Downregulation of ceramide synthase-6 during epithelial-to-mesenchymal transition reduces plasma membrane fluidity and cancer cell motility.

Epithelial-to-mesenchymal transition (EMT) promotes cell motility, which is important for the metastasis of malignant cells, and blocks CD95-mediated apoptotic signaling triggered by immune cells and chemotherapeutic regimens. CD95L, the cognate ligand of CD95, can be cleaved by metalloproteases and released as a soluble molecule (cl-CD95L). Unlike transmembrane CD95L, cl-CD95L does not induce apoptosis but triggers cell motility. Electron paramagnetic resonance was used to show that EMT and cl-CD95L treatment both led to augmentation of plasma membrane fluidity that was instrumental in inducing cell migration. Compaction of the plasma membrane is modulated, among other factors, by the ratio of certain lipids such as sphingolipids in the membrane. An integrative analysis of gene expression in NCI tumor cell lines revealed that expression of ceramide synthase-6 (CerS6) decreased during EMT. Furthermore, pharmacological and genetic approaches established that modulation of CerS6 expression/activity in cancer cells altered the level of C16-ceramide, which in turn influenced plasma membrane fluidity and cell motility. Therefore, this study identifies CerS6 as a novel EMT-regulated gene that has a pivotal role in the regulation of cell migration.

EPR study of antioxidant activity of the iron chelators pyoverdin and hydroxypyrid-4-one in iron-loaded hepatocyte culture: comparison with that of desferrioxamine.

Iron supplementation of hepatocyte culture induced the production of lipid-derived radicals as shown by spin-trapping with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN). The EPR signal corresponding to POBN/lipid-derived radicals (aN = 15.6 G aH = 2.6 G) was concentration dependent on iron (Fe-NTA) added to the culture medium (50, 100, 200 microM). It was also incubation time dependent (0 to 24 h). The EPR signal could be used as a marker for iron-induced lipid peroxidation. The antioxidant activity of two iron chelators, pyoverdin (Pa) and hydroxypyrid-4-one derivative (CP20) was compared with that of desferrioxamine (DFO) on iron-loaded hepatocyte culture. These compounds (100 microM) were tested either in pretreatment or simultaneously with Fe-NTA (100 microM). In each procedure, the EPR signal obtained from the cells supplemented with iron was substantially reduced in the presence of either DFO or CP20 but not with Pa. Moreover, the DFO and CP20 but not Pa showed protective effect on the leakage of the intracellular enzyme lactate dehydrogenase into the culture medium. The present study described a specific spin-trapping technique in conjunction with EPR spectroscopy that is able to demonstrate the cytoprotective effect of iron chelators, as shown by the elimination of lipid-derived radicals in iron-loaded hepatocyte culture.

Repair of iron-induced DNA oxidation by the flavonoid myricetin in primary rat hepatocyte cultures.

Oxidative DNA damage and its repair in primary rat hepatocyte cultures was investigated following 4 h of incubation with the toxic iron chelate, ferric nitrilotriacetate (Fe-NTA), in the presence or absence of the potent protective flavonoid myricetin (25-50-100 microM). Seven DNA base oxidation products were quantified in DNA extracts by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring mode. Concomitantly, DNA repair capacity of hepatocytes was estimated by the release of oxidized-base products into culture media, using the same GC-MS method. A genotoxic effect of Fe-NTA (100 microM) in hepatocytes was evidenced by a severe increase in DNA oxidation over basal levels, with accumulation in cellular DNA of five oxidation products derived from both purines and pyrimidines. This prooxidant effect of iron was also noted by an induction of lipid peroxidation, estimated by free malondialdehyde production. Addition of increasing concentrations of myricetin (25-50-100 microM) simultaneously with iron prevented both lipid peroxidation and accumulation of oxidation products in DNA. Moreover, as an activation of DNA repair pathways, myricetin stimulated the release of DNA oxidation bases into culture media, especially of purine-derived oxidation products. This removal of highly mutagenic oxidation products from DNA of hepatocytes might correspond to an activation of DNA excision-repair enzymes by myricetin. This was verified by RNA blot analysis of DNA polymerase beta gene expression which was induced by myricetin in a dose-dependent manner. This represented a novel and original mechanism of cytoprotection by myricetin against iron-induced genotoxicity via stimulation of DNA repair processes. Since iron-induced DNA damage and inefficient repair in hepatocytes could be related to genotoxicity and most probably to hepatocarcinogenesis, modulation of these processes in vitro by myricetin might be relevant in further prevention of liver cancer derived from iron overload pathologies.

Antioxidant and free radical scavenging activities of the iron chelators pyoverdin and hydroxypyrid-4-ones in iron-loaded hepatocyte cultures: comparison of their mechanism of protection with that of desferrioxamine.

The protective effect on iron-supplemented hepatocyte cultures of three iron chelators, pyoverdin Pa and hydroxypyrid-4-one derivatives CP20 and CP22, was compared to that of the widely known desferrioxamine B (Desferal:DFO), on the basis of two criteria: (a) their effectiveness in inhibiting free malondialdehyde (MDA) production as an index of iron-induced lipid peroxidation; and (b) their ability to reduce intracellular enzyme leakage. In view of these two markers of iron toxicity, the protective effect of these chelators was classified as follows: DFO > CP20 > or = CP22 > Pa. The mechanism of cellular protection was elucidated by investigating both the iron-chelating activity and the free radical scavenging property of these agents. As concerns the iron chelation, DFO and Pa exerted the same rank order as for cytoprotection (DFO > Pa). The free radical scavenging property toward hydroxyl radical .OH and peroxyl radical ROO. was investigated in a cell-free experimental model. The two siderophores, DFO and Pa, appeared to have a lower antiradical activity toward .OH than hydroxypyrid-4-one CP22. This .OH scavenging activity was classified as follows: CP22 >> Pa > DFO. Moreover, the chelators exhibited for the quenching of ROO. the same order of effectiveness as that observed for cellular protection: DFO > CP20 > or = CP22 > Pa. These data indicate that, in addition to the iron-chelating activity which represents the most important property for determining the protection capacity of these iron chelators, their free radical scavenging ability also must be taken into account. This direct demonstration of a strong association between the free radical scavenging activity and the protective effect of iron chelators further increases the prospects for the development and clinical applications of new oral chelating drugs.

Involvement of phenoxyl radical intermediates in lipid antioxidant action of myricetin in iron-treated rat hepatocyte culture.

Supplementation of rat hepatocyte cultures with the flavonoid myricetin (300 microM) led to the formation of phenoxyl radical intermediates, as detected in intact cells by electron paramagnetic resonance (EPR) spectroscopy. These radicals corresponded to one-electron oxidation products of myricetin. The level of phenoxyl radicals was significantly reduced when myricetin-treated hepatocyte cultures were also supplemented with iron (Fe-NTA 100 microM). This suggested that iron could accelerate the oxidation flux of myricetin. Moreover, myricetin was found to be able to inhibit lipid peroxidation induced by iron in hepatocyte culture. Free malondialdehyde (MDA) levels and the amount of radicals derived from oxidized lipids were greatly reduced when myricetin was added to iron-treated cultures. This showed that myricetin was a good inhibitor of lipid peroxidation in this model and that the intermediate generation of phenoxyl radicals might contribute to the antioxidant mechanism of myricetin.

Antioxidant and iron-chelating activities of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat hepatocyte cultures.

The cytoprotective effect of three flavonoids, catechin, quercetin and diosmetin, was investigated on iron-loaded hepatocyte cultures, considering two parameters: the prevention of iron-increased lipid peroxidation and the inhibition of intracellular enzyme release. These two criteria of cytoprotection allowed the calculation of mean inhibitory concentrations (IC50) which revealed that the effectiveness of these flavonoids could be classified as follows: catechin > quercetin > diosmetin. These IC50 values have been related to structural characteristics of the flavonoids tested. Moreover, the investigation of the capacity of these flavonoids to remove iron from iron-loaded hepatocytes revealed a good relationship between this iron-chelating ability and the cytoprotective effect. The cytoprotective activity of catechin, quercetin and diosmetin could thus be ascribed to their widely known antiradical property but also to their iron-chelating effectiveness. These findings increase further the prospects for the development and clinical application of these potent antioxidants.

Simultaneous measurements of conjugated dienes and free malondialdehyde, used as a micromethod for the evaluation of lipid peroxidation in rat hepatocyte cultures.

Membrane lipid peroxidation in rat hepatocyte cultures was induced by a 5-h incubation with either ethanol (50 mM) or the chelate iron-nitrilotriacetic acid (Fe-NTA) (100 microM). To test the oxidative stress, two indices were measured simultaneously on the same sample: extracellular free malondialdehyde (MDA) measured by HPLC with a size exclusion column, and conjugated dienes (CD) determined by second derivative spectroscopy. With ethanol, both CD and MDA gave nearly the same values of lipid peroxidation, about 135% of the control value. With Fe-NTA, both indices indicated a higher lipid peroxidation, but the MDA and CD values were different. Iron lipid peroxidation evaluated by free MDA and CD was, 290 and 230%, respectively, of the control. This discrepancy could be ascribed to an increased decomposition of hydroperoxides by iron. In addition, the ratio of cis,trans and trans,trans conjugated dienes, which reflects the cellular redox status, remained unchanged after 5 h of lipid peroxidation induced either by ethanol or iron.

Increase in cellular pool of low-molecular-weight iron during ethanol metabolism in rat hepatocyte cultures. Relationship with lipid peroxidation.

Ethanol-induced lipid peroxidation was studied in primary rat hepatocyte cultures supplemented with ethanol at the concentration of 50 mM. Lipid peroxidation was assessed by two indices: (1) conjugated dienes by second-derivative UV spectroscopy in lipid extract of hepatocytes (intracellular content), and (2) free malondialdehyde (MDA) by HPLC-UV detection and quantitation for the incubation medium (extracellular content). In cultures supplemented with ethanol, free MDA increased significantly in culture media, whereas no elevation of conjugated diene level was observed in the corresponding hepatocytes. The cellular pool of low-mol-wt (LMW) iron was also evaluated in the hepatocytes using an electron spin resonance procedure. An early increase of intracellular LMW iron (< or = 1 hr) was observed in ethanol-supplemented cultures; it was inhibited by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, whereas alpha-tocopherol, which prevented lipid peroxidation, did not inhibit the increase of LMW iron. Therefore, the LMW iron elevation was the result of ethanol metabolism and was not secondarily induced by lipid hydroperoxides. Thus, ethanol caused lipid peroxidation in rat hepatocytes as shown by the increase of free MDA, although no conjugated diene elevation was detected. During ethanol metabolism, an increase in cellular LMW iron was observed that could enhance conjugated diene degradation.

Alternariol induces abnormal nuclear morphology and cell cycle arrest in murine RAW 264.7 macrophages.

The mycotoxin alternariol (AOH), a frequent contaminant in fruit and cereal products, is known to induce DNA damage with subsequent cell cycle arrest. Here we elucidated the effects of AOH on stages of cell cycle progression using the RAW 264.7 macrophage model. AOH resulted in an accumulation of cells in the G2/M-phase (4N). Most cells exhibited a large G2 nucleus whereas numbers of true mitotic cells were reduced relative to control. Both cyclin B1 and p-cdc2 levels increased, while cyclin B1 remained in the cytoplasm; suggesting arrest in the G2/M transition point. Remarkably, after exposure to AOH for 24h, most of the cells exhibited abnormally shaped nuclei, as evidenced by partly divided nuclei, nuclear blebs, polyploidy and micronuclei (MN). AOH treatment also induced abnormal Aurora B bridges, suggesting that cytokinesis was interfered within cells undergoing karyokinesis. A minor part of the resultant G1 tetraploid (4N) cells re-entered the S-phase and progressed to 8N cells.

Effects of the observation method (direct v. from video) and of the presence of an observer on behavioural results in veal calves.

This study aimed at assessing the effect of the observation method (direct or from video) and the effect of the presence of an observer on the behavioural results in veal calves kept on a commercial farm. To evaluate the effect of the observation method, 20 pens (four to five calves per pen) were observed by an observer for 60 min (two observation sessions of 30 min) and video-recorded at the same time. To evaluate the effect of the presence of the observer in front of the pen, 24 pens were video-recorded on 4 consecutive days and an observer was present in front of each pen for 60 min (two observation sessions of 30 min) on the third day. Behaviour was recorded using instantaneous scan sampling. For the study of the observer's effect, the analysis was limited to the posture, abnormal oral behaviour and manipulation of substrates. The two observation methods gave similar results for the time spent standing, but different results for all other behaviours. The presence of an observer did not affect the behaviour of calves at day level; however, their behaviour was affected when the observer was actually present in front of the pens. A higher percentage of calves were standing and were manipulating substrate in the presence of the observer, but there was no effect on abnormal oral behaviour. In conclusion, direct observations are a more suitable observation method than observations from video recordings for detailed behaviours in veal calves. The presence of an observer has a short-term effect on certain behaviours of calves that will have to be taken into consideration when monitoring these behaviours.

TRAIL induces necroptosis involving RIPK1/RIPK3-dependent PARP-1 activation.

Although TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand) is a well-known apoptosis inducer, we have previously demonstrated that acidic extracellular pH (pHe) switches TRAIL-induced apoptosis to regulated necrosis (or necroptosis) in human HT29 colon and HepG2 liver cancer cells. Here, we investigated the role of RIPK1 (receptor interacting protein kinase 1), RIPK3 and PARP-1 (poly (ADP-ribose) polymerase-1) in TRAIL-induced necroptosis in vitro and in concanavalin A (Con A)-induced murine hepatitis. Pretreatment of HT29 or HepG2 with pharmacological inhibitors of RIPK1 or PARP-1 (Nec-1 or PJ-34, respectively), or transient transfection with siRNAs against RIPK1 or RIPK3, inhibited both TRAIL-induced necroptosis and PARP-1-dependent intracellular ATP depletion demonstrating that RIPK1 and RIPK3 were involved upstream of PARP-1 activation and ATP depletion. In the mouse model of Con A-induced hepatitis, where death of mouse hepatocytes is dependent on TRAIL and NKT (Natural Killer T) cells, PARP-1 activity was positively correlated with liver injury and hepatitis was prevented both by Nec-1 or PJ-34. These data provide new insights into TRAIL-induced necroptosis with PARP-1 being active effector downstream of RIPK1/RIPK3 initiators and suggest that pharmacological inhibitors of RIPKs and PARP-1 could be new treatment options for immune-mediated hepatitis.

[Alcohol and oxidative stress]. / Alcool et stress oxydatif.

There is accumulating evidence pointing oxidative stress as a mechanism of ethanol toxicity. Oxidative stress takes place when the balance between the antioxidant defenses and the generation of reactive oxygen species (ROS) is tipped in favour of the latter. Ethanol metabolism is directly involved in the production of ROS, but ethanol also participated to the formation of an environment favourable to oxidative stress such as hypoxia, endotoxemia and cytokine release. Following ethanol intoxication, balance between prooxidants and antioxidants is disturbed to such an extent that it results in an oxidative damage of biomolecules. The ability of ethanol to induce peroxidation of membrane lipids is widely reviewed in literature. More recently it has also been described that ethanol can oxidize proteins and ADN. In this review, is also discussed the impairment of cellular function resulting from this situation of oxidative stress.

Oxidative stress induced by ethanol in rat hepatocyte cultures.

Many controversies still exist with regard to the relationship between alcoholic intoxication and the occurrence of an oxidative stress. To attempt to resolve this question, first we investigated the induction by acute ethanol intoxication of lipid peroxidation in primary rat hepatocyte cultures using simultaneously two indices for each sample. When considering conjugated-diene indice, any lipid peroxidation elevation could be observed, whereas a net increase of extracellular free malondialdehyde was noted at 5 hours of incubation. These results led us to estimate the intracellular pool of low molecular weight iron which is known to be the iron species catalytically active in hydroperoxide degradation. An early enhancement of +20-30% of cellular low molecular weight iron was observed. Thus the discrepancy between conjugated dienes and malondialdehyde could be ascribed to an increase of hydroperoxide degradation into malondialdehyde by the transient cellular pool of low molecular weight iron. Lipid peroxidation and low molecular weight iron augmentation were linked to ethanol metabolism, since both were suppressed by the addition of 4-methylpyrazole, an alcohol dehydrogenase inhibitor. Superoxide dismutase activity was increased in the early incubation time (1 hour) and then markedly reduced. We conclude that ethanol metabolism can induce a lipid peroxidation accompanied by an elevation of intracellular pool of low molecular weight iron and a decrease of superoxide dismutase activity.

Ultraviolet and infrared spectroscopy for microdetermination of oxidized and unoxidized fatty acyl esters in cells.

New methods based on ultraviolet and infrared spectroscopy were developed to quantify oxidized and unoxidized fatty acyl esters (FAE) in cells. For this study, rat hepatocyte cultures (2.5 x 10(6) cells) were submitted to an oxidative stress by a 5-h incubation with iron(III) chelated with nitrilotriacetic acid (100 microM). Control hepatocytes were incubated under the same conditions except in the absence of iron. After cell lipid extraction, oxidized FAE were evaluated by the second derivative of the conjugated-diene (CD) spectrum, which exhibited minima at 233 and 242 nm ascribed to trans,trans (t,t) and cis,trans (c,t) CD isomers, respectively. These minima were quantified in arbitrary units as d2 A/d lambda 2; hydroperoxide concentration was determined using a linear regression curve obtained from autoxidized linoleic acid micelles. Total (oxidized and unoxidized) FAE were measured by Fourier transform infrared spectroscopy using the absorption band at 1740 cm-1. A highly significant correlation coefficient (r = 0.992) was found for the standard curve performed with glycerol trioleate expressed as nanomoles fatty acid equivalents. The extent of lipid oxidation could be estimated by the sum of minima at 233 and 242 nm which allowed the calculation of hydroperoxide concentrations. The amount of oxidized FAE was related to the amount of total FAE in the same sample. The ratio of minima at 242 nm (c,t isomers) and 233 nm (t,t isomers) could provide an evaluation of cell antioxidant capacity. A decrease of this ratio would indicate a large depletion of radical termination antioxidants.

Activated macrophages increase the susceptibility of rat hepatocytes to ethanol-induced oxidative stress: conflicting effects of nitric oxide.

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 gamma (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 N(g)-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.

EPR determination of low molecular weight iron content applied to whole rat hepatocytes.

Electron paramagnetic resonance (EPR) has been described as suitable for the evaluation of low molecular weight (LMW) iron in liver homogenates after chelation by desferrioxamine. LMW iron is a highly toxic iron species incriminated in free radical production. The first aim of the study was to evaluate the conditions of EPR application for LMW iron content determination in whole rat hepatocytes. For this purpose, LMW iron was simultaneously quantified by EPR and by atomic absorption spectrometry, EPR determination of LMW iron needed a preincubation of hepatocyte cultures with the iron chelator for at least on hr. Deferiprone as LMW iron chelator was revealed to be more suited than desferrioxamine. Secondly, we showed the applicability of this methods for evaluating the prooxidant status during an oxidative stress. As an example, oxidative stress induced by ethanol in hepatocytes was studied during inflammatory circumstances, well-known to lead to nitric oxide production. In hepatocyte cultures supplemented with ethanol, an evaluation of LMW iron content was observed in cells. But when nitric oxide donors or a supplementation constituted of lipopolysaccharide and gamma-interferon, able to induce nitric oxide synthase, were added, LMW iron content decreased. Thus EPR determination of LMW iron content in whole hepatocytes could give some insight about the mechanism of induction or inhibition of a oxidative stress.