Lupeol prevents acetaminophen-induced in vivo hepatotoxicity by altering the Bax/Bcl-2 and oxidative stress-mediated mitochondrial signaling cascade

AimsLupeol, a triterpene, possesses numerous pharmacological activities, including anti-malarial, anti-arthritic and anti-carcinogenic properties. The present study was conducted to explore the hepatoprotective potential of lupeol against acetaminophen (AAP)-induced hepatotoxicity in Wistar rats.Main methodsRats were given a prophylactic treatment of lupeol (150 mg/kg body weight, p.o., for 30 consecutive days) with a co-administration of AAP (1 g/kg body weight). The modulatory effects of lupeol on AAP-induced hepatotoxicity were investigated by assaying oxidative stress biomarkers, serum liver toxicity markers, pro/anti apoptotic proteins, DNA fragmentation and by the histopathological examination of the liver.Key findingsLupeol significantly prevented hepatic damage as evident from the histopathological studies and significant decline in serum trans-aminases. The alterations in cellular redox status (p < 0.01) and antioxidant enzyme activities together with the enhanced lipid peroxidation and protein carbonyl levels were also observed in the AAP-treated rats. In addition, significant ROS generation and mitochondrial depolarization were observed in this group. Co-administration of lupeol significantly decreased the level of serum transaminases, MDA and protein carbonyl content. It also prevented ROS generation and mitochondrial depolarization. Furthermore, lupeol enhanced the mitochondrial antioxidant and redox status and inhibited DNA damage and cell death by preventing the downregulation of Bcl-2, upregulation of Bax, release of cytochrome c and the activation of caspase 9/3.SignificanceThe conclusion of this study is that lupeol when co-administered with AAP effectively reduces oxidative stress and prevents AAP-induced hepatotoxicity by inhibiting critical control points of apoptosis.     

A novel proanthocyanidin IH636 grape seed extract increases in vivo Bcl-XL expression and prevents acetaminophen-induced programmed and unprogrammed cell death in mouse liver.

Several molecular events in the apoptotic or necrotic death of hepatocytes induced by acetaminophen (AAP) now appear to be well defined. Recent studies also indicate that select expression of bcl-Xl is possibly modified during AAP-induced liver injury. The purpose of this study was several-fold: (i) to examine the hepatoprotective ability of short-term (3-day) and long-term (7-day) exposures of a grape seed proanthocyanidin extract (GSPE) on AAP-induced liver injury and animal lethality; (ii) to monitor effects of GSPE on one of the prime targets of AAP, i.e., hepatocellular genomic DNA and associated apoptotic and necrotic death; and (iii) to unravel changes in the pattern of expression of an antiapoptotic gene, bcl-Xl in the liver. In order to investigate these events, male ICR mice (30-40 g) were administered nontoxic doses of GSPE (3 or 7 days, 100 mg/kg, po), followed by hepatotoxic doses of AAP (400 and 500 mg/kg, ip), and sacrificed 24 h later. Serum was analyzed for alanine aminotransferase activity (ALT) and the liver for histopathological diagnosis of apoptosis/necrosis. The ability of AAP to promote apoptotic DNA fragmentation and its counteraction by GSPE in the liver was also evaluated quantitatively (by a sedimentation assay) and qualitatively (by agarose gel electrophoresis). Portions of livers were also subjected to Western blot analysis (27,000g fraction of liver homogenates) to examine the pattern of expression of cell death inhibitory gene bcl-Xl. Results indicate that 7-day GSPE preexposure induced dramatic protection and markedly decreased liver injury and animal lethality culminated by AAP, when compared to a short-term 3-day exposure. Abrogation of toxicity was also mirrored in DNA fragmentation. Histopathological evaluation of liver sections showed remarkable counteraction of AAP-toxicity by this novel GSPE and substantial inhibition of both apoptotic and necrotic liver cell death. Agarose gel electrophoresis revealed that 7-day GSPE preexposure prior to AAP administration completely blocked Ca(2+)/Mg(2+)-Ca(2+)/Mg(2+)-dependent-endonuclease-mediated ladder-like fragmentation of genomic DNA and significantly altered the bcl-Xl expression. The most dramatic changes observed in this study were: (i) substantial increase in the expression of bcl-Xl in the liver by 7-day GSPE exposure alone; (ii) significant modification bcl-Xl expression by AAP alone; and (iii) dramatic inhibition of AAP-induced modification of bcl-Xl (phosphorylation?) expression by GSPE. In summary, these observations demonstrate that GSPE preexposure may significantly attenuate AAP-induced hepatic DNA damage, apoptotic and necrotic cell death of liver cells, and, most remarkably, antagonize the influence of AAP-induced changes in bcl-Xl expression in vivo.     

Role of caspases in acetaminophen-induced liver injury

The mode of cell death after acetaminophen (AAP) overdose is controversially discussed. A recent study reported a protective effect of the pancaspase inhibitor Z-VAD-fmk against AAP toxicity in vivo but the mechanism of protection remained unclear. Therefore, the objective of this investigation was to assess if Z-VAD-fmk or the low doses of dimethyl sulfoxide (DMSO) used as solvent were responsible for the protection. Treatment with 10 mg/kg Z-VAD-fmk or diluted DMSO (0.25 ml/kg) for 15 min before but not 2.5 h after AAP prevented the oxidant stress (hepatic glutathione disulfide content; nitrotyrosine staining), DNA fragmentation (anti-histone ELISA, TUNEL assay) and liver injury (plasma ALT activities) at 6 h after administration of 300 mg/kg AAP. Even a lower dose (0.1 ml/kg) of DMSO was partially effective. DMSO pretreatment also attenuated the initial decline in hepatic glutathione levels. On the other hand, 10 microM Z-VAD-fmk was unable to prevent AAP-induced cell death in primary cultured mouse hepatocytes. We conclude that Z-VAD-fmk does not protect against AAP-induced liver injury and, therefore, caspases are not involved in the mechanism of AAP-induced liver injury. In contrast, the protection in vivo is caused by the diluted DMSO, which is used to solubilize the inhibitor Z-VAD-fmk. The results emphasize that even very low doses of DMSO, which are generally necessary to dissolve water-insoluble inhibitors, can have a profound impact on the toxicity of drugs and chemicals when metabolic activation is a critical aspect of the mechanism of cell injury.     

Ca(2+)-calmodulin antagonist chlorpromazine and poly(ADP-ribose) polymerase modulators 4-aminobenzamide and nicotinamide influence hepatic expression of BCL-XL and P53 and protect against acetaminophen-induced programmed and unprogrammed cell death in mice.

Acetaminophen (AAP), the analgesic hepatotoxicant, is a powerful inducer of oxidative stress, DNA fragmentation, and apoptosis. The anti-apoptotic oncogene bcl-XL, and the pro-apoptotic oncogene p53 are two key regulators of cell cycle progression and/or apoptosis subsequent to DNA damage in vitro and in vivo. This study investigated the effect of AAP on the expression of these oncogenes and whether agents that modulate DNA fragmentation (chlorpromazine, CPZ) and DNA repair through poly(ADP-Ribose) polymerase (PARP) activity (4-AB: 4-aminobenzamide) can protect against AAP-induced hepatotoxicity by inhibiting oxidative stress, DNA fragmentation, and/or by altering the expression of bcl-XL and p53. In addition, the protective effect of supplemental nicotinamide (NICO), known to be depleted in cells with high PARP activity during DNA repair, is similarly evaluated. Male ICR mice (3 months old) were administered vehicle alone; nontoxic doses of 4-AB (400 mg/kg, ip), NICO (250 mg/kg, ip) or CPZ (25 mg/kg, ip), hepatotoxic dose of AAP alone (500 mg/kg, ip), or AAP plus one of the protective agents 1 h later. All animals were sacrificed 24 h following AAP administration. Serum alanine aminotransferase activity (ALT), hepatic histopathology and lipid peroxidation, DNA damage, and expression of bcl-XL and p53 (western blot analysis) were compared in various groups. All of the three agents significantly prevented AAP-induced liver injury, lipid peroxidation, DNA damage, and associated apoptotic and necrotic cell deaths, 4-AB being the most effective and NICO the least. Compared to control, there was a considerable decrease in bcl-XL expression, and an increase in p53 expression in AAP-exposed livers. The effect of AAP on bcl-XL was antagonized and that on p53 was synergized by the PARP-modulator 4-AB as well as NICO, whereas the endonuclease inhibitor CPZ was without effect on either bcl-XL or p53 expression. These results suggest that the hepatotoxic effect of AAP involves multiple mechanisms including oxidative stress, upregulation of endonuclease (or caspase-activated DNAse) and alteration of pro- and anti-apoptotic oncogenes. The observed antagonism of AAP-induced hepatocellular apoptosis and/or necrosis by modulators of multiple processes including DNA repair suggests the likelihood that a more effective therapy against AAP intoxication should involve a combination of antidotes.     

Protective effect of a fermented substance from Saccharomyces cerevisiae on liver injury in mice caused by acetaminophen

The protective effect of a fermented substance from Saccharomyces cerevisiae (FSSC) on liver injury caused by acetaminophen (AAP) was studied in mice. Mice were pretreated with FSSC (0.5-2.0 g/kg, p.o.) for 4 d, and on the fourth day, the mice received an overdose of AAP (500 mg/kg, i.p.). Subsequently, they were sacrificed at 7 h, and blood was drawn from the abdominal vein and liver samples were collected. Histological and biochemical examinations revealed that the administration of AAP caused liver injury in the mice, including increases in plasma alanine aminotransferase and asparate aminotransferase activities and decreases in the hepatic reduced form of glutathione (GSH) content and antioxidant enzyme activities. Prior to AAP treatment, the mice pretreated with FSSC showed significantly reduced levels of alanine aminotransferase (ALT) and aspirate aminotransferase (AST) activity. Liver histology in the FSSC-pretreated mice was significant. In these mice, pretreatment with FSSC also served to reduce hepatic GSH depletion and the inhibition of antioxidant enzyme activity caused by AAP overdose. In conclusion, oral administration of FSSC significantly reduced AAP-induced hepatic injury in the mice.     

Protective Effect of a Fermented Substance from Saccharomyces cerevisiae on Liver Injury in Mice Caused by Acetaminophen

The protective effect of a fermented substance from Saccharomyces cerevisiae (FSSC) on liver injury caused by acetaminophen (AAP) was studied in mice. Mice were pretreated with FSSC (0.5–2.0 g/kg, p.o.) for 4 d, and on the fourth day, the mice received an overdose of AAP (500 mg/kg, i.p.). Subsequently, they were sacrificed at 7 h, and blood was drawn from the abdominal vein and liver samples were collected. Histological and biochemical examinations revealed that the administration of AAP caused liver injury in the mice, including increases in plasma alanine aminotransferase and asparate aminotransferase activities and decreases in the hepatic reduced form of glutathione (GSH) content and antioxidant enzyme activities. Prior to AAP treatment, the mice pretreated with FSSC showed significantly reduced levels of alanine aminotransferase (ALT) and aspirate aminotransferase (AST) activity. Liver histology in the FSSC-pretreated mice was significant. In these mice, pretreatment with FSSC also served to reduce hepatic GSH depletion and the inhibition of antioxidant enzyme activity caused by AAP overdose. In conclusion, oral administration of FSSC significantly reduced AAP-induced hepatic injury in the mice.     

Assessment of hepatoprotective and nephroprotective potential of withaferin A on bromobenzene-induced injury in Swiss albino mice: possible involvement of mitochondrial dysfunction and inflammation

Bromobenzene is a well-known environmental toxin which causes liver and kidney damage through CYP450-mediated bio-activation to generate reactive metabolites and, consequently, oxidative stress. The present study aimed to evaluate the possible protective role of withaferin A against bromobenzene-induced liver and kidney damage in mice. Withaferin A (10 mg/kg) was administered orally to the mice for 8 days before intragastric intubation of bromobenzene (10 mmol/kg). As results of this experiment, the levels of liver and kidney functional markers, lipid peroxidation, and cytokines (TNF-α and IL-1β) presented an increase and there was a decrease in anti-oxidant activity in the bromobenzene-treated group of mice. Pre-treatment with withaferin A not only significantly decreased the levels of liver and kidney functional markers and cytokines but also reduced oxidative stress, as evidenced by improved anti-oxidant status. In addition, the mitochondrial dysfunction shown through the decrease in the activities of mitochondrial enzymes and imbalance in the Bax/Bcl-2 expression in the livers and kidneys of bromobenzene-treated mice was effectively prevented by pre-administration of withaferin A. These results validated our conviction that bromobenzene caused liver and kidney damage via mitochondrial pathway and withaferin A provided significant protection against it. Thus, withaferin A may have possible usage in clinical liver and kidney diseases in which oxidative stress and mitochondrial dysfunction may be existent.     

Ebselen attenuates cyclophosphamide-induced oxidative stress and DNA damage in mice

The role of selenium, a trace element in the human diet, has been extensively studied against cancer, immunity and infectious/inflammatory diseases. The purpose of the present study was to investigate the beneficial effect of ebselen, an organo-selenium compound, against cyclophosphamide-induced oxidative stress and DNA damage in mice. Malondialdehyde and total glutathione were estimated in the liver to detect the oxidative stress induced by cyclophosphamide. Standard and modified comet assays (the latter incorporated lesion-specific enzymes, formamidopyrimidine-DNA glycosylase and endonuclease-III) were used to detect the normal and oxidative stress-induced DNA damage by cyclophosphamide in the mouse bone marrow and the peripheral blood lymphocytes. In addition, a micronucleus assay capable of detecting DNA damage was also carried out in the mouse bone marrow and the peripheral blood reticulocytes induced by cyclophosphamide. The results confirm that pre-treatment with ebselen (2.5, 5 and 10 mg/kg) for 5 consecutive days decreased the oxidative stress induced by cyclophosphamide (100 mg/kg) based on the restoration in concentration of malondialdehyde and glutathione in the liver and decreased DNA damage and micronuclei count in the bone marrow and the peripheral blood. It is concluded that pre-treatment with ebselen attenuates cyclophosphamide-induced oxidative stress and the resultant DNA damage in mice.     

xCT deficiency aggravates acetaminophen-induced hepatotoxicity under inhibition of the transsulfuration pathway

Cystine, an oxidized form of cysteine (Cys), is imported into cells via the protein xCT, which is also associated with the export of glutamate as the counter amino acid. In the current study, we attempted to rationalize roles of xCT in the livers of male mice. While xCT was not expressed in the livers of ordinary mice, it was induced under conditions of glutathione depletion, caused by the administration of acetaminophen (AAP). To differentiate the role between xCT and the transsulfuration pathway on the supply of Cys, we employed an inhibitor of the enzyme cystathionine γ-lyase, propargylglycine (PPG). This inhibitor caused a marked aggravation in AAP-induced hepatic damage and the mortality of the xCT^?/? mice was increased to a greater extent than that for the xCT^+/+ mice. While a PPG pretreatment had no effect on liver condition or Cys levels, the administration of AAP to the PPG-pretreated mice reduced the levels of Cys as well as glutathione to very low levels in both the xCT^+/+ and xCT^?/? mice. These findings indicate that the transsulfuration pathway plays a major role in replenishing Cys when glutathione levels are low. Moreover, an ascorbic acid insufficiency, induced by Akr1a ablation, further aggravated the AAP-induced liver damage in the case of the xCT deficiency, indicating that glutathione and ascorbic acid function cooperatively in protecting the liver. In conclusion, while the transsulfuration pathway plays a primary role in supplying Cys to the redox system in the liver, xCT is induced in cases of emergencies, by compensating for Cys supply systems.     

Endogenous mitochondrial oxidative stress: neurodegeneration, proteomic analysis, specific respiratory chain defects, and efficacious antioxidant therapy in superoxide dismutase 2 null mice

Oxidative stress and mitochondrial dysfunction have been linked to neurodegenerative disorders such as Parkinson's and Alzheimer's disease. However, it is not yet understood how endogenous mitochondrial oxidative stress may result in mitochondrial dysfunction. Most prior studies have tested oxidative stress paradigms in mitochondria through either chemical inhibition of specific components of the respiratory chain, or adding an exogenous insult such as hydrogen peroxide or paraquat to directly damage mitochondria. In contrast, mice that lack mitochondrial superoxide dismutase (SOD2 null mice) represent a model of endogenous oxidative stress. SOD2 null mice develop a severe neurological phenotype that includes behavioral defects, a severe spongiform encephalopathy, and a decrease in mitochondrial aconitase activity. We tested the hypothesis that specific components of the respiratory chain in the brain were differentially sensitive to mitochondrial oxidative stress, and whether such sensitivity would lead to neuronal cell death. We carried out proteomic differential display and examined the activities of respiratory chain complexes I, II, III, IV, V, and the tricarboxylic acid cycle enzymes alpha-ketoglutarate dehydrogenase and citrate synthase in SOD2 null mice in conjunction with efficacious antioxidant treatment and observed differential sensitivities of mitochondrial proteins to oxidative stress. In addition, we observed a striking pattern of neuronal cell death as a result of mitochondrial oxidative stress, and were able to significantly reduce the loss of neurons via antioxidant treatment.     

Intervention of astaxanthin against cyclophosphamide-induced oxidative stress and DNA damage: A study in mice

Astaxanthin, a natural and nutritional red carotenoid pigment, is used as a dietary supplement. The intention of the present study was to investigate the beneficial effects of dietary pigment astaxanthin, against cyclophosphamide-induced oxidative stress and DNA damage. The end points of evaluation of the study included: (a) malondialdehyde, glutathione and superoxide dismutase concentration in liver to detect oxidative stress; (b) normal and modified alkaline comet assays (the latter includes lesion-specific enzymes formamidopyrimidine-DNA glycosylase and endonuclease-III) to detect normal and oxidative stress-induced DNA damage by cyclophosphamide in the mouse bone marrow and the peripheral blood lymphocytes. In addition, micronucleus assay and chromosomal aberration test capable of detecting the DNA damage were also carried out in peripheral blood and bone marrow of mice. Cyclophosphamide (100 mg/kg intra-peritoneal) treatment led to significant increase in liver malondialdehyde and decreased the antioxidant enzymes glutathione and superoxide dismutase. Further, cyclophosphamide also significantly increased the DNA damage as observed from normal and modified comet assays as well as micronucleus and chromosomal aberration assay. Pre-treatment with astaxanthin (12.5, 25 and 50 mg/kg/day for 5 days per oral) resulted in the restoration of oxidative stress markers such as malondialdehyde, glutathione and superoxide dismutase in liver. The amelioration of oxidative stress with astaxanthin pre-treatment correlated well with the decreased DNA damage as evident from normal and modified alkaline comet assays of bone marrow cells and peripheral blood lymphocytes. Further astaxanthin pre-treatment also reduced the frequency of chromosomal breakage and micronucleus formation in the mouse bone marrow cells and peripheral blood reticulocytes. It is thus concluded that pre-treatment with astaxanthin attenuates cyclophosphamide-induced oxidative stress and subsequent DNA damage in mice and it can be used as a chemoprotective agent against the toxicity of anticancer drug cyclophosphamide.     

The damage-associated molecular pattern HMGB1 is released early after clinical hepatic ischemia/reperfusion

Objective and backgroundActivation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice.MethodsPlasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury.ResultsIn patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects.ConclusionHMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage.     

Mitochondrial DNA Damage and Dysfunction,and Oxidative Stress Are Associated with Endoplasmic Reticulum Stress,Protein Degradation and Apoptosis in High Fat Diet-Induced Insulin Resistance Mice

Background

Recent studies showed a link between a high fat diet (HFD)-induced obesity and lipid accumulation in non-adipose tissues, such as skeletal muscle and liver, and insulin resistance (IR). Although the mechanisms responsible for IR in those tissues are different, oxidative stress and mitochondrial dysfunction have been implicated in the disease process. We tested the hypothesis that HFD induced mitochondrial DNA (mtDNA) damage and that this damage is associated with mitochondrial dysfunction, oxidative stress, and induction of markers of endoplasmic reticulum (ER) stress, protein degradation and apoptosis in skeletal muscle and liver in a mouse model of obesity-induced IR.

Methodology/Principal Findings

C57BL/6J male mice were fed either a HFD (60% fat) or normal chow (NC) (10% fat) for 16 weeks. We found that HFD-induced IR correlated with increased mtDNA damage, mitochondrial dysfunction and markers of oxidative stress in skeletal muscle and liver. Also, a HFD causes a change in the expression level of DNA repair enzymes in both nuclei and mitochondria in skeletal muscle and liver. Furthermore, a HFD leads to activation of ER stress, protein degradation and apoptosis in skeletal muscle and liver, and significantly reduced the content of two major proteins involved in insulin signaling, Akt and IRS-1 in skeletal muscle, and Akt in liver. Basal p-Akt level was not significantly influenced by HFD feeding in skeletal muscle and liver.

Conclusions/Significance

This study provides new evidence that HFD-induced mtDNA damage correlates with mitochondrial dysfunction and increased oxidative stress in skeletal muscle and liver, which is associated with the induction of markers of ER stress, protein degradation and apoptosis.     

Iron-sulfur enzyme mediated mitochondrial superoxide toxicity in experimental Parkinson's disease

Mitochondrial oxidative stress is thought to be an important pathological mediator of neuronal death in Parkinson's disease. However, the precise mechanism by which mitochondrial oxidative stress mediates the death of dopaminergic neurons of the substantia nigra remains unclear. We tested the idea that neuronal damage in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease results, in part, from superoxide radical toxicity via inactivation of an iron-sulfur (Fe-S) protein, mitochondrial aconitase. Administration of MPTP in mice resulted in inactivation of mitochondrial aconitase, but not fumarase in the substantia nigra. MPTP treatment mobilized an early mitochondrial pool of iron detectable by bleomycin chelation that coincided with mitochondrial aconitase inactivation. MPTP-induced mitochondrial aconitase inactivation, iron accumulation and dopamine depletion were significantly attenuated in transgenic mice overexpressing mitochondrial Sod2 and exacerbated in partial deficient Sod2 mice. These results suggest that mitochondrial aconitase may be an important early source of mitochondrial iron accumulation in experimental Parkinson's disease, and that superoxide radical toxicity manifested by oxidative inactivation of mitochondrial aconitase may play a pathogenic role in Parkinson's disease.     

Protective effects of ginkgo biloba against acetaminophen-induced toxicity in mice

Background: The analgesic acetaminophen (AAP) causes a potentially fatal, hepatic centrilobular necrosis when taken in overdose. It was reported that these toxic effects of AAP are due to oxidative reactions that take place during its metabolism. Objective: In this study, we aimed to investigate the possible beneficial effect of Ginkgo biloba (EGb), an antioxidant agent, against AAP toxicity in mice. Methods: Balb/c mice were injected i.p. with: (1) vehicle, control (C) group; (2) a single dose of 50 mg/kg Ginkgo biloba extract, EGb group; (3) a single dose of 900 mg/kg i.p. acetaminophen, AAP group, and (4) EGb, in a dose of 50 mg/kg after AAP injection, AAP + EGb group. Serum ALT, AST, and tumor necrosis factor-alpha (TNF-α) levels in blood and glutathione (GSH), malondialdehyde (MDA) levels, myeloperoxidase (MPO) activity, and collagen contents in liver tissues were measured. Formation of reactive oxygen species in hepatic tissue samples was monitored by using chemiluminescence (CL) technique with luminol and lusigenin probe. Tissues were also examined microscopically. Results: ALT, AST levels, and TNF-α were increased significantly (p < 0.001) after AAP treatment, and reduced with EGb. Acetaminophen caused a significant (p < 0.05–0.001) decrease in GSH levels while MDA levels and MPO activity were increased (p < 0.001) in liver tissues. These changes were reversed by EGb treatment. Furthermore, luminol and lusigenin CL levels in the AAP group increased dramatically compared to control and reduced by EGb treatment (p < 0.01). Conclusion: Our results implicate that AAP causes oxidative damage in hepatic tissues and Ginkgo biloba extract, by its antioxidant effects protects the tissues. Therefore, its therapeutic role as a “tissue injury-limiting agent” must be further elucidated in drug-induced oxidative damage.     

Increased mitochondrial antioxidative activity or decreased oxygen free radical propagation prevent mutant SOD1-mediated motor neuron cell death and increase amyotrophic lateral sclerosis-like transgenic mouse survival

The molecular mechanisms of selective motor neuron degeneration in human amyotrophic lateral sclerosis (ALS) disease remain largely unknown and effective therapies are not currently available. Mitochondrial dysfunction is an early event of motor neuron degeneration in transgenic mice overexpressing mutant superoxide dismutase (SOD)1 gene and mitochondrial abnormality is observed in human ALS patients. In an in vitro cell culture system, we demonstrated that infection of mouse NSC-34 motor neuron-like cells with adenovirus containing mutant G93A-SOD1 gene increased cellular oxidative stress, mitochondrial dysfunction, cytochrome c release and motor neuron cell death. Cells pretreated with highly oxidizable polyunsaturated fatty acid elevated lipid peroxidation and synergistically exacerbated motor neuron-like cell death with mutant G93A-SOD1 but not with wild-type SOD1. Similarly, overexpression of mitochondrial antioxidative genes, MnSOD and GPX4 by stable transfection significantly increased NSC-34 motor neuron-like cell resistance to mutant SOD1. Pre-incubation of cells with spin trapping molecule, 5',5'-dimethylpryrroline-N-oxide (DMPO), prevented mutant SOD1-mediated mitochondrial dysfunction and cell death. Furthermore, treatment of mutant G93A-SOD1 transgenic mice with DMPO significantly delayed paralysis and increased survival. These findings suggest a causal relationship between enhanced oxidative stress and mutant SOD1-mediated motor neuron degeneration, considering that enhanced oxygen free radical production results from the SOD1 structural alterations. Molecular approaches aimed at increasing mitochondrial antioxidative activity or effectively blocking oxidative stress propagation can be potentially useful in the clinical management of human ALS disease.     

The fungal nephrotoxin orellanine simultaneously increases oxidative stress and down-regulates cellular defenses

Confusion of various nephrotoxic Cortinarius species with edible mushrooms occurs every year throughout Europe and North America. The toxin, orellanine (OR), accumulates selectively in renal tubular epithelium with ensuing renal failure after several days as the only clinical manifestation. This study was performed to clarify the mechanisms behind the kidney damage. Sprague-Dawley rats, 100 g bw, received various doses of purified OR ip (0-5 mg/kg bw). One week later, renal function (GFR) was determined (51Cr-EDTA), ascorbyl radicals in venous blood were analyzed using electron spin resonance, and oxidative protein damage was evaluated immunohistochemically. One OR-treated group (3.5 mg/kg) simultaneously received superoxide dismutase (SOD) targeted to tubular epithelium (HC-SOD; 10 mg/kg ip daily for 5 days). RT-PCR was used for analysis of mRNA expression of genes related to oxidative stress. OR caused a dose-dependent decrease in GFR, paralleled by increased levels of ascorbyl radicals and oxidative protein damage. Antioxidant treatment with HC-SOD decreased renal function even more and also increased tissue damage and mortality. Renal mRNA levels for key components in the antioxidative defense were strongly decreased, whereas those for several cytokines were increased. The data strongly suggest that OR nephrotoxicity in vivo is mediated by oxidative stress, including a virtual shutdown of important antioxidative enzymes. We interpret the unexpected effect of HC-SOD in terms of unbalanced SOD and catalase levels in the presence of OR, leading to massive generation of *OH and cell death.     

Efficiency of propolis extract against mitochondrial stress induced by antineoplasic agents (doxorubicin and vinblastin) in rats

To assess the oxidative stress and mitochondrial dysfunction associated with disease, toxic process and aging, in vivo and in vitro preventive effect of propolis extract against mitochondrial oxidative stress induced by two anticancer drugs (doxorubicin and vinblastin) have been investigated in female wistar rat using liver and heart mitochondria. The results show that doxorubicin and vinblastin altered mitochondrial functions as observed by a decrease in respiratory control value, an activation of swelling and overproduction of superoxide anion. Myocardial tissue from doxorubicin treated rats showed a marked increase in malondialdehyde production, a depletion of reduced glutathione contents and an inhibition of catalase and superoxide dismutase activities. Similar results were also observed in liver tissue. Pretreatment of rats with propolis extract (100 mg/kg/day po) (10(-4) M ip) administered 4 days prior to doxorubicin (20 mg/kg) and/or vinblastin (2 mg/kg) injection, substantially reduced the peroxidative damage in myocardium and hepatic tissues and markedly restored the tissues catalase and SOD activities. The results strongly suggest that propolis extract protects heart and liver tissues from oxidative stress by protecting the mitochondria.     

Nimesulide-induced hepatic mitochondrial injury in heterozygous Sod2(+/-) mice

Nimesulide, a preferential COX-2 inhibitor, has been associated with rare idiosyncratic hepatotoxicity. The underlying mechanisms of liver injury are unknown, but experimental evidence has identified oxidative stress as a potential hazard and mitochondria as a target. The aim of this study was to explore whether genetic mitochondrial abnormalities, resulting in impaired mitochondrial function and mildly increased oxidative stress, might sensitize mice to the hepatic adverse effects of nimesulide. We used heterozygous superoxide dismutase 2 (Sod2(+/-)) mice as a model, as these mice develop clinically silent mitochondrial stress but otherwise appear normal. Nimesulide was administered for 4 weeks (10 mg/kg, ip, bid), at a dose equivalent to human therapeutic dosage. We found that the drug potentiated hepatic mitochondrial oxidative injury (decreased aconitase activity, increased protein carbonyls) in Sod2(+/-), but not wild-type, mice. Furthermore, the nimesulide-treated mutant mice exhibited increased hepatic cytosolic levels of cytochrome c and caspase-3 activity, as well as increased numbers of apoptotic hepatocytes. Finally, nimesulide in vitro caused a concentration-dependent net increase in superoxide anion in mitochondria from Sod2(+/-), but not Sod2(+/+) mice. In conclusion, repeated administration of nimesulide can superimpose an oxidant stress, potentiate mitochondrial damage, and activate proapoptotic factors in mice with genetically compromised mitochondrial function.     

Calorie restriction reduces oxidative stress by SIRT3-mediated SOD2 activation

A major cause of aging and numerous diseases is thought to be cumulative oxidative stress, resulting from the production of reactive oxygen species (ROS) during respiration. Calorie restriction (CR), the most robust intervention to extend life span and ameliorate various diseases in mammals, reduces oxidative stress and damage. However, the underlying mechanism is unknown. Here, we show that the protective effects of CR on oxidative stress and damage are diminished in mice lacking SIRT3, a mitochondrial deacetylase. SIRT3 reduces cellular ROS levels dependent on superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. SIRT3 deacetylates two critical lysine residues on SOD2 and promotes its antioxidative activity. Importantly, the ability of SOD2 to reduce cellular ROS and promote oxidative stress resistance is greatly enhanced by SIRT3. Our studies identify a defense program that CR provokes to reduce oxidative stress and suggest approaches to combat aging and oxidative stress-related diseases.