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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. [ABSTRACT FROM AUTHOR]

Succinate dehydrogenase (SDH) inhibitors (SDHIs) are used worldwide to limit the proliferation of molds on plants and plant products. However, as SDH, also known as respiratory chain (RC) complex II, is a universal component of mitochondria from living organisms, highly conserved through evolution, the specificity of these inhibitors toward fungi warrants investigation. We first establish that the human, honeybee, earthworm and fungal SDHs are all sensitive to the eight SDHIs tested, albeit with varying IC50 values, generally in the micromolar range. In addition to SDH, we observed that five of the SDHIs, mostly from the latest generation, inhibit the activity of RC complex III. Finally, we show that the provision of glucose ad libitum in the cell culture medium, while simultaneously providing sufficient ATP and reducing power for antioxidant enzymes through glycolysis, allows the growth of RC-deficient cells, fully masking the deleterious effect of SDHIs. As a result, when glutamine is the major carbon source, the presence of SDHIs leads to time-dependent cell death. This process is significantly accelerated in fibroblasts derived from patients with neurological or neurodegenerative diseases due to RC impairment (encephalopathy originating from a partial SDH defect) and/or hypersensitivity to oxidative insults (Friedreich ataxia, familial Alzheimer's disease). [ABSTRACT FROM AUTHOR]

Even if the comet assay has been widely used for decades, there is still a need for controlled studies and good mathematical models to assess the variability of the different versions of this assay and in particular to assess potential intra‐experimental variability of the high‐throughput comet assay. To address this point, we further validate a high‐throughput comet assay that uses hydrophilic polyester film (Gelbond®). Experiments were performed using human peripheral blood mononuclear cells (PBMC) either untreated or treated with different concentration of MMS (methyl methanesulfonate). A positive control for the Fpg (Formamidopyrimidine DNA glycosylase)‐modified comet assay (Ro 19‐8022 with light) was also included. To quantify the sources of variability of the assay, including intradeposit variability, instead of summarizing DNA damage on 50 cells from a deposit by the mean or median of their percentage DNA tail, we analyzed all logit‐transformed data with a linear mixed model. The main source of variation in our experimental data is between cells within the same deposit, suggesting genuine variability in the response of the cells rather than variation caused by technical treatment of cell samples. The second source of variation is the inter‐experimental variation (day‐to‐day experiment); the coefficient of this variation for the control was 13.6%. The variation between deposits in the same experiment is negligible. Moreover, there is no systematic bias because of the position of samples on the Gelbond® film nor the position of the films in the electrophoresis tank. This high‐throughput comet assay is thus reliable for various applications. Environ. Mol. Mutagen. 59:595–602, 2018. © 2018 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Epidemiological studies have associated red meat intake with risk of colorectal cancer. Experimental studies explain this positive association by the oxidative properties of heme iron released in the colon. This latter is a potent catalyst for lipid peroxidation, resulting in the neoformation of deleterious aldehydes in the fecal water of heme-fed rats. The toxicity of fecal water of heme-fed rats was associated to such lipid peroxidation. This study demonstrated that fecal water of hemoglobinand beef-fed rats preferentially induced apoptosis in mouse normal colon epithelial cells than in those carrying mutation on Apc (Adenomatous polyposis coli) gene, considered as preneoplastic. Highlighting the importance of lipid peroxidation and neoformation of secondary aldehydes like 4-hydroxy-2-nonenal (HNE), we optimized the depletion of carbonyl compounds in the fecal water which turned out to abolish the differential apoptosis in both cell lines. To explain the resistance of preneoplastic cells towards fecal water toxicity, we focused on Nrf2, known to be activated by aldehydes, including HNE. Fecal water activated Nrf2 in both cell lines, associated with the induction of Nrf2-target genes related to aldehydes detoxification. However, the antioxidant defense appeared to be higher in preneoplastic cells, favoring their survival, as evidenced by Nrf2 inactivation. Taken together, our results suggest that Nrf2-dependent antioxidant response was involved in the resistance of preneoplastic cells upon exposure to fecal water of hemoglobin- and beef-fed rats. This difference could explain the promoting effect of red meat and heme-enriched diet on colorectal cancer, by initiating positive selection of preneoplastic cells. [ABSTRACT FROM AUTHOR]

The composition of the human microbiota influences tumorigenesis, notably in colorectal cancer (CRC). Pathogenic Escherichia coli possesses a variety of virulent factors, among them the Cytolethal Distending Toxin (CDT). CDT displays dual DNase and phosphatase activities and induces DNA double strand breaks, cell cycle arrest and apoptosis in a broad range of mammalian cells. As CDT could promote malignant transformation, we investigated the cellular outcomes induced by acute and chronic exposures to E. coli CDT in normal human colon epithelial cells (HCECs). Moreover, we conducted a comparative study between isogenic derivatives cell lines of the normal HCECs in order to mimic the mutation of three major genes found in CRC genetic models: APC, KRAS, and TP53. Our results demonstrate that APC and p53 deficient cells showed impaired DNA damage response after CDT exposure, whereas HCECs expressing oncogenic KRASV12 were more resistant to CDT. Compared to normal HCECs, the precancerous derivatives exhibit hallmarks of malignant transformation after a chronic exposure to CDT. HCECs defective in APC and p53 showed enhanced anchorage independent growth and genetic instability, assessed by the micronucleus formation assay. In contrast, the ability to grow independently of anchorage was not impacted by CDT chronic exposure in KRASV12 HCECs, but micronucleus formation is dramatically increased. Thus, CDT does not initiate CRC by itself, but may have promoting effects in premalignant HCECs, involving different mechanisms in function of the genetic alterations associated to CRC. [ABSTRACT FROM AUTHOR]

Key points Acid extrusion from ventricular myocytes typically occurs via Na+/H+ exchange (NHE1) and Na+-HCO3− co-transporters (NBC). This maintains intracellular pH at ∼7.2: The membrane distribution of these transporters is uncertain., Immunofluorescence indicates that: NBC isoforms are located in lateral sarcolemma, intercalated discs and transverse tubules, whereas NHE1 is densely expressed at intercalated discs., Functional experiments with detubulated myocytes indicate reduced acid extrusion on NBC but no effect on NHE1 activity, confirming exclusion of NHE1 function from transverse tubules., Stimulating NHE1 activity induces sub-sarcolemmal [H+]i depletion (forming local pHi microdomains), particularly at intercalated discs, while stimulating NBC activity induces no pHi microdomains., Our results provide the first demonstration that pHi in ventricular myocytes is locally controlled through selective trafficking of membrane ion transporters. NHE1 preferentially controls pHi at intercalated discs, where cell-to-cell gap-junctional channels are located, while NBC influences pHi adjacent to transverse tubules, where key proteins for excitation-contraction coupling are located., Abstract Membrane acid extrusion by Na+/H+ exchange (NHE1) and Na+-HCO3− co-transport (NBC) is essential for maintaining a low cytoplasmic [H+] (∼60 n m, equivalent to an intracellular pH (pHi) of 7.2). This protects myocardial function from the high chemical reactivity of H+ ions, universal end-products of metabolism. We show here that, in rat ventricular myocytes, fluorescent antibodies map the NBC isoforms NBCe1 and NBCn1 to lateral sarcolemma, intercalated discs and transverse tubules (t-tubules), while NHE1 is absent from t-tubules. This unexpected difference matches functional measurements of pHi regulation (using AM-loaded SNARF-1, a pH fluorophore). Thus, myocyte detubulation (by transient exposure to 1.5 m formamide) reduces global acid extrusion on NBC by 40%, without affecting NHE1. Similarly, confocal pHi imaging reveals that NBC stimulation induces spatially uniform pHi recovery from acidosis, whereas NHE1 stimulation induces pHi non-uniformity during recovery (of ∼0.1 units, for 2-3 min), particularly at the ends of the cell where intercalated discs are commonly located, and where NHE1 immunostaining is prominent. Mathematical modelling shows that this induction of local pHi microdomains is favoured by low cytoplasmic H+ mobility and long H+ diffusion distances, particularly to surface NHE1 transporters mediating high membrane flux. Our results provide the first evidence for a spatial localisation of [H+]i regulation in ventricular myocytes, suggesting that, by guarding pHi, NHE1 preferentially protects gap junctional communication at intercalated discs, while NBC locally protects t-tubular excitation-contraction coupling. [ABSTRACT FROM AUTHOR]

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]Pinduced 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. [ABSTRACT FROM AUTHOR]

The Na+/H+ exchanger 1, which plays an essential role in intracellular pH regulation in most tissues, is also known to be a key actor in both proliferative and apoptotic processes. Its activation by H+ is best described by the Monod–Wyman–Changeux model: the dimeric NHE-1 oscillates between a low and a high affinity conformation, the balance between the two forms being defined by the allosteric constant L0. In this study, influence of cholesterol- and caveolin-rich microdomains on NHE-1 activity was examined by using cholesterol depleting agents, including methyl-β-cyclodextrin (MBCD). These agents activated NHE-1 by modulating its L0 parameter, which was reverted by cholesterol repletion. This activation was associated with NHE-1 relocation outside microdomains, and was distinct from NHE-1 mitogenic and hormonal stimulation; indeed MBCD and serum treatments were additive, and serum alone did not change NHE-1 localization. Besides, MBCD activated a serum-insensitive, constitutively active mutated NHE-1 (625KDKEEEIRK635 into KNKQQQIRK). Finally, the membrane-dependent NHE-1 regulation occurred independently of Mitogen Activated Protein Kinases, especially Extracellular Regulated Kinase activation, although this kinase was activated by MBCD. In conclusion, localization of NHE-1 in membrane cholesterol- and caveolin-rich microdomains constitutes a novel physiological negative regulator of NHE-1 activity. J. Cell. Physiol. 216: 207–220, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[ a]yrene (B[ a]P) constitute a widely distributed class of environmental pollutants, responsible for highly toxic effects. Elucidating the intracellular mechanisms of this cytotoxicity thus remains a major challenge. Besides the activation of the p53 apoptotic pathway, we have previously found in F258 hepatic cells that the B[ a]P (50 nM)-induced apoptosis was also dependent upon the transmembrane transporter NHE1, whose activation might result from membrane alterations in our model. We here demonstrate that: (1) B[ a]P induces a membrane fluidization surprisingly linked to NHE1 activation; (2) membrane stabilization by exogenous cholesterol protects cells from B[ a]P-induced apoptosis, via an effect on late acidification and iron uptake. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene (B[a]P), are ubiquitous genotoxic environmental pollutants. Their DNA-damaging effects lead to apoptosis induction, through similar pathways to those identified after exposure to other DNA-damaging stimuli with activation of p53-related genes and the involvement of the intrinsic apoptotic pathway. However, at a low concentration of B[a]P (50 nM), our previous results pointed to the involvement of intracellular pH (pHi) variations during B[a]P-induced apoptosis in a rat liver epithelial cell line (F258). In the present work, we identified the mitochondrial F0F1-ATPase activity reversal as possibly responsible for pHi decrease. This acidification not only promoted executive caspase activation, but also activated leucocyte elastase inhibitor/leucocyte-derived DNase II (LEI/L-DNase II) pathway. p53 appeared to regulate mitochondria homeostasis, by initiating F0F1-ATPase reversal and endonuclease G (Endo G) release. In conclusion, a low dose of B[a]P induced apoptosis by recruiting a large panel of executioners apparently depending on p53 phosphorylation and, for some of them, on acidification. J. Cell. Physiol. 208: 527–537, 2006. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

How tumor microenvironment, more specifically low extracellular pH (6.5), alters cell response to TNF-related apoptosis-inducing ligand (TRAIL)-based cancer therapy has yet to be determined. The aim of the current work was to test the effect of acidic extracellular pH on TRAIL-induced cell death in human HT29 colon carcinoma and HepG2 hepatocarcinoma cell lines as well as in human primary hepatocytes. We found an increase in TRAIL sensitivity at low extracellular pH, which is partially inhibited by Bcl-2 expression in HT29 cells. At low extracellular pH, TRAIL induced a new form of cell death, sharing necrotic and apoptotic features in tumor cells. By contrast, human primary hepatocytes were resistant to TRAIL-induced cell death even at acidic extracellular pH. [ABSTRACT FROM AUTHOR]

How pHi changes, more specifically alkalinization, affect the apoptotic cascade has yet to be determined. The aim of the present work was to test the involvement of mitochondria in the apoptotic cascade triggered by benzo(a)pyrene [B(a)P] and to determine the role of pHi changes and p53 relative to mitochondria. Our results indicate that B(a)P-induced apoptosis might rely upon a p53-dependent and a pH-sensitive mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Simple Summary: One's environment, including diet, play a major role in the occurrence and the development of colorectal cancer (CRC). In this study, we are interested in two western diet associated food contaminants: 4-hydroxynonenal (HNE), a major lipid peroxidation product neoformed during digestion, and a mixture of pesticides to which we are commonly exposed to via fruit and vegetable consumption. The aim of this study was to analyse the impact of acute and long-term exposure to these contaminants, alone or in combination, on colorectal carcinogenesis. We used in vitro models of human colonic cells, either exhibiting or not different genetic susceptibilities to CRC. After acute exposure, we did not observe major alteration. However, long-term exposure to contaminants induce malignant transformation with different cellular mechanisms, depending on genetic susceptibility and contaminants alone or in mixtures. To investigate environmental impacts upon colorectal carcinogenesis (CRC) by diet, we assessed two western diet food contaminants: 4-hydroxynonenal (HNE), a major lipid peroxidation product neoformed during digestion, and a mixture of pesticides. We used human colonic cell lines ectopically eliciting varied genetic susceptibilities to CRC: the non-transformed human epithelial colonic cells (HCECs) and their five isogenic cell lines with the loss of APC (Adenomatous polyposis coli) and TP53 (Tumor protein 53) and/or ectopic expression of mutated KRAS (Kristen-ras). These cell lines have been exposed for either for a short time (2–24 h) or for a long period (3 weeks) to 1 µM HNE and/or 10 µM pesticides. After acute exposure, we did not observe any cytotoxicity or major DNA damage. However, long-term exposure to pesticides alone and in mixture with HNE induced clonogenic transformation in normal HCECs, as well as in cells representing later stages of carcinogenesis. It was associated with genotoxic and non-genomic mechanisms (cell growth, metabolic reprogramming, cell mobility and epithelial-mesenchymal transition) depending on genetic susceptibility. This study demonstrated a potential initiating and promoting effect of food contaminants on CRC after long-term exposure. It supports that these contaminants can accelerate carcinogenesis when mutations in oncogenes or tumor suppressor genes occur. [ABSTRACT FROM AUTHOR]

Presents a study on the effects of polycyclic aromatic hydrocarbons (PAH) on intracellular pH in liver epithelial cells. Induction of an early intracellular alkalinization by PAH; Involvement of NHE-1-related transient alkalinization in benzo(a)pyrene-induced apoptosis of liver epithelial cells; Conclusion of the study.

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. [ABSTRACT FROM AUTHOR]

The Na+/H+ exchanger 1 NHE-1 is a ubiquitous membrane transporter mainly involved in pH and cell volume regulation. NHE-1 activation by H+ is carried out according to the Monod-Wyman-Changeux model in which the dimeric NHE-1 oscillates between a low and a high affinity conformation, the balance of which being defined by the ratio between the two forms L0. Influence of cholesterol- and caveolin-rich microdomains on NHE-1 activity is examined, by using a cholesterol depleting agent, methyl-β-cyclodextrin (MBCD). MBCD induce NHE-1 activation, by modulating its allosteric balance, these effects being reverted by cholesterol repletion. This activation appears to be associated with NHE-1 relocalization outside caveolin-rich microdomains and distinct from mitogenic stimulation of NHE-1, since MBCD and serum cotreatments are additive; besides, serum alone stimulates this transporter without modifying its localization. Finally, MBCD activate a serum-insensitive mutated NHE-1 form and this process occurs independently of MAP kinase phosphorylation. In conclusion, the membrane localization of NHE-1 in or out microdomains seems to be an essentiel regulator of NHE1 activity, independently of mitogenic action. [ABSTRACT FROM AUTHOR]

Na+-H+ exchange (NHE) is the principal H+-extrusion mechanism from cardiac cells. We have investigated the spatial location of functionally active NHE in rat isolated ventricular myocytes. Cells were subjected to transient hypertonic shock using 1.5M formamide (Brette et al, Am J Physiol 283; 2002) to disrupt the t-system, and then assayed for NHE-mediated H+-extrusion (using SNARF to monitor pHi recovery following an NH4Cl-prepulse; Hepes-buffered superfusates). Successful detubulation was checked in some cells by membrane staining with di-8-Anneps, and in others by monitoring the electrically evoked Ca2+i-transient (confocal linescan using fluo-3; a sub-sarcolemmal Ca2+i-increase in detubulated myocytes). Both intrinsic buffer capacity and H+-extrusion at a given pHi (7.2-6.2) were unaffected by detubulation, suggesting NHE activity is principally at the sarcolemmal surface. Immunofluorescent staining with NHE1 mouse monoclonal antibody 611775, clone 54 (BD Transduction Laboratories) revealed limited expression at the surface sarcolemma, widespread punctate expression at intracellular sites, but no appearance at t-system sites. We conclude that H+extrusion from the ventricular myocyte is achieved functionally at surface rather than t-system membranes. [ABSTRACT FROM AUTHOR]