Protective effect of lipoic acid against methotrexate-induced oxidative stress in liver mitochondria

Methotrexate (MTX) is a folic acid antagonist widely used as a cytotoxic chemotherapeutic agent for leukemia and other malignancies. The purpose of this study was to investigate the damage caused by MTX on liver mitochondria and its protection by using antioxidant properties of lipoic acid. MTX substantially affects mitochondrial function by reducing glutathione levels leading to disturbances in antioxidant enzyme defense system. Lipoic acid occurs naturally in mitochondria as a coenzyme. In various studies lipoic acid has been convincingly shown to exhibit an antioxidant role when supplemented exogenously. We studied the effect of lipoic acid pre-treatment on the toxicity of MTX in mouse liver mitochondria focusing specifically on the oxidative stress. MTX caused a significant rise in the mitochondrial lipid peroxidation (LPO), protein carbonyl (PC) content and superoxide radical generation. It also affected the mitochondrial thiol profile. Pre-treatment of mice with lipoic acid (35 mg/kg) markedly lowered mitochondrial LPO, PC content and superoxide radical generation. It also restored decreased enzymatic and non-enzymatic antioxidants of mitochondria. It is suggested that lipoic acid has a potential role in suppressing MTX-induced mitochondrial toxicity, and it affords protection either by reversing the decline of antioxidants or by the directly scavenging the free radicals.     

Taurine protects rat testes against NaAsO2-induced oxidative stress and apoptosis via mitochondrial dependent and independent pathways

Arsenic (As) is a well known toxicity inducer. Recent investigations, however, showed that it might have some therapeutic application in cancer treatment. These dual roles of arsenic have attracted a renewed research in organ pathophysiology. In this study, we report that As administration (in the form of NaAsO₂ at a dose of 10 mg/kg body weight for 2 days, orally) induces apoptosis in testicular tissue of the experimental rats by the activation of caspase-3 and reciprocal regulation of Bcl-2/Bad with the concomitant reduction of mitochondrial membrane potential and increased level of cytosolic cytochrome C. Arsenite has also been shown to induce activation of mitogen-activated protein kinases (MAPKs), Akt as well as NF-κB (p65) in testicular tissue. In addition, As significantly decreased testicular Δ⁵-3β-HSD and 17β-HSD activities and reduced the plasma testosterone level, testicular sperm count and sperm motility. Besides, arsenite exposure increased the levels of reactive oxygen species (ROS), serum TNF-α, As accumulation and lipid peroxidation and decreased the activities of the antioxidant enzymes and glutathione in the testicular tissue. Oral administration of taurine (at a dose of 100 mg/kg body weight for 5 days) was found to be effective in counteracting As-induced oxidative stress, attenuation of testicular damages and amelioration of apoptosis in testicular tissue by controlling the reciprocal regulation of Bcl-2/Bad, phospho-ERK1/2, phospho-p38, phospho-Akt and NF-κB. Taurine was also found to play similar beneficial role via mitochondrial dependent pathways in As-induced testicular damages leading to apoptotic cell death.     

D-saccharic acid-1,4-lactone ameliorates alloxan-induced diabetes mellitus and oxidative stress in rats through inhibiting pancreatic β-cells from apoptosis via mitochondrial dependent pathway

Oxidative stress plays a vital role in diabetic complications. To suppress the oxidative stress mediated damage in diabetic pathophysiology, a special focus has been given on naturally occurring antioxidants present in normal diet. d-saccharic acid 1,4-lactone (DSL), a derivative of d-glucaric acid, is present in many dietary plants and is known for its detoxifying and antioxidant properties. The aim of the present study was to evaluate the beneficial role of DSL against alloxan (ALX) induced diabetes in the pancreas tissue of Swiss albino rats. A dose-dependent study for DSL (20-120 mg/kg body weight) was carried out to find the effective dose of the compound in ALX-induced diabetic rats. ALX exposure elevated the blood glucose, glycosylated Hb, decreased the plasma insulin and disturbed the intra-cellular antioxidant machineries whereas oral administration of DSL at a dose of 80 mg/kg body weight restored these alterations close to normal. Investigating the mechanism of the protective activity of DSL we observed that it prevented the pancreatic β-cell apoptosis via mitochondria-dependent pathway. Results showed decreased mitochondrial membrane potential, enhanced cytochrome c release in the cytosol and reciprocal regulation of Bcl-2 family proteins in the diabetic rats. These events were also found to be associated with increased level of Apaf-1, caspase 9, and caspase 3 that ultimately led to pancreatic β-cell apoptosis. DSL treatment, however, counteracted these changes. In conclusion, DSL possesses the capability of ameliorating the oxidative stress in ALX-induced diabetes and thus could be a promising approach in lessening diabetic complications.     

Taurine exerts hypoglycemic effect in alloxan-induced diabetic rats, improves insulin-mediated glucose transport signaling pathway in heart and ameliorates cardiac oxidative stress and apoptosis

Hyperlipidemia, inflammation and altered antioxidant profiles are the usual complications in diabetes mellitus. In the present study, we investigated the therapeutic potential of taurine in diabetes associated cardiac complications using a rat model. Rats were made diabetic by alloxan (ALX) (single i.p. dose of 120 mg/kg body weight) and left untreated or treated with taurine (1% w/v, orally, in water) for three weeks either from the day of ALX exposure or after the onset of diabetes. Animals were euthanized after three weeks. ALX-induced diabetes decreased body weight, increased glucose level, decreased insulin content, enhanced the levels of cardiac damage markers and altered lipid profile in the plasma. Moreover, it increased oxidative stress (decreased antioxidant enzyme activities and GSH/GSSG ratio, increased xanthine oxidase enzyme activity, lipid peroxidation, protein carbonylation and ROS generation) and enhanced the proinflammatory cytokines levels, activity of myeloperoxidase and nuclear translocation of NFκB in the cardiac tissue of the experimental animals. Taurine treatment could, however, result to a decrease in the elevated blood glucose and proinflammatory cytokine levels, diabetes-evoked oxidative stress, lipid profiles and NFκB translocation. In addition, taurine increased GLUT 4 translocation to the cardiac membrane by enhanced phosphorylation of IR and IRS1 at tyrosine and Akt at serine residue in the heart. Results also suggest that taurine could protect cardiac tissue from ALX induced apoptosis via the regulation of Bcl2 family and caspase 9/3 proteins. Taken together, taurine supplementation in regular diet could play a beneficial role in regulating diabetes and its associated complications in the heart.     

Ameliorative role of conjugated linolenic acid isomers against oxidative DNA damage induced by sodium arsenite in rat model

The present study was undertaken to evaluate the ameliorative role of α-eleostearic acid and punicic acid, isomers of conjugated linolenic (CLnA) acid, against oxidative stress induced DNA damage. Male albino rats were divided into six groups. Group 1 and 2 were normal control and sodium arsenite treated (Sa; 10 mg/kg BW) control respectively. Group 3–6 were orally treated with different doses of two fatty acids (0.5% and 1.0% of total lipid given for each isomer) along with sodium arsenite (Sa; 10 mg/kg BW). Comet assay of blood leukocytes showed that administration of CLnA reduced DNA damage significantly (P < 0.05) which was determined by tail DNA percent and olive tail moment. Results showed that activity of antioxidant enzymes viz. catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx) and reduced glutathione (GSH) in plasma, liver and erythrocyte lysate decreased and activity of nitric oxide synthase in plasma and liver increased significantly due to oxidative stress generated by sodium arsenite. Administration of CLnA isomers restored all the altered parameters and also reduced lipid peroxidation and leakage of transaminase enzymes from liver to blood due to liver injury. α-Eleostearic acid was more efficient antioxidant than punicic acid against oxidative DNA damage.     

Taurine suppresses doxorubicin-triggered oxidative stress and cardiac apoptosis in rat via up-regulation of PI3-K/Akt and inhibition of p53, p38-JNK

The objective of the present study was to investigate the signaling mechanisms involved in the beneficial role of taurine against doxorubicin-induced cardiac oxidative stress. Male rats were administered doxorubicin. Hearts were collected 3 weeks after the last dose of doxorubicin and were analyzed. Doxorubicin administration retarded the growth of the body and the heart and caused injury in the cardiac tissue because of increased oxidative stress. Similar experiments with doxorubicin showed reduced cell viability, increased ROS generation, intracellular Ca²⁺ and DNA fragmentation, disrupted mitochondrial membrane potential and apoptotic cell death in primary cultured neonatal rat cardiomyocytes. Signal transduction studies showed that doxorubicin increased p53, JNK, p38 and NFκB phosphorylation; decreased the levels of phospho ERK and Akt; disturbed the Bcl-2 family protein balance; activated caspase 12, caspase 9 and caspase 3; and induced cleavage of the PARP protein. However, taurine treatment or cardiomyocyte incubation with taurine suppressed all of the adverse effects of doxorubicin. Studies with several inhibitors, including PS-1145 (an IKK inhibitor), SP600125 (a JNK inhibitor), SB203580 (a p38 inhibitor) and LY294002 (a PI3-K/Akt inhibitor), demonstrated that the mechanism of taurine-induced cardio protection involves activation of specific survival signals and PI3-K/Akt as well as the inhibition of p53, JNK, p38 and NFκB. These novel findings suggest that taurine might have clinical implications for the prevention of doxorubicin-induced cardiac oxidative stress.     

Oxidative stress and hepatic stellate cell activation are key events in arsenic induced liver fibrosis in mice

Arsenic is an environmental toxicant and carcinogen. Exposure to arsenic is associated with development of liver fibrosis and portal hypertension through ill defined mechanisms. We evaluated hepatic fibrogenesis after long term arsenic exposure in a murine model. BALB/c mice were exposed to arsenic by daily gavages of 6 μg/gm body weight for 1 year and were evaluated for markers of hepatic oxidative stress and fibrosis, as well as pro-inflammatory, pro-apoptotic and pro-fibrogenic factors at 9 and 12 months. Hepatic NADPH oxidase activity progressively increased in arsenic exposure with concomitant development of hepatic oxidative stress. Hepatic steatosis with occasional collection of mononuclear inflammatory cells and mild portal fibrosis were the predominant liver lesion observed after 9 months of arsenic exposure, while at 12 months, the changes included mild hepatic steatosis, inflammation, necrosis and significant fibrosis in periportal areas. The pathologic changes in the liver were associated with markers of hepatic stellate cells (HSCs) activation, matrix reorganization and fibrosis including α-smooth muscle actin, transforming growth factor-β1, PDGF-Rβ, pro-inflammatory cytokines and enhanced expression of tissue inhibitor of metalloproteinase-1 and pro(α) collagen type I. Moreover, pro-apoptotic protein Bax was dominantly expressed and Bcl-2 was down-regulated along with increased number of TUNEL positive hepatocytes in liver of arsenic exposed mice. Furthermore, HSCs activation due to increased hepatic oxidative stress observed after in vivo arsenic exposure was recapitulated in co-culture model of isolated HSCs and hepatocytes exposed to arsenic. These findings have implications not only for the understanding of the pathology of arsenic related liver fibrosis but also for the design of preventive strategies in chronic arsenicosis.     

Influence of repeated preexposure to arsenic on acetaminophen-induced oxidative stress in liver of male rats

We evaluated whether repeated arsenic preexposure can increase acetaminophen-induced hepatic oxidative stress. Rats were exposed to arsenic (25 ppm; rat equivalent concentration of maximum groundwater contamination level) via drinking water for 28 days. Next day, they were given single oral administration of acetaminophen (420 or 1000 mg/kg b.w.). Hepatotoxicity was evaluated by assessing serum biomarkers, cytochrome-P450 (CYP) content, CYP3A4- and CYP2E1-dependent enzymes, lipid peroxidation and antioxidants. Arsenic or acetaminophen increased serum ALT and AST activities and depleted CYP. Arsenic decreased, but acetaminophen increased CYP-dependent enzyme activities. These agents independently increased lipid peroxidation and decreased antioxidants. Arsenic did not alter the effects of acetaminophen on serum biomarkers, caused further CYP depletion and decreased acetaminophen-mediated induction of drug-metabolizing enzymes. Arsenic enhanced the lower dose of acetaminophen-mediated lipid peroxidation and glutathione depletion with no further alterations in enzymatic antioxidants. However, arsenic attenuated the higher dose-mediated lipid peroxidation and glutathione depletion with improvement in glutathione peroxidase and glutathione reductase activities, further decrease in catalase and no alterations in superoxide dismutase and glutathione-S-transferase activities. Results show that arsenic preexposure increased the susceptibility of rats to hepatic oxidative stress induced by the lower dose of acetaminophen, but reduced the oxidative stress induced by the higher dose.     

A role for glutathione,independent of oxidative stress,in the developmental toxicity of methanol

Oxidative stress and reactive oxygen species (ROS) have been implicated in the teratogenicity of methanol (MeOH) in rodents, both in vivo and in embryo culture. We explored the ROS hypothesis further in vivo in pregnant C57BL/6J mice. Following maternal treatment with a teratogenic dose of MeOH, 4 g/kg via intraperitoneal (ip) injection on gestational day (GD) 12, there was no increase 6 h later in embryonic ROS formation, measured by 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) fluorescence, despite an increase observed with the positive control ethanol (EtOH), nor was there an increase in embryonic oxidatively damaged DNA, quantified as 8-oxo-2′-deoxyguanosine (8-oxodG) formation. MeOH teratogenicity (primarily ophthalmic anomalies, cleft palate) also was not altered by pre- and post-treatment with varying doses of the free radical spin trapping agent alpha-phenyl-N-tert-butylnitrone (PBN). In contrast, pretreatment with l-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, depleted maternal hepatic and embryonic GSH, and enhanced some new anomalies (micrognathia, agnathia, short snout, fused digits, cleft lip, low set ears), but not the most common teratogenic effects of MeOH (ophthalmic anomalies, cleft palate) in this strain. These results suggest that ROS did not contribute to the teratogenic effects of MeOH in this in vivo mouse model, in contrast to results in embryo culture from our laboratory, and that the protective effect of GSH in this model may arise from its role as a cofactor for formaldehyde dehydrogenase in the detoxification of formaldehyde.     

Clerodendron glandulosum.Coleb extract ameliorates high fat diet/fatty acid induced lipotoxicity in experimental models of non-alcoholic steatohepatitis

This study evaluates the protective role of Clerodendron glandulosum.Coleb (CG) aqueous extract against high fat diet/fatty acid induced lipotoxicity in experimental models of non-alcoholic steatohepatitis (NASH). Supplementation of NASH mice with CG extract (1% and 3% in high fat diet for 16 weeks) prevented high fat diet induced elevation in liver enzymes, plasma and hepatic lipids, mitochondrial oxidative stress and compromised enzymatic and non-enzymatic antioxidant status and histopathological damage to hepatocytes. Furthermore, results from in vitro study indicates, addition of CG extract (20–200 μg/ml for 24 h) to HepG2 cells minimizes oleic acid induced lipid accumulation, higher lipid peroxidation, cytotoxicity and reduced cell viability. These in vivo and in vitro studies suggest that CG extract has the potential of preventing high fat/fatty acid induced NASH.     

Evaluation of cytotoxicity and oxidative DNA damaging effects of di(2-ethylhexyl)-phthalate (DEHP) and mono(2-ethylhexyl)-phthalate (MEHP) on MA-10 Leydig cells and protection by selenium

Di(2-ethylhexyl)-phthalate (DEHP) is the most abundantly used phthalate derivative, inevitable environmental exposure of which is suspected to contribute to the increasing incidence of testicular dysgenesis syndrome in humans. Oxidative stress and mitochondrial dysfunction in germ cells are suggested to contribute to phthalate-induced disruption of spermatogenesis in rodents, and Leydig cells are one of the main targets of phthalates’ testicular toxicity. Selenium is known to be involved in the modulation of intracellular redox equilibrium, and plays a critical role in testis, sperm, and reproduction. This study was aimed to investigate the oxidative stress potential of DEHP and its consequences in testicular cells, and examine the possible protective effects of selenium using the MA-10 mouse Leydig tumor cell line as a model. In the presence and absence of selenium compounds [30 nM sodium selenite (SS), and 10 μM selenomethionine (SM)], the effects of exposure to DEHP and its main metabolite mono(2-ethylhexyl)-phthalate (MEHP) on the cell viability, enzymatic and non-enzymatic antioxidant status, ROS production, p53 expression, and DNA damage by alkaline Comet assay were investigated. The overall results of this study demonstrated the cytotoxicity and genotoxicity potential of DEHP, where MEHP was found to be more potent than the parent compound. SS and SM produced almost the same level of protection against antioxidant status modifying effects, ROS and p53 inducing potentials, and DNA damaging effects of the two phthalate derivatives. It was thus shown that DEHP produced oxidative stress in MA-10 cells, and selenium supplementation appeared to be an effective redox regulator in the experimental conditions used in this study, emphasizing the critical importance of the appropriate selenium status.