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Oxidative DNA damage has been implicated in the aging process and in some of its features such as telomere shortening and replicative senescence. Poly(ADP-ribosyl)ation is involved in many molecular and cellular processes, including DNA damage detection and repair, chromatin modification, transcription, and cell death pathways. We decided to examine the behavior of poly(ADP-ribosyl)ation in centenarians, i.e., those subjects who represent the best example of longevity having reached a very advanced age avoiding the main age-associated diseases. In this study we investigated the relationship between DNA repair capacity and poly(ADP-ribose) polymerase activity in Epstein-Barr virus-immortalized B lymphocyte cell lines from subjects of three different groups of age, including centenarians. Our data show that cells from centenarians have characteristics typical of cells from young people both in their capability of priming the mechanism of repair after H(2)O(2) sublethal oxidative damage and in poly(ADP-ribosyl)ation capacity, while in cells from old subjects these phenomena are delayed or decreased. Moreover, cells from old subjects show a constitutive expression level of both parp 1 and parp 2 genes reduced by a half, together with a reduced presence of modified PARP 1 and other poly(ADP-ribosyl)ated chromatin proteins in comparison to cells from young subjects and centenarians. Our data support the hypothesis that this epigenetic modification is an important regulator of the aging process in humans and it appears to be rather preserved in healthy centenarians, the best example of successful aging. [ABSTRACT FROM AUTHOR]

We tested seven physical education students whether 30-s sprint anaerobic exercise (Wingate test) would result in oxidative stress (evaluated by lipid radical levels) sufficient to alter plasma non-enzymatic antioxidant status (plasma uric acid, ascorbic acid, a-tocopherol, b-carotene). This study demonstrates that 1) Wingate test increases plasma uric and ascorbic acid concentrations (p < .05), and decreases plasma a-tocopherol and b-carotene levels (p < .05); 2) lipid radical levels at rest and sprint performance are negatively correlated with resting plasma uric acid and a-tocopherol concentrations (p < .05). In conclusion, this study 1) demonstrates that a 30-s sprint anaerobic exercise is associated with acute changes in plasma non-enzymatic antioxidant status, 2) indicates that the subjects with largest leg peak power are those who exhibit the lowest plasma antioxidant status at rest (uric acid and a-tocopherol), 3) and suggests that antioxidant intake by maintaining plasma antioxidant concentration at rest in the normal range might protect athletes against oxidative stress induced by exercise. [ABSTRACT FROM AUTHOR]

The aim of this study was to examine how macrophages could act on ethanol-induced oxidative stress in rat hepatocytes during inflammatory conditions, well-known to induce nitric oxide (NO) synthase. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocyte cultures. Co-cultures were then supplemented with lipopolysaccharide (LPS) and interferon γ (IFN) for 18 h, in order to induce NO synthase before the addition of 50 mM ethanol. In cultures of hepatocytes alone, the addition of LPS and IFN protected from ethanol-induced oxidative stress. It has been shown previously that NO generated in hepatocytes was responsible for this effect. When macrophages were added to primary rat hepatocyte cultures supplemented with LPS and IFN, protection provided by NO against ethanol-induced oxidative stress in hepatocytes ceased. Using a pretreatment of macrophages with Ng-monomethyl-l-arginine, a NO synthase inhibitor, it was concluded that NO generated by macrophages was responsible for macrophage toxicity. Taken together, our observations suggest that NO biosynthesis in hepatocytes protects them from ethanol-induced oxidative stress, whereas NO production in macrophages deprives hepatocytes of this NO protection. [ABSTRACT FROM PUBLISHER]

Oltipraz (4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione) (OPZ) is recognized as a potent chemoprotective agent against chemical-induced carcinogenesis in several animal models and is thought to act mainly by inducing phase II conjugating together with inhibiting phase I detoxication enzymes. The present study was undertaken to determine whether oltipraz can also influence expression of genes encoding antioxidant enzymes. In rat hepatocytes in primary culture, this compound was found to selectively induce the transcription of the manganese superoxide dismutase (Mn-SOD) gene while it had no effect on copper/zinc-SOD and glutathione peroxidase genes. Oltipraz increased Mn-SOD gene expression in a time- and dose-dependent manner by 2- to 3-fold and enhanced the binding activity of the nuclear factor kappa B within 30 min. Moreover, the increase in Mn-SOD gene transcription was associated with a 2- to 3-fold increase of free malondialdehyde and conjugated dienes, two markers of lipid peroxidation, an index of oxidative stress. These results suggest that in rat hepatocytes, oltipraz induced a production of reactive oxygen species that probably acted as second messengers in order to trigger the transcription of many genes. Such a mechanism of action of OPZ and other dithiolethiones would account for the broad spectrum of action of these anticarcinogenic compounds. [ABSTRACT FROM PUBLISHER]

A growing body of evidences indicate the major role of extracellular vesicles (EVs) as players of cell communication in the pathogenesis of liver diseases. EVs are membrane-enclosed vesicles released by cells into the extracellular environment. Oxidative stress is also a key component of liver disease pathogenesis, but no role for hepatocyte-derived EVs has yet been described in the development of this process. Recently, some polycyclic aromatic hydrocarbons (PAHs), widespread environmental contaminants, were demonstrated to induce EV release from hepatocytes. They are also well-known to trigger oxidative stress leading to cell death. Therefore, the aim of this work was to investigate the involvement of EVs derived from PAHs-treated hepatocytes (PAH-EVs) in possible oxidative damages of healthy recipient hepatocytes, using both WIF-B9 and primary rat hepatocytes. We first showed that the release of EVs from PAHs -treated hepatocytes depended on oxidative stress. PAH-EVs were enriched in proteins related to oxidative stress such as NADPH oxidase and ferritin. They were also demonstrated to contain more iron. PAH-EVs could then induce oxidative stress in recipient hepatocytes, thereby leading to apoptosis. Mitochondria and lysosomes of recipient hepatocytes exhibited significant structural alterations. All those damages were dependent on internalization of EVs that reached lysosomes with their cargoes. Lysosomes thus appeared as critical organelles for EVs to induce apoptosis. In addition, pro-oxidant components of PAH-EVs, e.g. NADPH oxidase and iron, were revealed to be necessary for this cell death., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Extracellular vesicles (EVs) are membrane-enclosed nanostructures released by cells into the extracellular environment. As major actors of physiological intercellular communication, they have been shown to be pathogenic mediators of several liver diseases. Extracellular vesicles also appear to be potential actors of drug-induced liver injury but nothing is known concerning environmental pollutants. We aimed to study the impact of polycyclic aromatic hydrocarbons (PAHs), major contaminants, on hepatocyte-derived EV production, with a special focus on hepatocyte death. Three PAHs were selected, based on their presence in food and their affinity for the aryl hydrocarbon receptor (AhR): benzo[a]pyrene (BP), dibenzo[a,h]anthracene (DBA), and pyrene (PYR). Treatment of primary rat and WIF-B9 hepatocytes by all 3 PAHs increased the release of EVs, mainly comprised of exosomes, in parallel with modifying exosome protein marker expression and inducing apoptosis. Moreover, PAH treatment of rodents for 3 months also led to increased EV levels in plasma. The EV release involved CYP metabolism and the activation of the transcription factor, the AhR, for BP and DBA and another transcription factor, the constitutive androstane receptor, for PYR. Furthermore, all PAHs increased cholesterol levels in EVs but only BP and DBA were able to reduce the cholesterol content of total cell membranes. All cholesterol changes very likely participated in the increase in EV release and cell death. Finally, we studied changes in cell membrane fluidity caused by BP and DBA due to cholesterol depletion. Our data showed increased cell membrane fluidity, which contributed to hepatocyte EV release and cell death., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Exposure to herbicides can induce long-term chronic adverse effects such as respiratory diseases, malignancies and neurodegenerative diseases. Oxadiazon, a pre-emergence or early post-emergence herbicide, despite its low acute toxicity, may induce liver cancer and may exert adverse effects on reproductive and on endocrine functions. Unlike other herbicides, there are no indications on neurotoxicity associated with long-term exposure to oxadiazon. Therefore, we have analyzed in primary neuronal precursor cells isolated from human striatal primordium the effects of non-cytotoxic doses of oxadiazon on neuronal cell differentiation and migration, and on the expression and activity of the mitochondrial aldehyde dehydrogenase 2 (ALDH2) and of the acylphosphatase (ACYP). ALDH2 activity protects neurons against neurotoxicity induced by toxic aldehydes during oxidative stress and plays a role in neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease. ACYP is involved in ion transport, cell differentiation, programmed cell death and cancer, and increased levels of ACYP have been revealed in fibroblasts from patients affected by Alzheimer's disease. In this study we demonstrated that non-cytotoxic doses of oxadiazon were able to inhibit neuronal striatal cell migration and FGF2- and BDNF-dependent differentiation towards neuronal phenotype, and to inhibit the expression and activity of ALDH2 and to increase the expression and activity of ACYP2. In addition, we have provided evidence that in human primary neuronal precursor striatal cells the inhibitory effects of oxadiazon on cell migration and differentiation towards neuronal phenotype were achieved through modulation of ACYP2. Taken together, our findings reveal for the first time that oxadiazon could exert neurotoxic effects by impairing differentiative capabilities of primary neuronal cells and indicate that ALDH2 and ACYP2 are relevant molecular targets for the neurotoxic effects of oxadiazon, suggesting a potential role of this herbicide in the onset of neurodegenerative diseases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The rise in prevalence of non-alcoholic fatty liver disease (NAFLD) constitutes an important public health concern worldwide. Including obesity, numerous risk factors of NAFLD such as benzo[a]pyrene (B[a]P) and ethanol have been identified as modifying the physicochemical properties of the plasma membrane in vitro thus causing membrane remodeling-changes in membrane fluidity and lipid-raft characteristics. In this study, the possible involvement of membrane remodeling in the in vivo progression of steatosis to a steatohepatitis-like state upon co-exposure to B[a]P and ethanol was tested in obese zebrafish larvae. Larvae bearing steatosis as the result of a high-fat diet were exposed to ethanol and/or B[a]P for seven days at low concentrations coherent with human exposure in order to elicit hepatotoxicity. In this condition, the toxicant co-exposure raised global membrane order with higher lipid-raft clustering in the plasma membrane of liver cells, as evaluated by staining with the fluoroprobe di-4-ANEPPDHQ. Involvement of this membrane's remodeling was finally explored by using the lipid-raft disruptor pravastatin that counteracted the effects of toxicant co-exposure both on membrane remodeling and toxicity. Overall, it can be concluded that B[a]P/ethanol co-exposure can induce in vivo hepatotoxicity via membrane remodeling which could be considered as a good target mechanism for developing combination therapy to deal with steatohepatitis., Competing Interests: The authors declare no conflict of interest.

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

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

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.

Several epidemiologic studies have shown an interactive effect of heavy smoking and heavy alcohol drinking on the development of hepatocellular carcinoma. It has also been recently described that chronic hepatocyte death can trigger excessive compensatory proliferation resulting later in the formation of tumors in mouse liver. As we previously demonstrated that both benzo[a]pyrene (B[a]P), an environmental agent found in cigarette smoke, and ethanol possess similar targets, especially oxidative stress, to trigger death of liver cells, we decided to study here the cellular and molecular mechanisms of the effects of B[a]P/ethanol coexposure on cell death. After an 18-h incubation with 100nM B[a]P, primary rat hepatocytes were supplemented with 50mM ethanol for 5 or 8h. B[a]P/ethanol coexposure led to a greater apoptotic cell death that could be linked to an increase in lipid peroxidation. Plasma membrane remodeling, as depicted by membrane fluidity elevation and physicochemical alterations in lipid rafts, appeared to play a key role, because both toxicants acted with specific complementary effects. Membrane remodeling was shown to induce an accumulation of lysosomes leading to an important increase in low-molecular-weight iron cellular content. Finally, ethanol metabolism, but not that of B[a]P, by providing reactive oxygen species, induced the ultimate toxic process. Indeed, in lysosomes, ethanol promoted the Fenton reaction, lipid peroxidation, and membrane permeabilization, thereby triggering cell death. To conclude, B[a]P exposure, by depleting hepatocyte membrane cholesterol content, would constitute a favorable ground for a later toxic insult such as ethanol intoxication. Membrane stabilization of both plasma membrane and lysosomes might be a potential target for further investigation considering cytoprotective strategies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Plasma membrane is an early target of polycyclic aromatic hydrocarbons (PAH). We previously showed that the PAH prototype, benzo[a]pyrene (B[a]P), triggers apoptosis via DNA damage-induced p53 activation (genotoxic pathway) and via remodeling of the membrane cholesterol-rich microdomains called lipid rafts, leading to changes in pH homeostasis (non-genotoxic pathway). As omega-3 (n-3) fatty acids can affect membrane composition and function or hamper in vivo PAH genotoxicity, we hypothesized that addition of physiologically relevant levels of polyunsaturated n-3 fatty acids (PUFAs) might interfere with B[a]P-induced toxicity. The effects of two major PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), were tested on B[a]P cytotoxicity in the liver epithelial cell line F258. Both PUFAs reduced B[a]P-induced apoptosis. Surprisingly, pre-treatment with DHA increased the formation of reactive B[a]P metabolites, resulting in higher levels of B[a]P-DNA adducts. EPA had no apparent effect on B[a]P metabolism or related DNA damage. EPA and DHA prevented B[a]P-induced apoptotic alkalinization by affecting Na(+)/H(+) exchanger 1 activity. Thus, the inhibitory effects of omega-3 fatty acids on B[a]P-induced apoptosis involve a non-genotoxic pathway associated with plasma membrane remodeling. Our results suggest that dietary omega-3 fatty acids may have marked effects on the biological consequences of PAH exposure., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Previously, we demonstrated that eicosapentaenoic acid enhanced ethanol-induced oxidative stress and cell death in primary rat hepatocytes via an increase in membrane fluidity and lipid raft clustering. In this context, another n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), was tested with a special emphasis on physical and chemical alteration of lipid rafts. Pretreatment of hepatocytes with DHA reduced significantly ethanol-induced oxidative stress and cell death. DHA protection could be related to an alteration of lipid rafts. Indeed, rafts exhibited a marked increase in membrane fluidity and packing defects leading to the exclusion of a raft protein marker, flotillin. Furthermore, DHA strongly inhibited disulfide bridge formation, even in control cells, thus suggesting a disruption of protein-protein interactions inside lipid rafts. This particular spatial organization of lipid rafts due to DHA subsequently prevented the ethanol-induced lipid raft clustering. Such a prevention was then responsible for the inhibition of phospholipase C-γ translocation into rafts, and consequently of both lysosome accumulation and elevation in cellular low-molecular-weight iron content, a prooxidant factor. In total, the present study suggests that DHA supplementation could represent a new preventive approach for patients with alcoholic liver disease based upon modulation of the membrane structures., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported to modulate lipid raft-dependent signaling, but not yet lipid raft-dependent oxidative stress. Previously, we have shown that ethanol-induced membrane remodeling, i.e., an increase in membrane fluidity and alterations in physical and biochemical properties of lipid rafts, participated in the development of oxidative stress. Thus, we decided to study n-3 PUFA effects in this context, by pretreating hepatocytes with eicosapentaenoic acid (EPA), a long-chain n-3 PUFA, before addition of ethanol. EPA was found to increase ethanol-induced oxidative stress through membrane remodeling. Addition of EPA resulted in a marked increase in lipid raft aggregation compared to ethanol alone. In addition, membrane fluidity of lipid rafts was markedly enhanced. Interestingly, EPA was found to preferentially incorporate into nonraft membrane regions, leading to raft cholesterol increase. Lipid raft aggregation by EPA enhanced phospholipase Cγ translocation into these microdomains. Finally, phospholipase Cγ was shown to participate in the potentiation of oxidative stress by promoting lysosome accumulation, a major source of low-molecular-weight iron. To conclude, the ability of EPA to modify lipid raft physical and chemical properties plays a key role in the enhancement, by this dietary n-3 PUFA, of ethanol-induced oxidative stress., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Poly(ADP-Ribose) polymerase (PARP) activity has been demonstrated fundamental in many cellular processes, including DNA repair, cell proliferation and differentiation. In particular, PARP activity has been recently found to affect proliferation, migration, and tube formation of human umbilical vein endothelial cells. In recent times, PARP inhibitors have entered in clinical trials to potentiate cancer treatments by preventing DNA repair, but little is known about the effects performed by different drug concentrations on neoangiogenesis, an essential step in tumor growth., Methods: Human umbilical vein endothelial cells were treated with 3 aminobenzamide (3ABA), a PARP inhibitor, and tested for several different cellular parameters., Results: Here we present in vitro evidence that a low concentration of 3ABA (50 μM), stimulates angiogenesis by decreasing fibrinolytic activity, carried out by urokinase-type plasminogen activator (uPA), and by enhancing matrix metalloprotease-2 (MMP-2) gelatinolytic activity, in fibroblast growth factor-2-stimulated endothelial cells. These unbalanced pathways modify in vitro angiogenic steps, inhibiting chemoinvasion and stimulating tubulogenic activity., Conclusions: Our results suggest that the proangiogenic effect of low concentrations of 3ABA alerts on the efficacy of PARP inhibitors to potentiate anticancer therapy. Moreover, they indicate that endothelial chemoinvasion and tubulogenesis depend on distinct proteolytic pathways.

The effect on angiogenesis of (-)-alpha-bisabolol [(-)-6-methyl-2-(4-methyl-3-cyclohexen-1-yl)-5-hepten-2-ol] (1), a widely distributed plant sesquiterpene alcohol, was investigated for the first time. Human endothelial cells treated with 1 were analyzed for their ability to differentiate and organize in microvessels and for their sensitivity to this compound in terms of cytotoxicity and cell growth inhibition. Within 24 h of the treatment with 5 microM 1, cells underwent massive death. Apoptosis induction was responsible for cytotoxicity triggered by 1 as revealed by the release of cytochrome c from the mitochondria, reduction of the Bcl-2/Bax ratio, and caspase 3 activation. At a lower, non-apoptotic concentration (0.25 microM), 1 showed a differentiating effect resulting in growth inhibition, invasiveness reduction, and tubule stabilization.

The capability of PARP activity inhibitors to prevent DNA damage recovery suggested the use of these drugs as chemo- and radio-sensitisers for cancer therapy. Our research, carried out on cultured human M14 melanoma cells, was aimed to examine if PJ-34, a potent PARP activity inhibitor of second generation, was per se able to affect the viability of these cancer cells without any DNA damaging agents. Using time-lapse videomicroscopy, we evidenced that 10 microM PJ-34 treatment induced severe mitotic defects leading to dramatic reduction of cell proliferation and to cell death. PJ-34 cytotoxic effect was further confirmed by analysis of cell viability and clonogenic assay. Absence of canonic apoptosis markers allowed us to exclude this kind of cell death. No single and/or double stranded DNA damage was evidenced. Immunofluorescence analysis showed an aberrant mitotic scenario in several cells and subsequent multinucleation suggesting an atypical way for cells to die: the mitotic catastrophe. The detection of aberrant accumulation of polymerised actin inside the nucleolus was noteworthy. Taken together, our results demonstrate that, targeting PARP activity by PJ-34, cancer cell survival is affected independently of DNA damage repair. Two findings are remarkable: (a) cisplatin concentration can be reduced by three quarters if it is followed by treatment with 10 microM PJ-34 for 24 h to obtain the same cytotoxic effect; (b) effects dependent on PJ-34 treatment are reversible. Our data suggest that, to reduce the harm done to non-tumour cells during chemotherapy with cisplatin, the latter could be coupled with PJ-34 treatment., ((c) 2009 Wiley-Liss, Inc.)

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

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

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.