatocyte Release of Extracellular Vesicles by Various Mechanisms of Action Depending on Their Affinity for the Aryl Hydrocarbon Receptor.

Extracellular vesicles (EVs) are membrane-enclosed nanostructures released by cells into the extracellular environment. As major actors of physiological intercellular communication, they have been shown to be pathogenic mediators of several liver diseases. Extracellular vesicles also appear to be potential actors of drug-induced liver injury but nothing is known concerning environmental pollutants. We aimed to study the impact of polycyclic aromatic hydrocarbons (PAHs), major contaminants, on hepatocyte-derived EV production, with a special focus on hepatocyte death. Three PAHs were selected, based on their presence in food and their affinity for the aryl hydrocarbon receptor (AhR): benzo[a]pyrene (BP), dibenzo[a,h]anthracene (DBA), and pyrene (PYR). Treatment of primary rat and WIF-B9 hepatocytes by all 3 PAHs increased the release of EVs, mainly comprised of exosomes, in parallel with modifying exosome protein marker expression and inducing apoptosis. Moreover, PAH treatment of rodents for 3 months also led to increased EV levels in plasma. The EV release involved CYP metabolism and the activation of the transcription factor, the AhR, for BP and DBA and another transcription factor, the constitutive androstane receptor, for PYR. Furthermore, all PAHs increased cholesterol levels in EVs but only BP and DBA were able to reduce the cholesterol content of total cell membranes. All cholesterol changes very likely participated in the increase in EV release and cell death. Finally, we studied changes in cell membrane fluidity caused by BP and DBA due to cholesterol depletion. Our data showed increased cell membrane fluidity, which contributed to hepatocyte EV release and cell death.

Membrane Remodeling as a Key Player of the Hepatotoxicity Induced by Co-Exposure to Benzo[a]pyrene and Ethanol of Obese Zebrafish Larvae.

The rise in prevalence of non-alcoholic fatty liver disease (NAFLD) constitutes an important public health concern worldwide. Including obesity, numerous risk factors of NAFLD such as benzo[a]pyrene (B[a]P) and ethanol have been identified as modifying the physicochemical properties of the plasma membrane in vitro thus causing membrane remodeling-changes in membrane fluidity and lipid-raft characteristics. In this study, the possible involvement of membrane remodeling in the in vivo progression of steatosis to a steatohepatitis-like state upon co-exposure to B[a]P and ethanol was tested in obese zebrafish larvae. Larvae bearing steatosis as the result of a high-fat diet were exposed to ethanol and/or B[a]P for seven days at low concentrations coherent with human exposure in order to elicit hepatotoxicity. In this condition, the toxicant co-exposure raised global membrane order with higher lipid-raft clustering in the plasma membrane of liver cells, as evaluated by staining with the fluoroprobe di-4-ANEPPDHQ. Involvement of this membrane's remodeling was finally explored by using the lipid-raft disruptor pravastatin that counteracted the effects of toxicant co-exposure both on membrane remodeling and toxicity. Overall, it can be concluded that B[a]P/ethanol co-exposure can induce in vivo hepatotoxicity via membrane remodeling which could be considered as a good target mechanism for developing combination therapy to deal with steatohepatitis.

Possible Involvement of Mitochondrial Dysfunction and Oxidative Stress in a Cellular Model of NAFLD Progression Induced by Benzo[a]pyrene/Ethanol CoExposure.

Exposure to xenobiotics could favor the transition of nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis in obese patients. Recently, we showed in different models of NAFL that benzo[a]pyrene (B[a]P) and ethanol coexposure induced a steatohepatitis-like state. One model was HepaRG cells incubated with stearate and oleate for 2 weeks. In the present study, we wished to determine in this model whether mitochondrial dysfunction and reactive oxygen species (ROS) overproduction could be involved in the occurrence of this steatohepatitis-like state. CRISPR/Cas9-modified cells were also used to specify the role of aryl hydrocarbon receptor (AhR), which is potently activated by B[a]P. Thus, nonsteatotic and steatotic HepaRG cells were treated with B[a]P, ethanol, or both molecules for 2 weeks. B[a]P/ethanol coexposure reduced mitochondrial respiratory chain activity, mitochondrial respiration, and mitochondrial DNA levels and induced ROS overproduction in steatotic HepaRG cells. These deleterious effects were less marked or absent in steatotic cells treated with B[a]P alone or ethanol alone and in nonsteatotic cells treated with B[a]P/ethanol. Our study also disclosed that B[a]P/ethanol-induced impairment of mitochondrial respiration was dependent on AhR activation. Hence, mitochondrial dysfunction and ROS generation could explain the occurrence of a steatohepatitis-like state in steatotic HepaRG cells exposed to B[a]P and ethanol.

Evidence of selective activation of aryl hydrocarbon receptor nongenomic calcium signaling by pyrene.

In its classical genomic mode of action, the aryl hydrocarbon receptor (AhR) acts as a ligand activated transcription factor regulating expression of target genes such as CYP1A1 and CYP1B1. Some ligands may also trigger more rapid nongenomic responses through AhR, including calcium signaling (Ca2+). In the present study we observed that pyrene induced a relatively rapid increase in intracellular Ca2+-concentrations ([Ca2+]i) in human microvascular endothelial cells (HMEC-1) and human embryonic kidney cells (HEK293) that was attenuated by AhR-inhibitor treatment and/or transient AhR knockdown by RNAi. In silico molecular docking based on homology models, suggested that pyrene is not able to bind to the human AhR in the agonist conformation. Instead, pyrene docked in the antagonist conformation of the AhR PAS-B binding pocket, although the interaction differed from antagonists such as GNF-351 and CH223191. Accordingly, pyrene did not induce CYP1A1 or CYP1B1, but suppressed CYP1-expression by benzo[a]pyrene (B[a]P) in HMEC-1 cells, confirming that pyrene act as an antagonist of AhR-induced gene expression. Use of pharmacological inhibitors and Ca2+-free medium indicated that the pyrene-induced AhR nongenomic [Ca2+]i increase was initiated by Ca2+-release from intracellular stores followed by a later phase of extracellular Ca2+-influx, consistent with store operated calcium entry (SOCE). These effects was accompanied by an AhR-dependent reduction in ordered membrane lipid domains, as determined by di-4-ANEPPDHQ staining. Addition of cholesterol inhibited both the pyrene-induced [Ca2+]i-increase and alterations in membrane lipid order. In conclusion, we propose that pyrene binds to AhR, act as an antagonist of the canonical genomic AhR/Arnt/CYP1-pathway, reduces ordered membrane lipid domains, and activates AhR nongenomic Ca2+-signaling from intracellular stores.

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.

We previously demonstrated that co-exposing pre-steatotic hepatocytes to benzo[a]pyrene (B[a]P), a carcinogenic environmental pollutant, and ethanol, favored cell death. Here, the intracellular mechanisms underlying this toxicity were studied. Steatotic WIF-B9 hepatocytes, obtained by a 48h-supplementation with fatty acids, were then exposed to B[a]P/ethanol (10 nM/5 mM, respectively) for 5 days. Nitric oxide (NO) was demonstrated to be a pivotal player in the cell death caused by the co-exposure in steatotic hepatocytes. Indeed, by scavenging NO, CPTIO treatment of co-exposed steatotic cells prevented not only the increase in DNA damage and cell death, but also the decrease in the activity of CYP1, major cytochrome P450s of B[a]P metabolism. This would then lead to an elevation of B[a]P levels, thus possibly suggesting a long-lasting stimulation of the transcription factor AhR. Besides, as NO can react with superoxide anion to produce peroxynitrite, a highly oxidative compound, the use of FeTPPS to inhibit its formation indicated its participation in DNA damage and cell death, further highlighting the important role of NO. Finally, a possible key role for AhR was pointed out by using its antagonist, CH-223191. Indeed it prevented the elevation of ADH activity, known to participate to the ethanol production of ROS, notably superoxide anion. The transcription factor, NFκB, known to be activated by ROS, was shown to be involved in the increase in iNOS expression. Altogether, these data strongly suggested cooperative mechanistic interactions between B[a]P via AhR and ethanol via ROS production, to favor cell death in the context of prior steatosis.

Co-exposure to benzo[a]pyrene and ethanol induces a pathological progression of liver steatosis in vitro and in vivo.

Hepatic steatosis (i.e. lipid accumulation) and steatohepatitis have been related to diverse etiologic factors, including alcohol, obesity, environmental pollutants. However, no study has so far analyzed how these different factors might interplay regarding the progression of liver diseases. The impact of the co-exposure to the environmental carcinogen benzo[a]pyrene (B[a]P) and the lifestyle-related hepatotoxicant ethanol, was thus tested on in vitro models of steatosis (human HepaRG cell line; hybrid human/rat WIF-B9 cell line), and on an in vivo model (obese zebrafish larvae). Steatosis was induced prior to chronic treatments (14, 5 or 7 days for HepaRG, WIF-B9 or zebrafish, respectively). Toxicity and inflammation were analyzed in all models; the impact of steatosis and ethanol towards B[a]P metabolism was studied in HepaRG cells. Cytotoxicity and expression of inflammation markers upon co-exposure were increased in all steatotic models, compared to non steatotic counterparts. A change of B[a]P metabolism with a decrease in detoxification was detected in HepaRG cells under these conditions. A prior steatosis therefore enhanced the toxicity of B[a]P/ethanol co-exposure in vitro and in vivo; such a co-exposure might favor the appearance of a steatohepatitis-like state, with the development of inflammation. These deleterious effects could be partly explained by B[a]P metabolism alterations.