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The formation of reactive oxygen species (ROS) and the change of the intracellular pH (pHi) are common phenomena during apoptosis. How they are interconnected, however, is poorly understood. Here we show that numerous anticancer drugs and cytokines such as Fas ligand and tumour necrosis factor α provoke intracellular acidification and cause the formation of mitochondrial ROS. In parallel, we found that the succinate:ubiquinone oxidoreductase (SQR) activity of the mitochondrial respiratory complex II is specifically impaired without affecting the second enzymatic activity of this complex as a succinate dehydrogenase (SDH). Only in this configuration is complex II an apoptosis mediator and generates superoxides for cell death. This is achieved by the pHi decline that leads to the specific dissociation of the SDHA/SDHB subunits, which encompass the SDH activity, from the membrane-bound components of complex II that are required for the SQR activity. [ABSTRACT FROM AUTHOR]

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these environmental chemicals, the interactions of 15 SDHIs with activities of main human drug transporters implicated in pharmacokinetics were investigated in vitro. 5/15 SDHIs, i.e., benzovindiflupyr, bixafen, fluxapyroxad, pydiflumetofen and sedaxane, were found to strongly reduce activity of the renal organic anion transporter (OAT) 3, in a concentration-dependent manner (with IC 50 values in the 1.0-3.9 μM range), without however being substrates for OAT3. Moreover, these 5/15 SDHIs decreased the membrane transport of estrone-3 sulfate, an endogenous substrate for OAT3, and sedaxane was predicted to inhibit in vivo OAT3 activity in response to exposure to the acceptable daily intake (ADI) dose. In addition, pydiflumetofen strongly inhibited the renal organic cation transporter (OCT) 2 (IC 50  = 2.0 μM) and benzovindiflupyr the efflux pump breast cancer resistance protein (BCRP) (IC 50  = 3.9 μM). Other human transporters, including organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 as well as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K were moderately or weakly inhibited by SDHIs, whereas P-glycoprotein, multidrug resistance-associated protein (MRP), OCT1 and OAT1 activities were not or only marginally impacted. Then, some human drug transporters, especially OAT3, constitute molecular targets for SDHIs. This could have toxic consequences, notably with respect to levels of endogenous compounds and metabolites substrates for the considered transporters or to potential SDHI-drug interactions. This could therefore contribute to putative health risk of these fungicides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these agrochemicals, the interactions of 15 SDHIs with expression and activity of human cytochrome P-450 3A4 (CYP3A4), a major hepatic drug metabolizing enzyme, were investigated in vitro. 12/15 SDHIs, i.e., bixafen, boscalid, fluopyram, flutolanil, fluxapyroxad, furametpyr, isofetamid, isopyrazam, penflufen, penthiopyrad, pydiflumetofen and sedaxane, were found to enhance CYP3A4 mRNA expression in human hepatic HepaRG cells and primary human hepatocytes exposed for 48 h to 10 µM SDHIs, whereas 3/15 SDHIs, i.e., benzovindiflupyr, carboxin and thifluzamide, were without effect. The inducing effects were concentrations-dependent for boscalid (EC 50 =22.5 µM), fluopyram (EC 50 =4.8 µM) and flutolanil (EC 50 =53.6 µM). They were fully prevented by SPA70, an antagonist of the Pregnane X Receptor, thus underlining the implication of this xenobiotic-sensing receptor. Increase in CYP3A4 mRNA in response to SDHIs paralleled enhanced CYP3A4 protein expression for most of SDHIs. With respect to CYP3A4 activity, it was directly inhibited by some SDHIs, including bixafen, fluopyram, fluxapyroxad, isofetamid, isopyrazam, penthiopyrad and sedaxane, which therefore appears as dual regulators of CYP3A4, being both inducer of its expression and inhibitor of its activity. The inducing effect nevertheless predominates for these SDHIs, except for isopyrazam and sedaxane, whereas boscalid and flutolanil were pure inducers of CYP3A4 expression and activity. Most of SDHIs appear therefore as in vitro inducers of CYP3A4 expression in cultured hepatic cells, when, however, used at concentrations rather higher than those expected in humans in response to environmental or dietary exposure to these agrochemicals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

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.

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)., Competing Interests: The authors have declared that no competing interests exist.

Despite the improvement of diagnostic methods and anticancer therapeutics, the human population is still facing an increasing incidence of several types of cancers. According to the World Health Organization, this growing trend would be partly linked to our environment, with around 20% of cancers stemming from exposure to environmental contaminants, notably chemicals like polycyclic aromatic hydrocarbons (PAHs). PAHs are widespread pollutants in our environment resulting from incomplete combustion or pyrolysis of organic material, and thus produced by both natural and anthropic sources; notably benzo[a]pyrene (B[a]P), i.e. the prototypical molecule of this family, that can be detected in cigarette smoke, diesel exhaust particles, occupational-related fumes, and grilled food. This molecule is a well-recognized carcinogen belonging to group 1 carcinogens. Indeed, it can target the different steps of the carcinogenic process and all cancer hallmarks. Interestingly, H + dynamics have been described as key parameters for the occurrence of several, if not all, of these hallmarks. However, information regarding the role of such parameters during environmental carcinogenesis is still very scarce. The present review will thus mainly give an overview of the impact of B[a]P on H + dynamics in liver cells, and will show how such alterations might impact different aspects related to the finely-tuned balance between cell death and survival processes, thereby likely favoring environmental carcinogenesis. In total, the main objective of this review is to encourage further research in this poorly explored field of environmental molecular toxicology., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Background: The World Health Organization classified processed and red meat consumption as "carcinogenic" and "probably carcinogenic", respectively, to humans. Haem iron from meat plays a role in the promotion of colorectal cancer in rodent models, in association with enhanced luminal lipoperoxidation and subsequent formation of aldehydes. Here, we investigated the short-term effects of this haem-induced lipoperoxidation on mucosal and luminal gut homeostasis including microbiome in F344 male rats fed with a haem-enriched diet (1.5 μmol/g) 14-21 days., Results: Changes in permeability, inflammation, and genotoxicity observed in the mucosal colonic barrier correlated with luminal haem and lipoperoxidation markers. Trapping of luminal haem-induced aldehydes normalised cellular genotoxicity, permeability, and ROS formation on a colon epithelial cell line. Addition of calcium carbonate (2%) to the haem-enriched diet allowed the luminal haem to be trapped in vivo and counteracted these haem-induced physiological traits. Similar covariations of faecal metabolites and bacterial taxa according to haem-induced lipoperoxidation were identified., Conclusions: This integrated approach provides an overview of haem-induced modulations of the main actors in the colonic barrier. All alterations were closely linked to haem-induced lipoperoxidation, which is associated with red meat-induced colorectal cancer risk.

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.

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., (© 2018 Wiley Periodicals, Inc.)

Resveratrol (RES), a polyphenol found in natural foods, displays anti-oxidant, anti-inflammatory and anti-proliferative properties potentially beneficial in cancers, in particular in the prevention of tumor growth. However, the rapid metabolism of resveratrol strongly limits its bioavailability. The molecular mechanisms sustaining the potential biological activity of low doses of resveratrol has not been extensively studied and, thus, needs better characterization. Here, we show that resveratrol (10 µM, 48 hr) induces both a cell growth arrest and a metabolic reprogramming in colon cancer cells. Resveratrol modifies the lipidomic profile, increases oxidative capacities and decreases glycolysis, in association with a decreased pentose phosphate activity and an increased ATP production. Resveratrol targets the pyruvate dehydrogenase (PDH) complex, a key mitochondrial gatekeeper of energy metabolism, leading to an enhanced PDH activity. Calcium chelation, as well as the blockade of the mitochondrial calcium uniport, prevents the resveratrol-induced augmentation in oxidative capacities and the increased PDH activity suggesting that calcium might play a role in the metabolic shift. We further demonstrate that the inhibition of the CamKKB or the downstream AMPK pathway partly abolished the resveratrol-induced increase of glucose oxidation. This suggests that resveratrol might improve the oxidative capacities of cancer cells through the CamKKB/AMPK pathway.

According to the World Health Organization, around 20% of all cancers would be due to environmental factors. Among these factors, several chemicals are indeed well recognized carcinogens. The widespread contaminant benzo[a]pyrene (B[a]P), an often used model carcinogen of the polycyclic aromatic hydrocarbons' family, has been suggested to target most, if not all, cancer hallmarks described by Hanahan and Weinberg. It is classified as a group I carcinogen by the International Agency for Research on Cancer; however, the precise intracellular mechanisms underlying its carcinogenic properties remain yet to be thoroughly defined. Recently, the pH homeostasis, a well known regulator of carcinogenic processes, was suggested to be a key actor in both cell death and Warburg-like metabolic reprogramming induced upon B[a]P exposure. The present review will highlight those data with the aim of favoring research on the role of H + dynamics in environmental carcinogenesis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 β2-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.

Cancer cells display alterations in many cellular processes. One core hallmark of cancer is the Warburg effect which is a glycolytic reprogramming that allows cells to survive and proliferate. Although the contributions of environmental contaminants to cancer development are widely accepted, the underlying mechanisms have to be clarified. Benzo[a]pyrene (B[a]P), the prototype of polycyclic aromatic hydrocarbons, exhibits genotoxic and carcinogenic effects, and it is a human carcinogen according to the International Agency for Research on Cancer. In addition to triggering apoptotic signals, B[a]P may induce survival signals, both of which are likely to be involved in cancer promotion. We previously suggested that B[a]P-induced mitochondrial dysfunctions, especially membrane hyperpolarization, might trigger cell survival signaling in rat hepatic epithelial F258 cells. Here, we further characterized these dysfunctions by focusing on energy metabolism. We found that B[a]P promoted a metabolic reprogramming. Cell respiration decreased and lactate production increased. These changes were associated with alterations in the tricarboxylic acid cycle which likely involve a dysfunction of the mitochondrial complex II. The glycolytic shift relied on activation of the Na(+)/H(+) exchanger 1 (NHE1) and appeared to be a key feature in B[a]P-induced cell survival related to changes in cell phenotype (epithelial-to-mesenchymal transition and cell migration).

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 hemoglobin- and 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., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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 KRAS (V12) 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 KRAS(V12) 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.

Benzo[a]pyrene (B[a]P), the prototype molecule of polycyclic aromatic hydrocarbons, exhibits genotoxic and carcinogenic effects, which has led the International Agency for Research on Cancer to recognize it as a human carcinogen. Besides the well-known apoptotic signals triggered by B[a]P, survival signals have also been suggested to occur, both signals likely involved in cancer promotion. Our previous work showed that B[a]P induced an hyperpolarization of mitochondrial membrane potential (ΔΨm) in rat hepatic epithelial F258 cells. Elevated ΔΨm plays a role in tumor development and progression, and nitric oxide (NO) has been suggested to be responsible for increases in ΔΨm. The present study therefore aimed at evaluating the impact of B[a]P on NO level in F258 cells, and at testing the putative role for NO as a survival signal, notably in link with ΔΨm. Our data demonstrated that B[a]P exposure resulted in an NO production which was dependent upon the activation of the inducible NO synthase. This enzyme activation involved AhR and possibly p53 activation. Preventing NO production not only increased B[a]P-induced cell death but also blocked mitochondrial hyperpolarization. This therefore points to a role for NO as a survival signal upon B[a]P exposure, possibly targeting ΔΨm., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Bisphenol A (BPA) is a suspected endocrine disruptor highly prevalent in our environment since it is used as monomer of polycarbonate plastics and epoxy resins. Recent epidemiological and animal studies have suggested that BPA exposure may influence the development of obesity and related pathologies such as type 2 diabetes, and cardiovascular diseases. However, experimental studies have often focused on short-term exposures. In this study, we investigated the effect of several months of BPA exposure on hepatic and plasma metabolic markers in adult mice. Male CD1 mice were exposed during 8 months to five different BPA doses below or equivalent to the current no observed adverse effect level (NOAEL: 5000 μg/kg/day) through drinking water. Plasma lipid profiles and liver transcriptomic analysis were performed in control and BPA-treated animals. We report a specific impact of BPA exposure on glycaemia, glucose tolerance and cholesterolemia. Consistent with the hypercholesterolemia in BPA-treated animals, RT-qPCR performed on hepatic mRNA from same animals demonstrated an overexpression of key genes involved in cholesterol biosynthesis, namely, Mvd, Lss Hmgcr, and Sqle. BPA also induced the expression of the sterol regulatory element-binding proteins 2, a master regulator of hepatic cholesterol biosynthesis. As shown by the plasma lathosterol to cholesterol ratio, a surrogate marker for cholesterol biosynthesis, whole body cholesterol de novo synthesis was also increased in BPA-exposed animals. These original results are consistent with many epidemiological studies reporting on a link between BPA exposure and the onset of cardiovascular diseases., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

During the last three decades, 4-hydroxy-2-nonenal (HNE), a major α,β-unsaturated aldehyde product of n-6 fatty acid oxidation, has been shown to be involved in a great number of pathologies such as metabolic diseases, neurodegenerative diseases and cancers. These multiple pathologies can be explained by the fact that HNE is a potent modulator of numerous cell processes such as oxidative stress signaling, cell proliferation, transformation or cell death. The main objective of this review is to focus on the different aspects of HNE-induced cell death, with a particular emphasis on apoptosis. HNE is a special apoptotic inducer because of its abilities to form protein adducts and to propagate oxidative stress. It can stimulate intrinsic and extrinsic apoptotic pathways and interact with typical actors such as tumor protein 53, JNK, Fas or mitochondrial regulators. At the same time, due to its oxidant status, it can also induce some cellular defense mechanisms against oxidative stress, thus being involved in its own detoxification. These processes in turn limit the apoptotic potential of HNE. These dualities can imbalance cell fate, either toward cell death or toward survival, depending on the cell type, the metabolic state and the ability to detoxify.

Membrane acid extrusion by Na(+)/H(+) exchange (NHE1) and Na(+)-HCO3(-) co-transport (NBC) is essential for maintaining a low cytoplasmic [H(+)] (∼60 nm, 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.

F1 domain of F(1)F(o)-ATPase was initially believed to be strictly expressed in the mitochondrial membrane. Interestingly, recent reports have shown that the F1 complex can serve as a cell surface receptor for apparently unrelated ligands. Here we show for the first time the presence of the F(1)-ATPase at the cell surface of normal or cancerous colonic epithelial cells. Using surface plasmon resonance technology and mass spectrometry, we identified a peptide hormone product of the gastrin gene (glycine-extended gastrin (G-gly)) as a new ligand for the F(1)-ATPase. By molecular modeling, we identified the motif in the peptide sequence (E(E/D)XY), that directly interacts with the F(1)-ATPase and the amino acids in the F(1)-ATPase that bind this motif. Replacement of the Glu-9 residue by an alanine in the E(E/D)XY motif resulted in a strong decrease of G-gly binding to the F(1)-ATPase and the loss of its biological activity. In addition we demonstrated that F(1)-ATPase mediates the growth effects of the peptide. Indeed, blocking F(1)-ATPase activity decreases G-gly-induced cell growth. The mechanism likely involves ADP production by the membrane F(1)-ATPase, which is induced by G-gly. These results suggest an important contribution of cell surface F(1)-ATPase in the pro-proliferative action of this gastrointestinal peptide.

Bisphenol A (BPA) is an endocrine-disrupting chemical that leaches from polycarbonate plastics that consequently leads to low-dose human exposure. In addition to its known xenoendocrine action, BPA exerts a wide variety of metabolic effects, but no data are available on its actions on the functions of liver mitochondrial. To assess these effects, HepG2 cells were exposed to BPA (10(-4)-10(-12)M) and physiological parameters were measured by flow cytometry. We demonstrated a significant mitochondrial dysfunction including ROS production, ΔΨ(M) hyperpolarization, lipid accumulation, lipoperoxidation and the release of pro-inflammatory cytokines. In conclusion, we showed that low concentrations of BPA promote lipid accumulation in hepatic cells triggered by disturbances in mitochondrial function, alterations in lipid metabolism and by inflammation that can therefore contribute to steatosis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The early apoptotic events induced by environmental pollutants with carcinogenic properties are poorly understood. Here, we focus on the early cytotoxic effects of benzo[a]pyrene (B[a]P). In F258 rat hepatic epithelial cells, B[a]P induces intrinsic apoptosis via a mitochondrial dysfunction characterized by the release of hexokinase II (HKII) from the mitochondria. Cancer cells often have an anomalous cell energy metabolism; since HKII dysfunction regulates B[a]P-induced apoptosis in F258 cells, but may also alter cell energy metabolism, HKII release from the mitochondria may represent an important B[a]P-related carcinogenic issue. Thus in the present study, we aimed at deciphering the mechanisms underlying HKII dysfunction upon B[a]P exposure. We show that while glycogen synthase kinase 3 beta (GSK3β) regulated the expression of HKII at the transcriptional level, glycogen synthase kinase 3 alpha (GSK3α) was involved in B[a]P-induced apoptosis via a decrease in c-Myc expression. The reduced level of c-Myc caused the relocation of HKII from the mitochondria to the cytosol, thereby being involved in the formation of reactive oxygen species and apoptosis. In conclusion, we show that the couple GSK3α/c-Myc plays a key role in B[a]P-induced early apoptotic cell signaling via HKII dysfunction., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are ubiquitous toxic environmental pollutants capable of inducing cell death. Intracellular pH plays a key role in the regulation of cell survival and death. Our previous works have demonstrated that intracellular alkalinization mediated by Na(+)/H(+) exchanger 1 (NHE-1) is a critical event involved in B[a]P-induced apoptosis. The aim of this study was to further elucidate the mechanisms of NHE-1 activation upon B[a]P exposure., Methods: We tested the effects of plasma membrane cholesterol enrichment or depletion on B[a]P-induced NHE-1 activation related to apoptosis. We isolated cholesterol-rich plasma membrane microdomains to assess NHE-1 submembrane location and immunoprecipitated NHE-1 from the different sub-membrane fractions obtained to examine NHE-1 protein interactions during B[a]P-induced apoptosis., Results: We found that NHE-1 is preferentially located in cholesterol-rich microdomains and that B[a]P activates NHE-1 via its relocation and binding of calmodulin outside these specialized plasma membrane microstructures; these events are necessary for the execution of the apoptosis-related intracellular alkalinization., Conclusion: Plasma membrane location of NHE-1 affects its protein interactions and apoptotic function., (Copyright © 2012 S. Karger AG, Basel.)

Animal and epidemiological studies suggest that dietary heme iron would promote colorectal cancer. Oxidative properties of heme could lead to the formation of cytotoxic and genotoxic secondary lipid oxidation products, such as 4-hydroxy-2(E)-nonenal (HNE). This compound is more cytotoxic to mouse wild-type colon cells than to isogenic cells with a mutation on the adenomatous polyposis coli (APC) gene. The latter thus have a selective advantage, possibly leading to cancer promotion. This mutation is an early and frequent event in human colorectal cancer. To explain this difference, the HNE biotransformation capacities of the two cell types have been studied using radiolabeled and stable isotope-labeled HNE. Apc-mutated cells showed better biotransformation capacities than nonmutated cells did. Thiol compound conjugation capacities were higher for mutated cells, with an important advantage for the extracellular conjugation to cysteine. Both cells types were able to reduce HNE to 4-hydroxynonanal, a biotransformation pathway that has not been reported for other intestinal cells. Mutated cells showed higher capacities to oxidize 4-hydroxynonanal into 4-hydroxynonanoic acid. The mRNA expression of different enzymes involved in HNE metabolism such as aldehyde dehydrogenase 1A1, 2 and 3A1, glutathione transferase A4-4, or cystine transporter xCT was upregulated in mutated cells compared with wild-type cells. In conclusion, this study suggests that Apc-mutated cells are more efficient than wild-type cells in metabolizing HNE into thiol conjugates and 4-hydroxynonanoic acid due to the higher expression of key biotransformation enzymes. These differential biotransformation capacities would explain the differences of susceptibility between normal and Apc-mutated cells regarding secondary lipid oxidation products.

Metabolomics experiments seldom achieve their aim of comprehensively covering the entire metabolome. However, important information can be gleaned even from sparse datasets, which can be facilitated by placing the results within the context of known metabolic networks. Here we present a method that allows the automatic assignment of identified metabolites to positions within known metabolic networks, and, furthermore, allows automated extraction of sub-networks of biological significance. This latter feature is possible by use of a gap-filling algorithm. The utility of the algorithm in reconstructing and mining of metabolomics data is shown on two independent datasets generated with LC-MS LTQ-Orbitrap mass spectrometry. Biologically relevant metabolic sub-networks were extracted from both datasets. Moreover, a number of metabolites, whose presence eluded automatic selection within mass spectra, could be identified retrospectively by virtue of their inferred presence through gap filling. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-009-0196-9) contains supplementary material, which is available to authorized users.

Benzo[alpha]pyrene (B[alpha]P) often serves as a model for mutagenic and carcinogenic polycyclic aromatic hydrocarbons (PAHs). Our previous work suggested a role of membrane fluidity in B[alpha]P-induced apoptotic process. In this study, we report that B[alpha]P modifies the composition of cholesterol-rich microdomains (lipid rafts) in rat liver F258 epithelial cells. The cellular distribution of the ganglioside-GM1 was markedly changed following B[alpha]P exposure. B[alpha]P also modified fatty acid composition and decreased the cholesterol content of cholesterol-rich microdomains. B[alpha]P-induced depletion of cholesterol in lipid rafts was linked to a reduced expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). Aryl hydrocarbon receptor (AhR) and B[alpha]P-related H(2)O(2) formation were involved in the reduced expression of HMG-CoA reductase and in the remodeling of membrane microdomains. The B[alpha]P-induced membrane remodeling resulted in an intracellular alkalinization observed during the early phase of apoptosis. In conclusion, B[alpha]P altered the composition of plasma membrane microstructures through AhR and H(2)O(2) dependent-regulation of lipid biosynthesis. In F258 cells, the B[alpha]P-induced membrane remodeling was identified as an early apoptotic event leading to an intracellular alkalinization., (Copyright 2009 Elsevier Inc. All rights reserved.)

NHE-1 is a ubiquitous, mitogen-activatable, mammalian Na+/H+ exchanger that maintains cytosolic pH and regulates cell volume. We have previously shown that the kinetics of NHE-1 positive cooperative activation by intracellular acidifications fit best with a Monod-Wyman-Changeux mechanism, in which a dimeric NHE-1 oscillates between a low- and a high-affinity conformation for intracellular protons. The ratio between these two forms, the allosteric equilibrium constant L0, is in favor of the low-affinity form, making the system inactive at physiological pH. Conversely the high-affinity form is stabilized by intracellular protons, resulting in the observed positive cooperativity. The aim of the present study was to investigate the kinetics and mechanism of NHE-1 regulation by osmotic shocks. We show that they modify the L0 parameter (865 +/- 95 and 3757 +/- 328 for 500 and 100 mOsM, respectively, vs 1549 +/- 57 in isotonic conditions).This results in an activation of NHE-1 by hypertonic shocks and, conversely, in an inhibition by hypotonic media. Quantitatively, this modulation of L0 follows an exponential distribution relative to osmolarity, that is, additive to the activation of NHE-1 by intracellular signaling pathways. These effects can be mimicked by the asymmetric insertion of amphiphilic molecules into the lipid bilayer. Finally, site-directed mutagenesis of NHE-1 shows that neither its association with membrane PIP2 nor its interaction with cortical actin are required for mechanosensation. In conclusion, NHE-1 allosteric equilibrium and, thus, its cooperative response to intracellular acidifications is extremely sensitive to modification of its membrane environment.

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 L(0). In this study, influence of cholesterol- and caveolin-rich microdomains on NHE-1 activity was examined by using cholesterol depleting agents, including methyl-beta-cyclodextrin (MBCD). These agents activated NHE-1 by modulating its L(0) 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 ((625)KDKEEEIRK(635) 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.

The present study compares and elucidates possible mechanisms why B[a]P induces different cell signals and triggers apparently different apoptotic pathways in two rather similar cell lines (hepatic epithelial cells of rodents). The rate and maximal capacity of metabolic activation, as measured by the formation of B[a]P-tetrols and B[a]P-DNA adducts, was much higher in mouse hepatoma Hepa1c1c7 cells than in rat liver epithelial F258 cells due to a higher induced level of cyp1a1. B[a]P increased intracellular pH in both cell lines, but this change modulated the apoptotic process only in F258 cells. In Hepa1c1c7 cells reactive oxygen species (ROS) production appeared to be a consequence of toxicity, unlike F258 cells in which it was an initial event. The increased mitochondrial membrane potential found in F258 cells was not observed in Hepa1c1c7 cells. Surprisingly, F258 cells cultured at low cell density were somewhat more sensitive to low (50nM) B[a]P concentrations than Hepa1c1c7 cells. This could be explained partly by metabolic differences at low B[a]P concentrations. In contrast to the Hepa1c1c7 model, no activation of cell survival signals including p-Akt, p-ERK1/2 and no clear inactivation of pro-apoptotic Bad was observed in the F258 model following exposure to B[a]P. Another important difference between the two cell lines was related to the role of Bax and cytochrome c. In Hepa1c1c7 cells, B[a]P exposure resulted in a "classical" translocation of Bax to the mitochondria and release of cytochrome c, whereas in F258 cells no intracellular translocation of these two proteins was seen. These results suggest that the rate of metabolism of B[a]P and type of reactive metabolites formed influence the resulting balance of pro-apoptotic and anti-apoptotic cell signaling, and hence the mechanisms involved in cell death and the chances of more permanent genetic damage.

Regulation of the balance between survival, proliferation, and apoptosis on carcinogenic polycyclic aromatic hydrocarbon (PAH) exposure is still poorly understood and more particularly the role of physiologic variables, including intracellular pH (pH(i)). Although the involvement of the ubiquitous pH(i) regulator Na(+)/H(+) exchanger isoform 1 (NHE1) in tumorigenesis is well documented, less is known about its role and regulation during apoptosis. Our previous works have shown the primordial role of NHE1 in carcinogenic PAH-induced apoptosis. This alkalinizing transporter was activated by an early CYP1-dependent H(2)O(2) production, subsequently promoting mitochondrial dysfunction leading to apoptosis. The aim of this study was to further elucidate how NHE1 was activated by benzo(a)pyrene (BaP) and what the downstream events were in the context of apoptosis. Our results indicate that the mitogen-activated protein kinase kinase 4/c-Jun NH(2)-terminal kinase (MKK4/JNK) pathway was a link between BaP-induced H(2)O(2) production and NHE1 activation. This activation, in combination with BaP-induced phosphorylated p53, promoted mitochondrial superoxide anion production, supporting the existence of a common target for NHE1 and p53. Furthermore, we showed that the mitochondrial expression of glycolytic enzyme hexokinase II (HKII) was decreased following a combined action of NHE1 and p53 pathways, thereby enhancing the BaP-induced apoptosis. Taken together, our findings suggest that, on BaP exposure, MKK4/JNK targets NHE1 with consequences on HKII protein, which might thus be a key protein during carcinogenic PAH apoptosis.

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a potential anticancer agent that induces apoptosis in cancer cells but not in most normal cells. How tumor physiology, particularly acidic extracellular pH (pH(e)), would modify sensitivity of cancer cells to TRAIL-induced cell death is not known. We have previously shown that cancer cells, resistant to TRAIL-induced apoptosis at physiologic pH(e) (7.4), could be sensitized to TRAIL at acidic pH(e) (6.5). However, at this acidic pH(e), cell death was necrotic. We show here that, in spite of a necrosis-like cell death morphology, caspases are activated and are necessary for TRAIL-induced cell death at acidic pH(e) in HT29 human colon cancer cells. Furthermore, we observed that, whereas receptor-interacting protein (RIP) was cleaved following TRAIL treatment at physiologic pH(e) (7.4), it was not cleaved following TRAIL treatment at acidic pH(e) (6.5). Moreover, RIP degradation by geldanamycin or decrease expression of RIP by small RNA interference transfection inhibited TRAIL-induced necrosis at acidic pH(e), showing that RIP was necessary for this necrotic cell death pathway. We also show that RIP kinase activity was essential for this cell death pathway. Altogether, we show that, under acidic pH(e) conditions, TRAIL induces a necrosis-like cell death pathway that depends both on caspases and RIP kinase activity. Thus, our data suggest for the first time that RIP-dependent necrosis might be a major death pathway in TRAIL-based therapy in solid tumors with acidic pH(e).

Exogenous treatment with monosialoganglioside GM1 has been described to afford protection against different apoptotic insults. However, the underlying mechanisms remain to be determined. In this study, we focused on the effect of GM1 on the apoptotic cascade induced by benzo[a]pyrene (B[a]P) in rat hepatic F258 epithelial cells. We first demonstrated that a co-treatment with GM1 (80 microM) reduced B[a]P (50 nM)-induced apoptosis as evidenced by a decrease of both cell population exhibiting nuclear fragmentation and caspase 3 cleavage and activity. We next showed that the p53 phosphorylation and nuclear translocation as well as the intracellular alkalinization related to Na+/H+ exchanger 1 (NHE1) activation, two early events of the apoptosis induced by B[a]P, were not inhibited by GM1. In contrast, the late mitochondria-dependent acidification elicited by B[a]P was inhibited by GM1 co-treatment, and an inhibition of the oxidative stress was also observed. Because GM1 has been shown to reduce the low-molecular weight iron content related to ethanol-induced oxidative stress, we finally investigated the involvement of iron under our conditions. Using the two iron chelators deferiprone and desferrioxamine, we clearly showed that iron played an important role in B[a]P-induced apoptosis in F258 cells, and that B[a]P-treatment resulted in a significant GM1-sensitive increase in (55)Fe uptake. In conclusion, our results indicate that exogenous GM1 partly prevents B[a]P-induced apoptosis by interfering with mitochondria-related intracellular acidification and iron transport.

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.

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.

The relationship between bulk membrane fluidizing effect of ethanol and its toxicity due to oxidative stress is still unknown. To elucidate this issue, membrane fluidity of primary rat hepatocytes was studied by measuring order parameter after inhibition of ethanol-induced oxidative stress. We showed that pretreating cells with either 4-methyl-pyrazole (to inhibit ethanol metabolism), thiourea [a reactive oxygen species (ROS) scavenger], or vitamin E (a free radical chain-breaking antioxidant) prevented the ethanol-induced increase in membrane fluidity, thus suggesting that ethanol metabolism and ROS formation were involved in this elevation. The effects of membrane stabilizing agents (ursodeoxycholic acid or ganglioside GM1), shown to prevent fluidification, next pointed to a role for this increase in membrane fluidity in the development of ethanol-induced oxidative stress. Indeed, ROS production, lipid peroxidation, and cell death were all inhibited by these agents. In contrast, the fluidizing compounds Tween 20 or 2-(2-methoxyethoxy) ethyl 8-(cis-2-n-octylcyclopropyl) octanoate, which increased the membrane fluidizing effect of ethanol, enhanced the related oxidative stress. Using electron paramagnetic resonance to determine low molecular weight iron, we finally demonstrated that membrane fluidity influence proceeded through an increase in low molecular weight iron to enhance oxidative stress. In conclusion, the present findings clearly highlight the pivotal role of membrane fluidity in ethanol-induced oxidative stress and the potential therapeutic effect of membrane stabilizing compounds.

We examined the effects of amiloride derivatives, especially 5-(N-ethyl-N-isopropyl)amiloride (EIPA), on the activity of cytochrome P450 (CYP) 1 isoforms, known to metabolize carcinogenic polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BP), into mutagenic metabolites and whose cellular expression can be induced through interaction of PAHs with the arylhydrocarbon receptor. EIPA was found to cause a potent and dose-dependent inhibition of CYP1-related ethoxyresorufine O-deethylase (EROD) activity in both liver cells and microsomes. It also markedly reduced activity of human recombinant CYP1A1 enzyme through a competitive mechanism; activities of other human CYP1 isoforms, i.e. CYP1A2 and CYP1B1, were also decreased. However, EIPA did not affect BP-mediated induction of CYP1A1 mRNA and protein levels in rat liver cells, likely indicating that EIPA does not block activation of the arylhydrocarbon receptor by PAHs. Inhibition of CYP1 activity by EIPA was associated with a decreased metabolism of BP, a reduced formation of BP-derived DNA adducts and a diminished BP-induced apoptosis in liver cells. The present data suggest that amiloride derivatives, such as EIPA, may be useful for preventing toxicity of chemical carcinogens, such as PAHs, through inhibition of CYP1 enzyme activity.

The ubiquitous environmental pollutants polycyclic aromatic hydrocarbons are responsible for important carcinogenic and apoptotic effects, whose mechanisms are still poorly understood, owing to the multiplicity of possible cellular targets. Among these mechanisms, alterations of ionic homeostasis have been suggested. In this work, the effects of benzo(a)pyrene [B(a)P] on pHi were tested in the rat liver F258 epithelial cell line, using the fluoroprobe carboxy-SNARF-1. After a 48-h treatment, B(a)P (50 nM) induced an alkalinization, followed by an acidification after 72 h and the development of apoptosis. Determinations of pH(i) recovery following an acid load showed an increased acid efflux at 48 h. Cariporide inhibited both the early alkalinization and the increased acid efflux, thus suggesting the involvement of Na+/H+ exchanger 1 (NHE1). Besides, alpha-naphtoflavone (alpha-NF), an inhibitor of CYP1A1-mediated B(a)P metabolism, prevented all pH(i) changes, and NHE1 activation was blocked by the antioxidant thiourea, which inhibited CYP1A1 metabolism-dependent H2O2 production. Regarding B(a)P-induced apoptosis, this was prevented by alpha-NF and bongkrekic acid, an inhibitor of mitochondria-dependent apoptosis. Interestingly, apoptosis was significantly reduced by cariporide. Taken together, our results indicate that B(a)P, via H2O2 produced by CYP1A1-dependent metabolism, induces an early activation of NHE1, resulting in alkalinization; this appears to play a significant role in mitochondria-dependent B(a)P-induced apoptosis.