Akkermansia muciniphila and Alcohol-Related Liver Diseases. A Systematic Review.

SCOPE: Akkermansia muciniphila (A. muciniphila) are Gram negative commensal bacteria, degrading mucin in the intestinal mucosa, modulating intestinal permeability and inflammation in the digestive tract, liver, and blood. Some components can promote the relative abundance of A. muciniphila in the gut microbiota, but lower levels of A. muciniphila are more commonly found in people with obesity, diabetes, metabolic syndromes, or inflammatory digestive diseases. Over-intake of ethanol can also induce a decrease of A. muciniphila, associated with dysregulation of microbial metabolite production, impaired intestinal permeability, induction of chronic inflammation, and production of cytokines. METHODS AND RESULTS: Using a PRISMA search strategy, a review is performed on the bacteriological characteristics of A. muciniphila, the factors capable of modulating its relative abundance in the digestive tract and its probiotic use in alcohol-related liver diseases (alcoholic hepatitis, cirrhosis, hepatocellular carcinoma, hepatic transplantation, partial hepatectomy). CONCLUSION: Several studies have shown that supplementation with A. muciniphila can improve ethanol-related hepatic pathologies, and highlight the interest in using this bacterial species as a probiotic.

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.

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.

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.

Differential influences of the BPA, BPS and BPF on in vitro IL-17 secretion by mouse and human T cells.

The endocrine disruptor and food contaminant bisphenol A (BPA) is frequently present in consumer plastics and can produce several adverse health effects participating in the development of inflammatory and autoimmune diseases. Regulatory restrictions have been established to prevent risks for human health, leading to the substitution of BPA by structural analogues, such as bisphenol S (BPS) and F (BPF). In this study, we aimed at comparing the in vitro impact of these bisphenols from 0.05 to 50,000 nM on Th17 differentiation, frequency and function in mouse systemic and intestinal immune T cells and in human blood T cells. This study reports the ability of these bisphenols, at low and environmentally relevant concentration, i.e, 0.05 nM, to increase significantly IL-17 production in mouse T cells but not in human T lymphocytes. The use of an aryl hydrocarbon receptor (AhR) specific inhibitor demonstrated its involvement in this bisphenol-induced IL-17 production. We also observed an increased IL-17 secretion by BPS and BPF, and not by BPA, in mouse naive T cells undergoing in vitro Th17 differentiation. In total, this study emphasizes the link between bisphenol exposures and the susceptibility to develop immune diseases, questioning thus the rational of their use to replace BPA.

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.

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.

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.

Nrf2 expression and activity in human T lymphocytes: stimulation by T cell receptor activation and priming by inorganic arsenic and tert-butylhydroquinone.

The transcription factor nuclear factor-erythroid 2-related-2 (Nrf2) controls cellular redox homeostasis and displays immunomodulatory properties. Nrf2 alters cytokine expression in murine T cells, but its effects in human T lymphocytes are unknown. This study investigated the expression and activity of Nrf2 in human activated CD4(+) T helper lymphocytes (Th cells) that mediate the adaptive immune response. Th cells were isolated from peripheral blood mononuclear cells and activated with antibodies against CD3 and CD28, mimicking physiologic Th cell stimulation by dendritic cells. Nrf2 is hardly detectable in unstimulated Th cells. Activation of Th cells rapidly and strongly increases the levels of Nrf2 protein by increasing NRF2 gene transcription. Th cell activation also enhances mRNA and protein levels of Nrf2 target genes encoding antioxidant enzymes. Blocking Nrf2 expression using chemical inhibitors or siRNAs prevents these gene inductions. Pretreatment with inorganic arsenic, a Nrf2 inducer that does not alter NRF2 gene expression, increases protein level and transcriptional activity of Nrf2 induced by Th cell stimulation. Inorganic arsenic enhances nuclear translocation of Nrf2, its interaction with the coactivator protein p300, and its DNA binding activity. Inhibition of Nrf2 expression abrogates the effects of inorganic arsenic on mRNA levels of antioxidant genes, but does not alter the expression of IL-2, TNF-α, interferon-γ, or IL-17 in Th cells activated in the absence or presence of the metalloid. In conclusion, this study demonstrates for the first time that stimulation of human Th cells increases transcription of the NRF2 gene and activity of the Nrf2 protein. However, modulation of Nrf2 levels does not modify the secretion of inflammatory cytokines from these T lymphocytes.

The aryl hydrocarbon receptor is functionally upregulated early in the course of human T-cell activation.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates immunosuppression caused by a variety of environmental contaminants, such as polycyclic aromatic hydrocarbons or dioxins. Recent evidence suggests that AhR plays an important role in T-cell-mediated immune responses by affecting the polarization and differentiation of activated T cells. However, the regulation of AhR expression in activated T cells remains poorly characterized. In the present study, we used purified human T cells stimulated with anti-CD3 and anti-CD28 Abs to investigate the effect of T-cell activation on AhR mRNA and protein expression. The expression of AhR mRNA increased significantly and rapidly after T-cell activation, identifying AhR as an immediate-early activation gene. AhR upregulation occurred in all of the T-cell subtypes, and is associated with its nuclear translocation and induction of the cytochromes P-450 1A1 and 1B1 mRNA expression in the absence of exogenous signals. In addition, the use of an AhR antagonist or siRNA-mediated AhR knockdown significantly inhibited IL-22 expression, suggesting that expression and functional activation of AhR is necessary for the secretion of IL-22 by activated T cells. In conclusion, our data support the idea that AhR is a major player in T-cell physiology.

Inorganic arsenic represses interleukin-17A expression in human activated Th17 lymphocytes.

Trivalent inorganic arsenic [As(III)] is an efficient anticancer agent used to treat patients suffering from acute promyelocytic leukemia. Recently, experimental studies have clearly demonstrated that this metalloid can also cure lymphoproliferative and/or pro-inflammatory syndromes in different murine models of chronic immune-mediated diseases. T helper (Th) 1 and Th17 lymphocytes play a central role in development of these diseases, in mice and humans, especially by secreting the potent pro-inflammatory cytokine interferon-γ and IL-17A, respectively. As(III) impairs basic functions of human T cells but its ability to modulate secretion of pro-inflammatory cytokines by differentiated Th lymphocytes is unknown. In the present study, we demonstrate that As(III), used at concentrations clinically achievable in plasma of patients, has no effect on the secretion of interferon-γ from Th1 cells but almost totally blocks the expression and the release of IL-17A from human Th17 lymphocytes co-stimulated for five days with anti-CD3 and anti-CD28 antibodies, in the presence of differentiating cytokines. In addition, As(III) specifically reduces mRNA levels of the retinoic-related orphan receptor (ROR)C gene which encodes RORγt, a key transcription factor controlling optimal IL-17 expression in fully differentiated Th17 cells. The metalloid also blocks initial expression of IL-17 gene induced by the co-stimulation, probably in part by impairing activation of the JNK/c-Jun pathway. In conclusion, our results demonstrate that As(III) represses expression of the major pro-inflammatory cytokine IL-17A produced by human Th17 lymphocytes, thus strengthening the idea that As(III) may be useful to treat inflammatory immune-mediated diseases in humans.

Aryl hydrocarbon receptor-dependent induction of the IgA receptor FcαRI by the environmental contaminant benzo(a)pyrene in human macrophages.

Polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BaP), are widely distributed toxic environmental contaminants well known to regulate gene expression through activation of the aryl hydrocarbon receptor (AhR). In the present study, we demonstrated that the IgA receptor FcαRI/CD89 constitutes a molecular target for PAHs. Indeed, in vitro exposure to BaP markedly increased mRNA and protein expression of FcαRI in primary human macrophages; intratracheal instillation of BaP to rats also enhanced mRNA expression of FcαRI in alveolar macrophages. BaP concomitantly increased activity of the previously uncharacterized -1734 to -42 fragment of the FcaRI promoter that we subcloned in a luciferase reporter vector. Three-methylcholanthrene, a PAH known to activate AhR like BaP, induced FcαRI expression, in contrast to benzo(e)pyrene, a PAH known to poorly interact with AhR. Moreover, FcαRI induction in BaP-exposed human macrophages was fully prevented by down-regulating AhR expression through small interference RNA transfection. In addition, BaP increased nuclear protein binding to a consensus AhR-related xenobiotic-responsive element found in the FcαRI gene promoter, as revealed by electrophoretic mobility shift assay. Overall, these data highlight an AhR-dependent up-regulation of FcαRI in response to BaP, which may contribute to the deleterious effects of environmental PAHs toward the immune/inflammatory response and which also likely emphasizes the role played by AhR in the regulation of genes involved in immunity and inflammation.

Predicting in vivo gene expression in macrophages after exposure to benzo(a)pyrene based on in vitro assays and toxicokinetic/toxicodynamic models.

Predictive toxicology aims at developing methodologies to relate the results obtained from in vitro experiments to in vivo exposure. In the case of polycyclic aromatic hydrocarbons (PAHs), a substantial amount of knowledge on effects and modes of action has been recently obtained from in vitro studies of gene expression. In the current study, we built a physiologically based toxicokinetic (PBTK) model to relate in vivo and in vitro gene expression in case of exposure to benzo(a)pyrene (BaP), a referent PAH. This model was calibrated with two toxicokinetic datasets obtained on rats exposed either through intratracheal instillation or through intravenous administration and on an in vitro degradation study. A good agreement was obtained between the model's predictions and the concentrations measured in target organs, such as liver and lungs. Our model was able to relate correctly the gene expression for two genes targeted by PAHs, measured in vitro on primary human macrophages and in vivo in rat macrophages after exposure to BaP. Combining in vitro studies and PBTK modeling is promising for PAH risk assessment, especially for mixtures which are more efficiently studied in vitro than in vivo.

Transcriptional signature of human macrophages exposed to the environmental contaminant benzo(a)pyrene.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed immunotoxic and carcinogenic environmental contaminants, known to affect macrophages. In order to identify their molecular targets in such cells, we have analyzed gene expression profile of primary human macrophages treated by the prototypical PAH benzo(a)pyrene (BaP), using pangenomic oligonucleotides microarrays. Exposure of macrophages to BaP for 8 and 24 h resulted in 96 and 1100 genes, differentially expressed by at least a twofold change factor, respectively. Some of these targets, including the chemokine receptor CXCR5, the G protein-coupled receptor 35 (GPR35), and the Ras regulator RASAL1, have not been previously shown to be affected by PAHs, in contrast to others, such as interleukin-1beta and the aryl hydrocarbon receptor (AhR) repressor. These BaP-mediated gene regulations were fully validated by reverse transcription-quantitative polymerase chain reaction assays for some selected genes. Their bioinformatic analysis indicated that biological functions linked to immunity, inflammation, and cell death were among the most affected by BaP in human macrophages and that the AhR and p53 signaling pathways were the most significant canonical pathways activated by the PAH. AhR and p53 implications were moreover fully confirmed by the prevention of BaP-related upregulation of some selected target genes by AhR silencing or the use of pifithrin-alpha, an inhibitor of PAH bioactivation-related DNA damage/p53 pathways. Overall, these data, through identifying genes and signaling pathways targeted by PAHs in human macrophages, may contribute to better understand the molecular basis of the immunotoxicity of these environmental contaminants.

Aryl hydrocarbon receptor-dependent induction of the NADPH oxidase subunit NCF1/p47 phox expression leading to priming of human macrophage oxidative burst.

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene (BaP) are toxic environmental contaminants known to regulate gene expression through activation of the aryl hydrocarbon receptor (AhR). In the present study, we demonstrated that acute treatment by BaP markedly increased expression of the NADPH oxidase subunit gene neutrophil cytosolic factor 1 (NCF1)/p47(phox) in primary human macrophages; NCF1 was similarly up-regulated in alveolar macrophages from BaP-instilled rats. NCF1 induction in BaP-treated human macrophages was prevented by targeting AhR, through its chemical inhibition or small interference RNA-mediated down-modulation of its expression. BaP moreover induced activity of the NCF1 promoter sequence, containing a consensus AhR-related xenobiotic-responsive element (XRE), and electrophoretic mobility shift assays and chromatin immunoprecipitation experiments indicated that BaP-triggered binding of AhR to this XRE. Finally, we showed that BaP exposure resulted in p47(phox) protein translocation to the plasma membrane and in potentiation of phorbol myristate acetate (PMA)-induced superoxide anion production in macrophages. This BaP priming effect toward NADPH oxidase activity was inhibited by the NADPH oxidase specific inhibitor apocynin and the chemical AhR inhibitor alpha-naphtoflavone. These results indicated that BaP induced NCF1/p47(phox) expression and subsequently enhanced superoxide anion production in PMA-treated human macrophages, in an AhR-dependent manner; such an NCF1/NADPH oxidase regulation by polycyclic aromatic hydrocarbons may participate in deleterious effects toward human health triggered by these environmental contaminants, including atherosclerosis and smoking-related diseases.

Global effects of inorganic arsenic on gene expression profile in human macrophages.

Inorganic arsenic, a major environmental contaminant, exerts immunosuppressive effects towards human cells. We previously demonstrated that relevant environmental concentrations of inorganic arsenic altered morphology and functions of human primary macrophages, suggesting interference with macrophage differentiation program. The goal of this study was to determine global effect of low concentrations of arsenic trioxide (As(2)O(3)) on gene expression profile in human primary macrophages, in order to identify molecular targets of inorganic arsenic, especially those relevant of macrophage differentiation process. Using a pan-genomic microarray, we demonstrate that exposure of human blood monocyte-derived macrophages to 1microM As(2)O(3) for 72h, a non-cytototoxic concentration, results in up-regulation of 32 genes and repression of 91 genes. Among these genes, 26 are specifically related to differentiation program of human macrophages. Particularly, we validated that As(2)O(3) strongly alters expression of MMP9, MMP12, CCL22, SPON2 and CXCL2 genes, which contribute to major macrophagic functions. Most of these metalloid effects were reversed when As(2)O(3)-treated macrophages were next cultured in arsenic-free medium. We also show that As(2)O(3) similarly regulates expression of this macrophagic gene subset in human alveolar macrophages, the phenotype of which closely resembles that of blood monocyte-derived macrophage. In conclusion, our study demonstrates that environmentally relevant concentrations of As(2)O(3) impair expression of macrophage-specific genes, which fully supports interference of metalloid with differentiation program of human macrophages.

Interleukin-8 induction by the environmental contaminant benzo(a)pyrene is aryl hydrocarbon receptor-dependent and leads to lung inflammation.

Benzo(a)pyrene (BP) is an environmental contaminant known to favor airway inflammation likely through up-regulation of pro-inflammatory cytokines. The present study was designed to characterize its effects toward interleukin-8 (IL-8), a well-established pulmonary inflammatory cytokine. In primary human macrophages, BP was shown to induce IL-8 expression at both mRNA and secretion levels in a dose-dependent manner. Such an up-regulation was likely linked to aryl hydrocarbon receptor (AhR)-activation since BP-mediated IL-8 induction was reduced after AhR expression knock-down through RNA interference. Moreover, electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation experiments showed BP-triggered binding of AhR to a consensus xenobiotic responsive element (XRE) found in the human IL-8 promoter. Finally, BP administration to mice led to over-expression of keratinocyte chemoattractant (KC), the murine functional homologue of IL-8, in lung. It also triggered the recruitment of neutrophils in bronchoalveolar lavage (BAL) fluids, which was however fully abolished in the presence of a chemical antagonist of the KC/IL-8 receptors CXCR1/CXCR2, thus supporting the functional and crucial involvement of KC in BP-induced lung inflammation. Overall, these data highlight an AhR-dependent regulation of IL-8 in response to BP that likely contributes to the airway inflammatory effects of this environmental chemical.

Potent inhibition of carcinogen-bioactivating cytochrome P450 1B1 by the p53 inhibitor pifithrin alpha.

Pifithrin alpha (PFTalpha) is a chemical compound that inhibits p53-mediated gene activation and apoptosis. It has also been recently shown to alter metabolism of carcinogenic polycyclic aromatic hydrocarbons (PAHs). This has led us to examine the effect of PFTalpha on the activity of cytochrome P-450 (CYP) 1 isoforms, known to metabolize PAHs, such as benzo(a)pyrene (BP), into mutagenic metabolites. We report that PFTalpha caused a potent inhibition of CYP1-related activity as measured by ethoxyresorufin O-deethylase activity in CYP1-containing MCF-7 cells and liver microsomes. It also directly affected the catalytic activity of human recombinant CYP1A1, CYP1A2 and CYP1B1 isoforms, with a potent inhibitory effect towards CYP1B1. The nature of this CYP1B1 inhibition by PFTalpha was mixed-type with an apparent K(i) of 4.38 nM. Blockage of CYP1 activity by PFTalpha was associated with a decreased metabolism of BP, a reduced formation of BP-derived adducts and a diminished BP-induced apoptosis in human cultured cells targets for PAHs like primary human macrophages and p53-negative KG1a leukaemia cells. These data further substantiate an unexpected and p53-independent action of PFTalpha for preventing toxicity of chemical carcinogens such as PAHs, through inhibition of CYP1 enzyme activities, especially that of CYP1B1.