SARS-CoV-2 receptor ACE2 is upregulated by fatty acids in human MASH.

Background & Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) results in steatosis, inflammation (steatohepatitis), and fibrosis. Patients with MASLD more likely develop liver injury in coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As viral RNA has been identified in liver tissues, we studied expression levels and cellular sources of the viral receptor angiotensin-converting enzyme 2 (ACE2) and coreceptors in MASLD and fibroinflammatory liver diseases. Methods: We built a transcriptomic MASLD meta-dataset (N = 243) to study SARS-CoV-2 receptor expression and verified results in 161 additional cases of fibroinflammatory liver diseases. We assessed the fibroinflammatory microenvironment by deconvoluting immune cell populations. We studied the cellular sources of ACE2 by multiplex immunohistochemistry followed by high-resolution confocal microscopy (N = 9 fatty livers; N = 7 controls), meta-analysis of two single-cell RNA sequencing datasets (N = 5 cirrhotic livers; N = 14 normal livers), and bulk transcriptomics from 745 primary cell samples. In vitro, we tested ACE2 mRNA expression in primary human hepatocytes treated with inflammatory cytokines, bacterial lipopolysaccharides, or long-chain fatty acids. Results: We detected ACE2 at the apical and basal poles of hepatocyte chords, in CLEC4M+ liver sinusoidal endothelial cells, the lumen of ABCC2+ bile canaliculi, HepPar-1+-TMPRSS2+ hepatocytes, cholangiocytes, and CD34+ capillary vessels. ACE2 steeply increased between 30 and 50 years of age; was related to liver fat area, inflammation, high immune reactivity, and fibrogenesis; and was upregulated in steatohepatitis. Although ACE2 mRNA was unmodified in alcoholic or viral hepatitis, it was upregulated in fibroinflammatory livers from overweight patients. In vitro, treatment of primary human hepatocytes with inflammatory cytokines alone downregulated but long chain fatty acids upregulated ACE2 mRNA expression. Conclusions: Lipid overload in fatty liver disease leads to an increased availability of ACE2 receptors. Impact and implications: COVID-19 can be a deadly disease in vulnerable individuals. Patients with fatty liver disease are at a higher risk of experiencing severe COVID-19 and liver injury. Recent studies have indicated that one of the reasons for this vulnerability is the presence of a key cell surface protein called ACE2, which serves as the main SARS-CoV-2 virus receptor. We describe the cellular sources of ACE2 in the liver. In patients with fatty liver disease, ACE2 levels increase with age, liver fat content, fibroinflammatory changes, enhanced positive immune checkpoint levels, and innate immune reactivity. Moreover, we show that long chain fatty acids can induce ACE2 expression in primary human hepatocytes. Understanding the cellular sources of ACE2 in the liver and the factors that influence its availability is crucial. This knowledge will guide further research and help protect potentially vulnerable patients through timely vaccination boosters, dietary adjustments, and improved hygiene practices.

Lung cancer associated with combustion particles and fine particulate matter (PM2.5) - The roles of polycyclic aromatic hydrocarbons (PAHs) and the aryl hydrocarbon receptor (AhR).

Air pollution is the leading cause of lung cancer after tobacco smoking, contributing to 20% of all lung cancer deaths. Increased risk associated with living near trafficked roads, occupational exposure to diesel exhaust, indoor coal combustion and cigarette smoking, suggest that combustion components in ambient fine particulate matter (PM2.5), such as polycyclic aromatic hydrocarbons (PAHs), may be central drivers of lung cancer. Activation of the aryl hydrocarbon receptor (AhR) induces expression of xenobiotic-metabolizing enzymes (XMEs) and increase PAH metabolism, formation of reactive metabolites, oxidative stress, DNA damage and mutagenesis. Lung cancer tissues from smokers and workers exposed to high combustion PM levels contain mutagenic signatures derived from PAHs. However, recent findings suggest that ambient air PM2.5 exposure primarily induces lung cancer development through tumor promotion of cells harboring naturally acquired oncogenic mutations, thus lacking typical PAH-induced mutations. On this background, we discuss the role of AhR and PAHs in lung cancer development caused by air pollution focusing on the tumor promoting properties including metabolism, immune system, cell proliferation and survival, tumor microenvironment, cell-to-cell communication, tumor growth and metastasis. We suggest that the dichotomy in lung cancer patterns observed between smoking and outdoor air PM2.5 represent the two ends of a dose-response continuum of combustion PM exposure, where tumor promotion in the peripheral lung appears to be the driving factor at the relatively low-dose exposures from ambient air PM2.5, whereas genotoxicity in the central airways becomes increasingly more important at the higher combustion PM levels encountered through smoking and occupational exposure.

Small RNA-sequencing reveals the involvement of microRNA-132 in benzo[a]pyrene-induced toxicity in primary human blood cells.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed environmental contaminants, triggering deleterious effects such as carcinogenicity and immunosuppression, and peripheral blood mononuclear cells (PBMCs) are among the main cell types targeted by these pollutants. In the present study, we sought to identify the expression profiles and function of miRNAs, gene regulators involved in major cellular processes recently linked to environmental pollutants, in PBMC-exposed to the prototypical PAH, benzo[a]pyrene (B[a]P). Using small RNA deep sequencing, we identified several B[a]P-responsive miRNAs. Bioinformatics analyses showed that their predicted targets could modulate biological processes relevant to cell death and survival. Further studies of the most highly induced miRNA, miR-132, showed that its up-regulation by B[a]P was time- and dose-dependent and required aryl hydrocarbon receptor (AhR) activation. By evaluating the role of miR-132 in B[a]P-induced cell death, we propose a mechanism linking B[a]P-induced miR-132 expression and cytochromes P-450 (CYPs) 1A1 and 1B1 mRNA levels, which could contribute to the apoptotic response of PBMCs. Altogether, this study increases our understanding of the roles of miRNAs induced by B[a]P and provides the basis for further investigations into the mechanisms of gene expression regulation by PAHs.

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.

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.

Microalgal carotenoids and phytosterols regulate biochemical mechanisms involved in human health and disease prevention.

Microalgae are photosynthetic microorganisms that produce numerous bioactive molecules that can be used as food supplement to prevent chronic disease installation. Indeed, they produce phycobiliproteins, polysaccharides, lipids, carotenoids and sterolic compounds. The use of microalgae in human nutrition provide a mixture of these molecules with synergistic effect. The aim of this review is to present the specific roles played by the xanthophylls, and specifically astaxanthin and fucoxanthin, two high added value carotenoids, and by microalgal phytosterols such as ß-sitosterol, campesterol and stigmasterol on several cell mechanisms involved in the prevention of cardiometabolic diseases and cancers. This review explains how these microalgal molecules modulate cell signaling pathways involved in carbohydrate and lipid metabolisms, inflammation, apoptosis, invasion and metastasis. Xanthophylls and phytosterols are involved in the reduction of inflammatory markers in relation with the regulation of the c-Jun N-terminal kinases and nuclear factor-kappa B signaling pathways, and suppression of production of pro-inflammatory mediators. Xanthophylls act on glucose and lipid metabolisms via both the upregulation of peroxisome proliferator-activated receptors (PPARs) and glucose transporters and its effects on the expression of enzymes involved in fatty acid synthesis and cholesterol metabolism. Their anti-cancer effects are related to the induction of intrinsic apoptosis due to down-regulation of key regulatory kinases. The anti-angiogenesis, anti-proliferative and anti-invasive effects are correlated with decreased production of endothelial growth factors and of matrix metalloproteinases. Phytosterols have a major role on cholesterol absorption via modification of the activities of Niemann-Pick C1 like 1 and ATP-binding cassette transporters and on cholesterol esterification. Their action are also related with the modulation of PPARs and sterol regulatory element-binding protein-1 activities.

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.

Combustion Particle-Induced Changes in Calcium Homeostasis: A Contributing Factor to Vascular Disease?

Air pollution is the leading environmental risk factor for disease and premature death in the world. This is mainly due to exposure to urban air particle matter (PM), in particular, fine and ultrafine combustion-derived particles (CDP) from traffic-related air pollution. PM and CDP, including particles from diesel exhaust (DEP), and cigarette smoke have been linked to various cardiovascular diseases (CVDs) including atherosclerosis, but the underlying cellular mechanisms remain unclear. Moreover, CDP typically consist of carbon cores with a complex mixture of organic chemicals such as polycyclic aromatic hydrocarbons (PAHs) adhered. The relative contribution of the carbon core and adhered soluble components to cardiovascular effects of CDP is still a matter of discussion. In the present review, we summarize evidence showing that CDP affects intracellular calcium regulation, and argue that CDP-induced impairment of normal calcium control may be a critical cellular event through which CDP exposure contributes to development or exacerbation of cardiovascular disease. Furthermore, we highlight in vitro research suggesting that adhered organic chemicals such as PAHs may be key drivers of these responses. CDP, extractable organic material from CDP (CDP-EOM), and PAHs may increase intracellular calcium levels by interacting with calcium channels like transient receptor potential (TRP) channels, and receptors such as G protein-coupled receptors (GPCR; e.g., beta-adrenergic receptors [ßAR] and protease-activated receptor 2 [PAR-2]) and the aryl hydrocarbon receptor (AhR). Clarifying a possible role of calcium signaling and mechanisms involved may increase our understanding of how air pollution contributes to CVD.

Organic chemicals from diesel exhaust particles affects intracellular calcium, inflammation and ß-adrenoceptors in endothelial cells.

Exposure to diesel exhaust particles (DEP) may contribute to endothelial dysfunction and cardiovascular disease. DEP, extractable organic material from DEP (DEP-EOM) and certain PAHs seem to trigger [Ca2+]i increase as well as inflammation via GPCRs like ßARs and PAR-2. In the present study we explored the involvement of ßARs and PAR-2 in effects of DEP-EOM on [Ca2+]i and expression of inflammation-associated genes in the endothelial cell-line HMEC-1. We exposed the human microvascular endothelial cell line HMEC-1 to DEP-EOM fractionated by sequential extraction with solvents of increasing polarity: n-hexane (n-Hex-EOM), dichloromethane (DCM-EOM), methanol (Methanol-EOM) and water (Water-EOM). While Methanol-EOM and Water-EOM had no marked effects, n-Hex-EOM and DCM-EOM enhanced [Ca2+]i (2-3 times baseline) and expression of inflammation-associated genes (IL-1α, IL-1ß, COX-2 and CXCL8; 2-15 times baseline) in HMEC-1. The expression of ßARs (60-80% of baseline) and ßAR-inhibitor carazolol suppressed the increase in [Ca2+]i induced by both n-Hex- and DCM-EOM. Carazolol as well as the Ca2+-channel inhibitor SKF-96365 reduced the DCM-EOM-induced pro-inflammatory gene-expression. Overexpression of ßARs increased DCM-EOM-induced [Ca2+]i responses in HEK293 cells, while ßAR-overexpression suppressed [Ca2+]i responses from n-Hex-EOM. Furthermore, the PAR-2-inhibitor ENMD-1068 attenuated [Ca2+]i responses to DCM-EOM, but not n-Hex-EOM in HMEC-1. The results suggest that ßAR and PAR-2 are partially involved in effects of complex mixtures of chemicals extracted from DEP on calcium signalling and inflammation-associated genes in the HMEC-1 endothelial cell-line.

Genome-Wide Transcriptional and Functional Analysis of Human T Lymphocytes Treated with Benzo[α]pyrene.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed environmental contaminants, known to affect T lymphocytes. However, the molecular targets and pathways involved in their immunotoxic effects in human T lymphocytes remain unknown. Here, we analyzed the gene expression profile of primary human T lymphocytes treated with the prototypical PAH, benzo[α]pyrene (B[α]P), using a microarray-based transcriptome analysis. After a 48 h exposure to B[α]P, we identified 158 genes differentially expressed in T lymphocytes, including not only genes well-known to be affected by PAHs such as the cytochromes P450 (CYP) 1A1 and 1B1, but also others not previously shown to be targeted by B[α]P such as genes encoding the gap junction beta (GJB)-2 and 6 proteins. Functional enrichment analysis revealed that these candidates were significantly associated with the aryl hydrocarbon (AhR) and interferon (IFN) signaling pathways; a marked alteration in T lymphocyte recruitment was also observed. Using functional tests in transwell migration experiments, B[α]P was then shown to significantly decrease the chemokine (C-X-C motif) ligand 12-induced chemotaxis and transendothelial migration of T lymphocytes. In total, this study opens the way to unsuspected responsive pathway of interest, i.e., T lymphocyte migration, thus providing a more thorough understanding of the molecular basis of the immunotoxicity of PAHs.

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.

Lipophilic components of diesel exhaust particles induce pro-inflammatory responses in human endothelial cells through AhR dependent pathway(s).

BACKGROUND: Exposure to traffic-derived particulate matter (PM), such as diesel exhaust particles (DEP), is a leading environmental cause of cardiovascular disease (CVD), and may contribute to endothelial dysfunction and development of atherosclerosis. It is still debated how DEP and other inhaled PM can contribute to CVD. However, organic chemicals (OC) adhered to the particle surface, are considered central to many of the biological effects. In the present study, we have explored the ability of OC from DEP to reach the endothelium and trigger pro-inflammatory reactions, a central step on the path to atherosclerosis. RESULTS: Exposure-relevant concentrations of DEP (0.12 µg/cm2) applied on the epithelial side of an alveolar 3D tri-culture, rapidly induced pro-inflammatory and aryl hydrocarbon receptor (AhR)-regulated genes in the basolateral endothelial cells. These effects seem to be due to soluble lipophilic constituents rather than particle translocation. Extractable organic material of DEP (DEP-EOM) was next fractionated with increasing polarity, chemically characterized, and examined for direct effects on pro-inflammatory and AhR-regulated genes in human microvascular endothelial (HMEC-1) cells and primary human endothelial cells (PHEC) from four healthy donors. Exposure-relevant concentrations of lipophilic DEP-EOM (0.15 µg/cm2) induced low to moderate increases in IL-1α, IL-1ß, COX2 and MMP-1 gene expression, and the MMP-1 secretion was increased. By contrast, the more polar EOM had negligible effects, even at higher concentrations. Use of pharmacological inhibitors indicated that AhR and protease-activated receptor-2 (PAR-2) were central in regulation of EOM-induced gene expression. Some effects also seemed to be attributed to redox-responses, at least at the highest exposure concentrations tested. Although the most lipophilic EOM, that contained the majority of PAHs and aliphatics, had the clearest low-concentration effects, there was no straight-forward link between chemical composition and biological effects. CONCLUSION: Lipophilic and semi-lipophilic chemicals seemed to detach from DEP, translocate through alveolar epithelial cells and trigger pro-inflammatory reactions in endothelial cells at exposure-relevant concentrations. These effects appeared to be triggered by AhR agonists, and involve PAR-2 signaling.

Lipophilic Chemicals from Diesel Exhaust Particles Trigger Calcium Response in Human Endothelial Cells via Aryl Hydrocarbon Receptor Non-Genomic Signalling.

Exposure to diesel exhaust particles (DEPs) affects endothelial function and may contribute to the development of atherosclerosis and vasomotor dysfunction. As intracellular calcium concentration [Ca2+]i is considered important in myoendothelial signalling, we explored the effects of extractable organic matter from DEPs (DEP-EOM) on [Ca2+]i and membrane microstructure in endothelial cells. DEP-EOM of increasing polarity was obtained by pressurized sequential extraction of DEPs with n-hexane (n-Hex-EOM), dichloromethane (DCM-EOM), methanol, and water. Chemical analysis revealed that the majority of organic matter was extracted by the n-Hex- and DCM-EOM, with polycyclic aromatic hydrocarbons primarily occurring in n-Hex-EOM. The concentration of calcium was measured in human microvascular endothelial cells (HMEC-1) using micro-spectrofluorometry. The lipophilic n-Hex-EOM and DCM-EOM, but not the more polar methanol- and water-soluble extracts, induced rapid [Ca2+]i increases in HMEC-1. n-Hex-EOM triggered [Ca2+]i increase from intracellular stores, followed by extracellular calcium influx consistent with store operated calcium entry (SOCE). By contrast, the less lipophilic DCM-EOM triggered [Ca2+]i increase via extracellular influx alone, resembling receptor operated calcium entry (ROCE). Both extracts increased [Ca2+]i via aryl hydrocarbon receptor (AhR) non-genomic signalling, verified by pharmacological inhibition and RNA-interference. Moreover, DCM-EOM appeared to induce an AhR-dependent reduction in the global plasma membrane order, as visualized by confocal fluorescence microscopy. DCM-EOM-triggered [Ca2+]i increase and membrane alterations were attenuated by the membrane stabilizing lipid cholesterol. In conclusion, lipophilic constituents of DEPs extracted by n-hexane and DCM seem to induce rapid AhR-dependent [Ca2+]i increase in HMEC-1 endothelial cells, possibly involving both ROCE and SOCE-mediated mechanisms. The semi-lipophilic fraction extracted by DCM also caused an AhR-dependent reduction in global membrane order, which appeared to be connected to the [Ca2+]i increase.

Benzo(a)pyrene triggers desensitization of ß2-adrenergic pathway.

Exposure to environmental polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (B(a)P), has been linked to several health-threatening risks. PAHs were also shown to hinder adrenergic receptor (ADR) responses. As we previously demonstrated that B(a)P can directly interact with the ß2ADR, we investigated here whether B(a)P could decrease ß2ADR responsiveness by triggering receptor desensitization phenomena. We firstly showed that exposure to B(a)P reduced ß2ADR-mediated epinephrine-induced induction of NR4A gene mRNAs and of intracellular cAMP. Analysis of ß2ADR protein expression demonstrated that B(a)P rapidly decreased membrane expression of ß2ADR with a subsequent degradation of receptor protein. B(a)P exposure concomitantly rapidly increased the ß2ADR mRNA levels. The use of the ß-blockers, propranolol and ICI 118.551, demonstrated the involvement of ß2ADR itself in this increase. However, sustained exposure to B(a)P induced a diminution of ß2ADR mRNA steady-state as a result of the acceleration of its degradation. Together, these results show that, beside the well-known activation of the aryl hydrocarbon receptor, PAH deleterious effects may involve the dysfunction of adrenergic responses through, in part, the desensitization of ß2ADR. This may be taken in consideration when ß2-agonists/antagonists are administered in patients exposed to important concentrations of PAHs, e.g. in cigarette smokers.

Benzo[a]pyrene-induced DNA damage associated with mutagenesis in primary human activated T lymphocytes.

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are widely distributed environmental contaminants exerting toxic effects such as genotoxicity and carcinogenicity, mainly associated with aryl hydrocarbon receptor (AhR) activation and the subsequent induction of cytochromes P-450 (CYP) 1-metabolizing enzymes. We previously reported an up-regulation of AhR expression and activity in primary cultures of human T lymphocyte by a physiological activation. Despite the suggested link between exposure to PAHs and the risk of lymphoma, the potential of activated human T lymphocytes to metabolize AhR exogenous ligands such as B[a]P and produce DNA damage has not been investigated. In the present study, we characterized the genotoxic response of primary activated T lymphocytes to B[a]P. We demonstrated that, following T lymphocyte activation, B[a]P treatment triggers a marked increase in CYP1 expression and activity generating, upon metabolic activation, DNA adducts and double-strand breaks (DSBs) after a 48-h treatment. At this time point, B[a]P also induces a DNA damage response with ataxia telangiectasia mutated kinase activation, thus producing a p53-dependent response and T lymphocyte survival. B[a]P activates DSB repair by mobilizing homologous recombination machinery but also induces gene mutations in activated human T lymphocytes which could consequently drive a cancer process. In conclusion, primary cultures of activated human T lymphocytes represent a good model for studying genotoxic effects of environmental contaminants such as PAHs, and predicting human health issues.

Role for the ATPase inhibitory factor 1 in the environmental carcinogen-induced Warburg phenotype.

Most tumors undergo metabolic reprogramming towards glycolysis, the so-called Warburg effect, to support growth and survival. Overexpression of IF1, the physiological inhibitor of the F0F1ATPase, has been related to this phenomenon and appears to be a relevant marker in cancer. Environmental contributions to cancer development are now widely accepted but little is known about the underlying intracellular mechanisms. Among the environmental pollutants humans are commonly exposed to, benzo[a]pyrene (B[a]P), the prototype molecule of polycyclic aromatic hydrocarbons (PAHs), is a well-known human carcinogen. Besides apoptotic signals, B[a]P can also induce survival signals in liver cells, both likely involved in cancer promotion. Our previous works showed that B[a]P elicited a Warburg-like effect, thus favoring cell survival. The present study aimed at further elucidating the molecular mechanisms involved in the B[a]P-induced metabolic reprogramming, by testing the possible involvement of IF1. We presently demonstrate, both in vitro and in vivo, that PAHs, especially B[a]P, strongly increase IF1 expression. Such an increase, which might rely on ß2-adrenergic receptor activation, notably participates to the B[a]P-induced glycolytic shift and cell survival in liver cells. By identifying IF1 as a target of PAHs, this study provides new insights about how environmental factors may contribute to related carcinogenesis.

Calcium signaling and ß2-adrenergic receptors regulate 1-nitropyrene induced CXCL8 responses in BEAS-2B cells.

Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are widespread environmental pollutants, generated from reactions between PAHs and nitrogen oxides during combustion processes. In the present study we have investigated the mechanisms of CXCL8 (IL-8) responses induced by 1-nitropyrene (1-NP) in human bronchial epithelial BEAS-2B cells, with focus on the possible importance of Ca(2+)-signaling and activation of ß2-adrenergic receptors (ß2AR). Ca(2+)-chelator treatment obliterated 1-NP-induced CXCL8 (IL-8) responses. 1-NP at 10µM (but not 1µM) induced a rapid and sustained increase in intracellular Ca(2+)-levels ([Ca(2+)]i). The early but not the later, sustained phase of 1-NP-induced [Ca(2+)]i was suppressed by beta-blocker treatment (carazolol). Moreover, inhibition of ß2AR by blocking-antibody, beta-blocker treatment (ICI 118551) or siRNA transfection attenuated CXCL8 responses induced by 1-NP. The results confirm that PAHs may induce Ca(2+)-signaling also in BEAS-2B cells, at least partly through activation of ß2AR, and suggest that both ß2AR- and Ca(2+)-signaling may be involved in 1-NP-induced CXCL8 responses in bronchial epithelial cells.

Induction of intracellular calcium concentration by environmental benzo(a)pyrene involves a ß2-adrenergic receptor/adenylyl cyclase/Epac-1/inositol 1,4,5-trisphosphate pathway in endothelial cells.

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (B(a)P) are widely distributed environmental contaminants, known as potent ligands of the aryl hydrocarbon receptor (AhR). These chemicals trigger an early and transient increase of intracellular calcium concentration ([Ca(2+)](i)), required for AhR-related effects of PAHs. The mechanisms involved in this calcium mobilization were investigated in the present study. We demonstrated that B(a)P-mediated [Ca(2+)](i) induction was prevented in endothelial HMEC-1 cells by counteracting ß2-adrenoreceptor (ß2ADR) activity using pharmacological antagonists, anti-ß2ADR antibodies, or siRNA-mediated knockdown of ß2ADR expression; by contrast, it was strongly potentiated by ß2ADR overexpression in human kidney HEK293 cells. B(a)P was shown, moreover, to directly bind to ß2ADR, as assessed by in vitro binding assays and molecular modeling. Pharmacological inhibition and/or siRNA-mediated silencing of various signaling actors acting downstream of ß2ADR in a sequential manner, such as G protein, adenylyl cyclase, Epac-1 protein, and inositol 1,4,5-trisphosphate (IP(3))/IP(3) receptor, were next demonstrated to prevent B(a)P-induced calcium signal. Inhibition or knockdown of these signaling elements, as well as the use of chemical ß-blockers, were finally shown to counteract B(a)P-mediated induction of cytochrome P-450 1B1, a prototypical AhR target gene. Taken together, our results show that B(a)P binds directly to ß2ADR and consequently utilizes ß2ADR machinery to mobilize [Ca(2+)](i), through activation of a G protein/adenylyl cyclase/cAMP/Epac-1/IP(3) pathway. This ß2ADR-dependent signaling pathway activated by PAHs may likely be crucial for PAH-mediated up-regulation of AhR target genes, thus suggesting a contribution of ß2ADR to the health-threatening effects of these environmental pollutants.

Aryl hydrocarbon receptor-independent up-regulation of intracellular calcium concentration by environmental polycyclic aromatic hydrocarbons in human endothelial HMEC-1 cells.

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (B(a)P) constitute a major family of widely-distributed environmental toxic contaminants, known as potent ligands of the aryl hydrocarbon receptor (AhR). B(a)P has been recently shown to trigger an early and transient increase of intracellular calcium concentration ([Ca(2+)](i)), involved in AhR-related up-regulation of target genes by B(a)P. This study was designed to determine whether AhR may play a role in [Ca(2+)](i) induction provoked by B(a)P. We demonstrated that, in addition to B(a)P, various PAHs, including pyrene and benzo(e)pyrene, known to not or only very poorly interact with AhR, similarly up-regulated [Ca(2+)](i) in human endothelial HMEC-1 cells. Moreover, α-naphthoflavone, a flavonoïd antagonist of AhR, was also able to induce [Ca(2+)](i). Knocking-down AhR expression in HMEC-1 cells through transfection of siRNAs, was finally demonstrated to not prevent B(a)P-mediated induction of [Ca(2+)](i), whereas it efficiently counteracted B(a)P-mediated induction of the referent AhR target gene cytochrome P-450 1B1. Taken together, these data demonstrate that environmental PAHs trigger [Ca(2+)](i) induction in an AhR-independent manner.

TNFalpha- and NF-kappaB-dependent induction of the chemokine CCL1 in human macrophages exposed to the atherogenic lipoprotein(a).

AIMS: CCL1 is a chemokine thought to contribute to cardiovascular diseases and recently reported to be regulated by the pro-atherogenic lipoprotein(a) (Lp(a)) and the ligand-activated aryl hydrocarbon receptor (AhR). The present study was designed to investigate molecular regulatory pathways involved in Lp(a)-mediated induction of CCL1. MAIN METHODS: CCL1 regulation was studied in Lp(a)-exposed human primary macrophages using mainly quantitative reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay and electrophoretic mobility shift assay (EMSA). KEY FINDINGS: Using the AhR antagonist alpha-napthtoflavone, the translational inhibitor cycloheximide and anti-tumor necrosis factor alpha (TNFalpha) neutralizing antibodies, we demonstrated that Lp(a)-mediated mRNA induction of CCL1 occurs in an AhR-independent manner and requires de novo protein synthesis of TNFalpha. Involvement of this cytokine was further underlined by the fact that it increased expression and secretion of CCL1 by itself in macrophages. DNA binding activity of NF-kappaB, a well-known molecular effector of TNFalpha, was moreover activated by Lp(a) in a TNFalpha-dependent manner and the use of the NF-kappaB inhibitor Bay 11-7082 blocked Lp(a)-triggered CCL1 induction. In addition, Lp(a) induced binding of NF-kappaB to a NF-kappaB consensus element on CCL1 promoter as assessed by EMSA. Co-exposure to Lp(a) and the AhR ligand benzo(a)pyrene was finally shown to superinduce CCL1 expression in human macrophages, supporting the conclusion that Lp(a) and AhR ligands act on CCL1 through independent ways. SIGNIFICANCE: These data suggest that Lp(a)-triggered induction of CCL1 expression is mediated by TNFalpha and subsequent activation of NF-kappaB, without AhR involvement.