Critical role of exposure time to endogenous oxidative stress in hepatocyte apoptosis

We have previously shown that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in sustained oxidative stress, followed by apoptosis. To examine the effects of duration of oxidative stress, ATZ and MS were removed from culture medium at 3, 6 and 9 h after treatment with both inhibitors. Oxidative stress was induced for periods of time by ATZ and MS exposures in primary hepatocytes. Treatment with ATZ and MS reduced catalase (CAT) and glutathione peroxidase (GPx) activities, and decreased CAT and GPx activities recovered to normal values upon withdrawal. Although oxidative stress of up to 6 h duration did not cause cell death, sustained oxidative stress (over 9 h) induced apoptosis. The increase in the glutathione disulfide/reduced glutathione ratio under oxidative stress up to 6 h was transient and reversible, while that due to sustained oxidative stress was irreversible. These results suggest that irreversible redox shifts resulting from sustained oxidative stress play a critical role in the induction of hepatocyte apoptosis in this experimental system.     

Critical role of exposure time to endogenous oxidative stress in hepatocyte apoptosis

Abstract

We have previously shown that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in sustained oxidative stress, followed by apoptosis. To examine the effects of duration of oxidative stress, ATZ and MS were removed from culture medium at 3, 6 and 9 h after treatment with both inhibitors. Oxidative stress was induced for periods of time by ATZ and MS exposures in primary hepatocytes. Treatment with ATZ and MS reduced catalase (CAT) and glutathione peroxidase (GPx) activities, and decreased CAT and GPx activities recovered to normal values upon withdrawal. Although oxidative stress of up to 6 h duration did not cause cell death, sustained oxidative stress (over 9 h) induced apoptosis. The increase in the glutathione disulfide/reduced glutathione ratio under oxidative stress up to 6 h was transient and reversible, while that due to sustained oxidative stress was irreversible. These results suggest that irreversible redox shifts resulting from sustained oxidative stress play a critical role in the induction of hepatocyte apoptosis in this experimental system.     

Direct contribution of inducible nitric oxide synthase expression to apoptosis induction in primary smooth chorion trophoblast cells of human fetal membrane tissues

We have previously demonstrated that apoptosis induction is observed only in smooth chorion laeve trophoblast cells, and not in amnion epithelial cells of human fetal membrane tissues prepared at the term. Apoptosis induction was suppressed by the presence of an inhibitor specific for inducible nitric oxide synthase (iNOS), suggesting that intracellular oxidative stress plays a critical role in this process. In this study, we transfected the iNOS gene into primary cultured chorion and amnion cells to examine the direct contribution of iNOS gene expression to the apoptosis induction in these cells. We identified a significant increase in the levels of iNOS protein expression and nitrite accumulation in both chorion and amnion cells after the iNOS gene transfection. However, the induction of apoptosis was observed in an approximately 70% of chorion cells transfected with iNOS gene. Transfection of the iNOS gene into chorion cells resulted in the activation of p38 mitogen-activated protein kinase (MAPK) and downregulation of hemeoxygenase-1 protein expression, whereas no such events were observed in the transfected amnion cells. These results suggest that apoptosis induced in the chorion trophoblast cells by the iNOS gene expression is closely linked to a physiological consequence, such as the rupture of fetal membranes.     

Primary hepatocyte apoptosis is unlikely to relate to caspase-3 activity under sustained endogenous oxidative stress

We previously showed that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in endogenous oxidative stress and apoptosis. For the present study, we determined whether this apoptosis involved activation of caspase-3, which is known to execute apoptosis in many cell types. ATZ and MS increased levels of reactive oxygen species (ROS) from 3-9 h, just before the onset of chromatin condensation (apoptosis) and decreases in protein thiols. Pretreatment with either SKF, a cytochrome P450 inhibitor, or L-ascorbic acid, an antioxidant, completely suppressed the increase in ROS levels and apoptosis, suggesting that the sustained ROS increases may cause the apoptosis. SKF also abolished the decrease in protein thiol content, further supporting the contribution of the P450 system to increased ROS levels. DEVD-CHO, a caspase-3 inhibitor, even at 1 mM had no effect on apoptosis. Caspase-3 activity remained unchanged and pro-caspase-3 processing was not detected during 18 h incubation with ATZ and MS. Moreover, the amount of unoxidized pro-caspase-3 decreased even below the level of untreated hepatocytes. These findings suggest that the sustained oxidative stress is a major cause for the hepatocyte apoptosis, which occurs independently of the caspase-3 related pathway.     

The protective effect of docosahexaenoic acid on the bilirubin neurotoxicity

Usually, all newborns demonstrate high serum unconjugated bilirubin (UCB) level. UCB may induce adverse effects in the central nervous system. We aimed to evaluate the cytotoxic effects of UCB and the protective effects of docosahexaenoic acid (DHA) on astrocyte cell cultures. The viability of astrocyte cells decreased after UCB treatment in a dose-dependent manner. Pre-incubation of DHA prevents the cells from UCB-mediated neurotoxicity. Our results shown that UCB leads to inhibition of antioxidant enzymes superoxide dismutase (SOD), catalase and GPx activity and induction of apoptosis. But only 4-h pretreatment of DHA can suppress of UCB-mediated inhibition of antioxidant enzymes SOD, catalase and GPx activity and induction of apoptosis in astrocytes. Our results strongly indicated that DHA has a protective effect on UCB-mediated neurotoxicity through inhibition apoptosis and antioxidant enzymes activity of SOD, CAT and GPx in rat primer astrocyte cell line     

Glutathione peroxidase-1 protects from CD95-induced apoptosis

Through the induction of apoptosis, CD95 plays a crucial role in the immune response and the elimination of cancer cells. Ligation of CD95 receptor activates a complex signaling network that appears to implicate the generation of reactive oxygen species (ROS). This study investigated the place of ROS production in CD95-mediated apoptosis and the role of the antioxidant enzyme glutathione peroxidase-1 (GPx1). Anti-CD95 antibodies triggered an early generation of ROS in human breast cancer T47D cells that was blocked by overexpression of GPx1 and inhibition of initiator caspase activation. Enforced expression of GPx1 also resulted in inhibition of CD95-induced effector caspase activation, DNA fragmentation, and apoptotic cell death. Resistance to CD95-mediated apoptosis was not due to an increased expression of anti-apoptotic molecules and could be reversed by glutathione-depleting agents. In addition, whereas the anti-apoptotic protein Bcl-xL prevented CD95-induced apoptosis in MCF-7 cells, it did not inhibit the early ROS production. Moreover, Bcl-xL but not GPx1 overexpression could suppress the staurosporine-induced late generation of ROS and subsequent cell death. Altogether, these findings suggest that GPx1 functions upstream of the mitochondrial events to inhibit the early ROS production and apoptosis induced by CD95 ligation. Finally, transgenic mice overexpressing GPx1 were partially protected from the lethal effect of anti-CD95, underlying the importance of peroxide formation (and GPx1) in CD95-triggered apoptosis.     

Changes in oxidative balance in rat pericytes exposed to diabetic conditions

Recent data indicate that the oxidative stress plays an important role in the pathogenesis of diabetes and its complications such as retinopathy, nephropathy and accelerated atherosclerosis. In diabetic retinopathy, it was demonstrated a selective loss of pericytes accompanied by capillary basement membrane thickening, increased permeability and neovascularization. This study was designed to investigate the role of diabetic conditions such as high glucose, AGE-Lysine, and angiotensin II in the modulation of antioxidant enzymes activities, glutathione level and reactive oxygen species (ROS) production in pericytes. The activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total glutathione (GSH) was measured spectrophotometrically. The production of ROS was detected by spectrofluorimetry and fluorescence microscopy after loading the cells with 2'-7' dichlorofluoresceine diacetate; as positive control H2O2 was used. Intracellular calcium was determined using Fura 2 AM assay. The results showed that the cells cultured in high glucose alone, do not exhibit major changes in the antioxidant enzyme activities. The presence of AGE-Lys or Ang II induced the increase of SOD activity. Their combination decreased significantly GPx activity and GSH level. A three times increase in ROS production and a significant impairment of intracellular calcium homeostasis was detected in cells cultured in the presence of the three pro-diabetic agents used. In conclusion, our data indicate that diabetic conditions induce in pericytes: (i) an increase of ROS and SOD activity, (ii) a decrease in GPx activity and GSH level, (iii) a major perturbation of the intracellular calcium homeostasis. The data may explain the structural and functional abnormalities of pericytes characteristic for diabetic retinopathy.     

Antioxidant proteins and reactive oxygen species are decreased in a murine epidermal side population with stem cell-like characteristics

Reactive oxygen species (ROS) and antioxidants are essential to maintain a redox balance within tissues and cells. Intracellular ROS regulate key cellular functions such as proliferation, differentiation and apoptosis through cellular signaling, and response to injury. The redox environment is particularly important for stem/progenitor cells, as their self-renewal and differentiation has been shown to be redox sensitive. However, not much is known about ROS and antioxidant protein function in freshly isolated keratinocytes, notably the different keratinocyte subpopulations. Immunostaining of neonatal cutaneous sections revealed that antioxidant enzymes [catalase, SOD2, gluthatione peroxidase-1 (GPx)] and ROS are localized predominantly to the epidermis. We isolated keratinocyte subpopulations and found lower levels of SOD2, catalase and GPx, as well as decreased SOD and catalase activity in an epidermal side population with stem cell-like characteristics (EpSPs) compared to more differentiated (Non-SP) keratinocytes. EpSPs also exhibited less mitochondrial area, fewer peroxisomes and produced lower levels of ROS than Non-SPs. Finally, EpSPs were more resistant to UV radiation than their progeny. Together, our data indicate ROS and antioxidant levels are decreased in stem-like EpSPs.     

Induction of antioxidant enzymes by FAK in a human leukemic cell line, HL-60

We have established several focal adhesion kinase (FAK) cDNA-transfected HL-60 (HL-60/FAK) cells which were highly resistant to oxidative stress-induced apoptosis. To identify target genes that are involved in HL-60/FAK cells, we performed cDNA microarray screening using apoptosis-chip. There, we identified the decrease of glutathione peroxidase (GPx). This result prompted us to investigate the changes of antioxidant enzymes. Here, we demonstrate that lipid peroxidation was suppressed after treatment with hydrogen peroxide in HL-60/FAK cells but not vector-transfected HL-60 (HL-60/Vect) cells. Furthermore, we demonstrate that HL-60/FAK cells have higher basal reactive oxygen species (ROS) levels than the parental HL-60 or HL-60/Vect cells, while ROS accumulation by hydrogen peroxide treatment was almost the same in these cells. Basal activity and mRNA expression of antioxidant enzymes, particularly of GSH reductase (GRe), phospholipid hydroperoxide glutathione peroxidase (PHGPx) were markedly elevated in HL-60/FAK cells. In contrast, GPx and catalase levels were decreased in HL-60/FAK cells. Further, a Src family kinases inhibitor, PP2, suppressed GRe and PHGPx mRNA by inactivation of FAK and c-Src in HL-60/FAK cells. These results suggest that FAK upregulates antioxidant enzymes and suppresses lipid peroxidation, resulting in the anti-apoptotic state for oxidative stress.     

Catalase characterization and implication in bleaching of a symbiotic sea anemone

Symbiotic cnidarians are marine invertebrates harboring photosynthesizing microalgae (named zooxanthellae), which produce great amounts of oxygen and free radicals upon illumination. Studying antioxidative balance is then crucial to understanding how symbiotic cnidarians cope with ROS production. In particular, it is suspected that oxidative stress triggers cnidarian bleaching, i.e., the expulsion of zooxanthellae from the animal host, responsible for symbiotic cnidarian mass mortality worldwide. This study therefore investigates catalase antioxidant enzymes and their role in bleaching of the temperate symbiotic sea anemone Anemonia viridis. Using specific separation of animal tissues (ectoderm and endoderm) from the symbionts (zooxanthellae), spectrophotometric assays and native PAGE revealed both tissue-specific and activity pattern distribution of two catalase electrophoretypes, E1 and E2. E1, expressed in all three tissues, presents high sensitivity to the catalase inhibitor aminotriazole (ATZ) and elevated temperatures. The ectodermal E1 form is responsible for 67% of total catalase activity. The E2 form, expressed only within zooxanthellae and their host endodermal cells, displays low sensitivity to ATZ and relative thermostability. We further cloned an ectodermal catalase, which shares 68% identity with mammalian monofunctional catalases. Last, 6 days of exposure of whole sea anemones to ATZ (0.5 mM) led to effective catalase inhibition and initiated symbiont expulsion. This demonstrates the crucial role of this enzyme in cnidarian bleaching, a phenomenon responsible for worldwide climate-change-induced mass mortalities, with catastrophic consequences for marine biodiversity.     

A comparative proteomic analysis for capsaicin-induced apoptosis between human hepatocarcinoma (HepG2) and human neuroblastoma (SK-N-SH) cells

The endogenous ROS levels were increased during HepG2 apoptosis, whereas they were decreased during SK-N-SH apoptosis in response to capsaicin treatments. We used 2-DE-based proteomics to analyze the altered protein levels in both cells, with special attention on oxidative stress proteins before and after capsaicin treatments. The 2-DE analysis demonstrated that 23 proteins were increased and 26 proteins were decreased significantly (fold change>1.4) in capsaicin-treated apoptotic HepG2 and SK-N-SH cells, respectively. The distinct effect of capsaicin-induced apoptosis on the expression pattern of HepG2 proteins includes the downregulation of some antioxidant enzymes including aldose reductase (AR), catalase, enolase 1, peroxiredoxin 1, but upregulation of peroxiredoxin 6, cytochrome c oxidase, and SOD2. In contrast, most antioxidant enzymes were increased in SK-N-SH cells in response to capsaicin, where catalase might play a pivotal role in maintenance of low ROS levels in the course of apoptosis. The global gene expression for oxidative stress and antioxidant defense genes revealed that 84 gene expressions were not significantly different in HepG2 cells between control and capsaicin-treated cells. In contrast, a number of oxidative genes were downregulated in SK-N-SH cells, supporting the evidence of low ROS environment in apoptotic SK-N-SH cells after capsaicin treatment. It was concluded that the different relationship between endogenous ROS levels and apoptosis of two cancer cells presumably resulted from complicated expression patterns of many oxidative stress and antioxidant genes, rather than the individual role of some classical antioxidant enzymes such as SOD and catalase.     

Dietary Antioxidant,Quercetin, Protects Sertoli‐Germ Cell Coculture from Atrazine‐Induced Oxidative Damage

Quercetin (QT), a dietary‐derived flavonoid, is ubiquitous in fruits and vegetables and plays an important role in human health by virtue of its antioxidant function. The present study was designed to examine the effects of QT on oxidative damage that was induced by the herbicide, atrazine (ATZ), in mixed cultures of Sertoli‐germ cells. Results showed that treatment with QT increased cell viability and decreased catalase activity, malondialdehyde, and reactive oxygen species (ROS) levels. QT treatment also increased the mRNA expression of glutathione peroxidase (GSH‐Px), glutathione reductase (GR), glutathione‐S‐transferase, and superoxide dismutase‐1 and could not reversed to the control levels ATZ‐induced steady‐state mRNA levels of these antioxidant genes as well as the level of glutathione and activities of GSH‐Px and GR. QT has protective effect against ATZ‐induced oxidative stress through a reduction in ROS levels and lipid peroxidation. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:477‐485, 2012; View this article online at wileyonlinelibrary.com . DOI 10:1002/jbt.21449     

Mitochondrial catalase and oxidative injury

Mitochondria dysfunction induced by reactive oxygen species (ROS) is related to many human diseases and aging. In physiological conditions, the mitochondrial respiratory chain is the major source of ROS. ROS could be reduced by intracellular antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase as well as some antioxidant molecules like glutathione and vitamin E. However, in pathological conditions, these antioxidants are often unable to deal with the large amount of ROS produced. This inefficiency of antioxidants is even more serious in mitochondria, because mitochondria in most cells lack catalase. Therefore, the excessive production of hydrogen peroxide in mitochondria will damage lipid, proteins and mDNA, which can then cause cells to die of necrosis or apoptosis. In order to study the important role of mitochondrial catalase in protecting cells from oxidative injury, a HepG2 cell line overexpressing catalase in mitochondria was developed by stable transfection of a plasmid containing catalase cDNA linked with a mitochondria leader sequence which would encode a signal peptide to lead catalase into the mitochondria. Mitochondria catalase was shown to protect cells from oxidative injury induced by hydrogen peroxide and antimycin A. However, it increased the sensitivity of cells to tumor necrosis factor-alpha-induced apoptosis by changing the redox-oxidative status in the mitochondria. Therefore, the antioxidative effectiveness of catalase when expressed in the mitochondrial compartment is dependent upon the oxidant and the locus of ROS production.     

Fas-stimulated generation of reactive oxygen species or exogenous oxidative stress sensitize cells to Fas-mediated apoptosis

Inhibition of Fas-mediated apoptosis in B cell lymphomas by thiol antioxidants (glutathione and N-acetylcysteine) supported previous studies, suggesting that Fas-stimulated ROS generation may play a role in Fas-mediated apoptosis. Thus, the goal of the current study was to determine if Fas stimulation could induce ROS generation and what role, if any, it played in apoptosis. Fas crosslinking induced rapid generation of ROS (within 15 min) well before the appearance of characteristic apoptotic changes. Overexpression of catalase or superoxide dismutase suggested that Fas induced production of both superoxide anion and hydrogen peroxide. ROS generation was only observed, however, in cells that were sensitive to apoptosis and not in B cells inherently resistant to anti-Fas or in those in which resistance was induced by B cell receptor crosslinking. The exogenous addition of 250 microM hydrogen peroxide could reverse the resistant phenotype and sensitize cells to Fas-induced apoptosis. In Fas-sensitive cells, depletion of endogenous antioxidant defenses with buthionine sulfoximine increased the sensitivity to Fas-induced apoptosis, while overexpression of antioxidant enzymes and antiapoptotic proteins suggested a role for Fas-induced production of hydrogen peroxide in apoptosis. Further analysis suggested a redox-sensitive step early in Fas signaling at the level of initiator caspase (caspase-8) activation. Thus, the data suggest that the level of oxidative stress, either from exogenous sources or generated endogenously upon receptor stimulation, regulates the sensitivity to Fas-mediated apoptosis.     

Reactive oxygen species are key mediators of the nitric oxide apoptotic pathway in anterior pituitary cells.

We previously showed that long-term exposure of anterior pituitary cells to nitric oxide (NO) induces apoptosis. The intracellular signals underlying this effect remained unclear. In this study, we searched for possible mechanisms involved in the early stages of the NO apoptotic cascade. Caspase 3 was activated by NO with no apparent disruption of mitochondrial membrane potential. NO caused a rapid increase of reactive oxygen species (ROS), and this increase seems to be dependent of mitochondrial electron transport chain. The antioxidant N-acetyl-cysteine avoided ROS increase, prevented the NO-induced caspase 3 activation, and reduced the NO apoptotic effect. Catalase was inactivated by NO, while glutathione peroxidase (GPx) activity and reduced glutathione (GSH) were not modified at first, but increased at later times of NO exposure. The increase of GSH level is important for the scavenging of the NO-induced ROS overproduction. Our results indicate that ROS have an essential role as a trigger of the NO apoptotic cascade in anterior pituitary cells. The permanent inhibition of catalase may strengthen the oxidative damage induced by NO. GPx activity and GSH level augment in response to the oxidative damage, though this increase seems not to be enough to rescue the cells from the NO effect.     

Involvement of endonuclease G in nucleosomal DNA fragmentation under sustained endogenous oxidative stress

We have previously shown that inhibition of catalase and glutathione peroxidase activities by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), respectively, in rat primary hepatocytes caused sustained endogenous oxidative stress and apoptotic cell death without caspase-3 activation. In this study, we investigated the mechanism of this apoptotic cell death in terms of nucleosomal DNA fragmentation. Treatment with ATZ+MS time-dependently increased the number of deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive nuclei from 12 h, resulting in clear DNA laddering at 24 h. The deoxyribonuclease (DNase) inhibitor, aurintricarboxylic acid (ATA), completely inhibited nucleosomal DNA fragmentation but the pan-caspase inhibitor, z-VAD-fmk was without effects; furthermore, the cleavage of inhibitor of caspase-activated DNase was not detected, indicating the involvement of DNase(s) other than caspase-activated DNase. Considering that endonuclease G (EndoG) reportedly acts in a caspase-independent manner, we cloned rat EndoG cDNA for the first time. Recombinant EndoG alone digested plasmid DNA and induced nucleosomal DNA fragmentation in isolated hepatocyte nuclei. Recombinant EndoG activity was inhibited by ATA but not by hydrogen peroxide, even at 10 mm. ATZ+MS stimulation elicited decreases in mitochondrial membrane potential and EndoG translocation from mitochondria to nuclei. By applying RNA interference, the mRNA levels of EndoG were almost completely suppressed and the amount of EndoG protein was decreased to approximately half the level of untreated cells. Under these conditions, decreases in TUNEL-positive nuclei were significantly suppressed. These results indicate that EndoG is responsible, at least in part, for nucleosomal DNA fragmentation under endogenous oxidative stress conditions induced by ATZ+MS.     

Receptor-interacting Protein 1 Increases Chemoresistance by Maintaining Inhibitor of Apoptosis Protein Levels and Reducing Reactive Oxygen Species through a microRNA-146a-mediated Catalase Pathway

Although receptor-interacting protein 1 (RIP1) is well known as a key mediator in cell survival and death signaling, whether RIP1 directly contributes to chemotherapy response in cancer has not been determined. In this report, we found that, in human lung cancer cells, knockdown of RIP1 substantially increased cytotoxicity induced by the frontline anticancer therapeutic drug cisplatin, which has been associated with robust cellular reactive oxygen species (ROS) accumulation and enhanced apoptosis. Scavenging ROS dramatically protected RIP1 knockdown cells against cisplatin-induced cytotoxicity. Furthermore, we found that, in RIP1 knockdown cells, the expression of the hydrogen peroxide-reducing enzyme catalase was dramatically reduced, which was associated with increased miR-146a expression. Inhibition of microRNA-146a restored catalase expression, suppressed ROS induction, and protected against cytotoxicity in cisplatin-treated RIP1 knockdown cells, suggesting that RIP1 maintains catalase expression to restrain ROS levels in therapy response in cancer cells. Additionally, cisplatin significantly triggered the proteasomal degradation of cellular inhibitor of apoptosis protein 1 and 2 (c-IAP1 and c-IAP2), and X-linked inhibitor of apoptosis (XIAP) in a ROS-dependent manner, and in RIP1 knockdown cells, ectopic expression of c-IAP2 attenuated cisplatin-induced cytotoxicity. Thus, our results establish a chemoresistant role for RIP1 that maintains inhibitor of apoptosis protein (IAP) expression by release of microRNA-146a-mediated catalase suppression, where intervention within this pathway may be exploited for chemosensitization.     

The down-regulation of glutathione peroxidase causes bovine luteal cell apoptosis during structural luteolysis

Prostaglandin (PG) F(2)a is known to initiate luteal cell apoptosis in the bovine corpus luteum (CL) via its specific receptor (FP) on the luteal membrane by inducing intracellular Ca(2+) mobilization and the activation of PKC. In order to identify the signaling components involved in cell apoptosis, mRNA levels and activities of antioxidative enzymes were analyzed using bovine CL at different stages of the estrous cycle. Northern blot analysis revealed that the levels of two isozymes of superoxide dismutase (SOD), the Mn and Cu/Zn types, and catalase are highly enriched in the middle estrous phase, whereas glutathione peroxidase (GPx) levels gradually decrease as the estrous cycle progresses. The incubation of bovine luteal cells with H(2)O(2) and mercaptosuccinate (MS), a specific inhibitor of GPx, resulted in an increase in chromatin DNA condensation and apoptotic DNA fragmentation. Analyses of the enzymatic activities of GPx and catalase support the RNA data, indicating that H(2)O(2) produced due to the lack of GPx is a potent inducer of luteal cell apoptosis.     

Taurine alleviates ochratoxin A-induced pyroptosis in PK-15 cells by inhibiting oxidative stress

Ochratoxin A (OTA) is one of the most harmful mycotoxins, which can cause multiple toxicological effects, especially nephrotoxicity in animals and humans. Taurine is an essential amino acid with various biological functions such as anti-inflammatory and anti-oxidation. However, the protective effect of taurine on OTA-induced nephrotoxicity and pyroptosis had not been reported. Our results showed that OTA exposure induced cytotoxicity and oxidative stress in PK-15 cells, including reactive oxygen species (ROS) accumulation, increased mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), and decreased mRNA levels of catalase (CAT), glutathione peroxidase 1 (GPx1), and glutathione peroxidase 4 (GPx4). In addition, OTA treatment induced pyroptosis by increasing the expressions of pyroptosis-related proteins NLRP3, GSDMD, Caspase-1 P20, ASC, Pro-caspase-1, and IL-1β. Meanwhile, taurine could alleviate OTA-induced pyroptosis and cytotoxicity, as well as reduce ROS level, COX-2, and iNOS mRNA levels, and increase the mRNA levels of the antioxidant enzyme in PK-15 cells. Taken together, taurine alleviated OTA-induced pyroptosis in PK-15 cells by inhibiting ROS generation and altering the activity of antioxidant enzymes, thereby attenuating its nephrotoxicity.     

Melatonin prevents oxidative stress and hepatocyte cell death induced by experimental cholestasis

The induction of oxidative stress precedes liver injury during experimental obstructive jaundice (OJ). In this sense, different evidences suggest that melatonin (MEL), as antioxidant, may be useful in the protection against apoptosis and necrosis during experimental cholestasis. In addition, we will also assess if MEL-dependent protection is related to a recovery of antioxidant status disturbances induced by OJ. Cholestasis was achieved by double ligature and sectioning of the principal bile duct. MEL was injected intraperitoneally (500 microg/kg/day). Lipid peroxidation was evaluated by the measurement of malondialdehyde (MDA) content in liver. Different parameters related to antioxidant status, such as reduced glutathione (GSH), glutathione peroxidase (GPx), catalase and superoxide dismutase (SOD) were determined in liver. Liver injury was assessed by alanine amino-transferase (ALT) in serum, histological examination, DNA fragmentation and TUNEL assay. The activation of perisinusoidal stellate cells was evaluated by immunohistochemical measurement of alpha-smooth muscle actin in liver sections. The induction of OJ increased all the parameters related to apoptosis and necrosis in liver. The induction of liver injury was associated with stellate cell activation, as well as an increase in MDA (p < 0.0001) and a reduction in GSH, GPx, catalase and SOD content (p < 0.0001) in liver. MEL reduced hepatic apoptosis and necrosis (p < 0.004) with a significant improvement in all oxidative stress markers. In conclusion, our results showed that MEL recovered the antioxidant status and reduced apoptosis and necrosis induced by experimental cholestasis.