Smriti Shukla

The involvement of nitric oxide in maneb-and paraquat-induced oxidative stress in rat polymorphonuclear leukocytes

Oxidative stress plays a crucial role in the manifestations of maneb (MB) and paraquat (PQ)-induced toxicity including MB+PQ-induced Parkinsons disease (PD). Polymorphonuclear leukocytes (PMNs) actively participate in the oxidative stress-mediated inflammation and organ toxicity. The present study was undertaken to investigate the MB- and/or PQ-induced alterations in the indices of oxidative stress in rat PMNs. Animals were treated with or without MB and/or PQ in an exposure time dependent manner. In some sets of experiments, the animals were pre-treated with NOS inhibitors NG-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (AG) along with respective controls. A significant increase in myeloperoxidase (MPO), superoxide dismutase (SOD), nitric oxide, iNOS expression and lipid peroxidation (LPO) was observed in PMNs of MB- and/or PQ-treated animals, while catalase and glutathione S-transferase (GST) activities were attenuated. L-NAME and AG significantly reduced the augmented nitrite content, iNOS expression and MPO activity to control level in MB and PQ exposed animals. Although the augmented LPO was also reduced significantly in L-NAME and AG treated rat PMNs, the level was still higher as compared with controls. Alterations induced in SOD and GST activities were not affected by NOS inhibitors. The results thus suggest that MB and/or PQ induce iNOS-mediated nitric oxide production, which in turn increases MPO activity and lipid peroxidation, thereby oxidative stress.

Biochemical and molecular mechanisms of N-acetyl cysteine and silymarin-mediated protection against maneb- and paraquat-induced hepatotoxicity in rats

Oxidative stress is one of the major players in the pathogenesis of maneb (MB) and paraquat (PQ)-induced disorders. N-acetyl cysteine (NAC), a glutathione (GSH) precursor and silymarin (SIL), a naturally occurring antioxidant, encounter oxidative stress-mediated cellular damage. The present study was aimed to investigate the effects of NAC and SIL against MB and/or PQ-induced hepatotoxicity in rats. The levels of hepatotoxicity markers - alanine aminotransaminase (ALT), aspartate aminotransaminase (AST) and total bilirubin, histological changes, oxidative stress indices, phase I and phase II xenobiotic metabolizing enzymes - cytochrome P450 (CYP) and glutathione S-transferase (GST) and pro-inflammatory molecules - inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were measured in animals treated with MB and/or PQ in the presence or absence of NAC and SIL. MB and/or PQ augmented ALT, AST, total bilirubin, lipid peroxidation and nitrite contents and catalytic activities of superoxide dismutase and glutathione peroxidase however, the GSH content was attenuated. NAC and SIL restored the above-mentioned alterations towards basal levels but the restorations were more pronounced in SIL treated groups. Similarly, MB and/or PQ-mediated histopathological symptoms and changes in the catalytic activities/expressions of CYP1A2, CYP2E1, iNOS, TNF-α, and IL-1β were alleviated by NAC and SIL. Conversely, MB and/or PQ-induced GSTA4-4 expression/activity was further increased by NAC/SIL and glutathione reductase activity was also increased. The results obtained thus suggest that NAC and SIL protect MB and/or PQ-induced hepatotoxicity by reducing oxidative stress, inflammation and by modulating xenobitic metabolizing machinery and SIL seems to be more effective.

Involvement of NADPH oxidase and glutathione in zinc-induced dopaminergic neurodegeneration in rats: similarity with paraquat neurotoxicity.

An association between excessive zinc (Zn) accumulation in brain and incidences of Parkinsons disease (PD) has been shown in several epidemiological and experimental investigations. The involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and glutathione (GSH) in the pathogenesis of PD has also been proposed in a few studies. Despite the implicated role of oxidative stress in PD, the entire mechanism of Zn-induced dopaminergic neurodegeneration has not yet been clearly understood. The present study aimed to investigate the involvement of NADPH oxidase and GSH in Zn-induced dopaminergic neurodegeneration and also to assess its similarity with paraquat (PQ)-induced rat model of PD. Male Wistar rats were treated either with Zn (20 mg/kg; i.p.) or PQ (5 mg/kg; i.p.) in the presence and absence of NADPH oxidase inhibitor, apocynin (10 mg/kg; i.p.) and a GSH precursor, N-acetyl cysteine (NAC; 200 mg/kg; i.p.) either alone or in combination along with the respective controls. Apocynin and/or NAC pre-treatment significantly alleviated Zn- and PQ-induced changes in neurobehavioral deficits, number of dopaminergic neurons and contents of the striatal dopamine and its metabolites. Apocynin and/or NAC also mitigated Zn- and PQ-induced alterations in oxidative stress, NADPH oxidase activation and cytochrome c release, caspases-9 and -3 activation and CD11b expression. The results obtained thus suggest that Zn induces oxidative stress via the activation of NADPH oxidase and depletion of GSH, which in turn activate the apoptotic machinery leading to dopaminergic neurodegeneration similar to PQ.

Effect of zinc and paraquat co-exposure on neurodegeneration: Modulation of oxidative stress and expression of metallothioneins, toxicant responsive and transporter genes in rats

Abstract Oxidative stress is implicated in Parkinsons disease (PD). Metallothioneins (MT), cytochrome P450 IIE1 (CYP2E1) and glutathione S-transferases alpha4-4 (GSTA4-4) are involved in oxidative stress-mediated damage. Altered dopamine transporter (DAT) and vesicular monoamine transporter-2 (VMAT-2) are also documented in PD. The present study was undertaken to investigate the effect of Zn and PQ co-exposure on neurodegeneration in rats. A significant reduction was observed in spontaneous locomotor activity (SLA), striatal dopamine (DA) levels, tyrosine hydroxylase (TH) immunoreactivity, glutathione reductase (GR) and catalase activity along with increased lipid peroxidation (LPO) and glutathione peroxidase (GPx) activity after Zn and/or PQ exposure. Zn and/or PQ exposure increased gene expression of DAT, CYP2E1, GSTA4-4, MT-I and MT-II, but reduced the expression of VMAT-2. Protein expression analysis of TH, VMAT-2 and DAT showed results similar to those obtained with gene expression study. Zn and PQ co-exposure caused a more pronounced effect than that of individual exposure. The results obtained in this study suggest that, similar to PQ, Zn induced neurodegeneration via alterations in oxidative stress and expression of the above-mentioned genes. However, the effect of Zn+PQ was only slightly higher than that of alone, indicating that probably Zn and PQ follow some different molecular events leading to neurodegeneration.

Maneb and paraquat-induced modulation of toxicant responsive genes in the rat liver: Comparison with polymorphonuclear leukocytes

Experimental studies have shown that toxicant responsive genes, cytochrome P450s (CYPs) and glutathione S-transferases (GSTs) play a critical role in pesticide-induced toxicity. CYPs play pro-oxidant role and GSTs offer protection in maneb (MB) and paraquat (PQ)-induced brain and lung toxicities. The present study aimed to investigate the effect of repeated exposures of MB and/or PQ on lipid peroxidation (LPO), glutathione content (GSH) and toxicant responsive genes, i.e., CYP1A1, 1A2, 2E1, GSTA4-4, GSTA1-1 and GSTA3-3 in the liver and to correlate the same with polymorphonuclear leukocytes (PMNs). A significant augmentation in LPO and reduction in GSH content was observed in a time of exposure dependent manner in the liver and PMNs of MB and/or PQ treated animals. The expression and catalytic activity of CYP2E1 and GSTA4-4 were significantly increased following MB and/or PQ exposure both in the liver and PMNs. Although the expression of GSTA3-3 was increased, the expression of GSTA1-1 was unaltered after MB and/or PQ treatment in both the liver and PMNs. MB augmented the expression and catalytic activity of CYP1A1 in the liver, however, CYP1A2 was unaffected. PQ, on the other hand, significantly increased hepatic CYP1A2 expression and catalytic activity. MB and/or PQ did not produce any significant changes in CYP1A1 and CYP1A2 in PMNs. The results of the study thus demonstrate that MB and PQ differentially regulate hepatic CYP1A1 and CYP1A2 while LPO, GSH, CYP2E1, GSTA4-4 and GSTA3-3 are modulated in the similar fashions both in the liver and PMNs.

NADPH oxidase mediated maneb- and paraquat-induced oxidative stress in rat polymorphs: Crosstalk with mitochondrial dysfunction

Oxidative stress is a key factor in Parkinsons disease (PD) pathogenesis. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and impaired mitochondrion regulate oxidative stress-mediated maneb (MB)- and paraquat (PQ)-induced Parkinsonism. However, their contribution in the MB- and PQ-induced toxicity in polymorphonuclear leukocytes (PMNs) is still elusive. The study investigated the role of NADPH oxidase and mitochondria in MB- and/or PQ-induced oxidative stress in the PMNs and the crossing point between the two. Animals were treated with MB and/or PQ for 1-3 weeks along with respective controls. In a few sets of experiments, rats were treated with/without NADPH oxidase inhibitor, apocynin, an hour prior to MB and/or PQ treatment. PMNs of MB and/or PQ treated animals were also treated with/without carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to assess the role of the mitochondria in superoxide and total free radical productions. MB and/or PQ were found to increase the level of total reactive oxygen species (ROS), superoxide radicals, catalytic activity and expression of NADPH oxidase and superoxide dismutase (SOD1/2) and mitochondrial ROS content in a time dependent manner. Conversely, catalase activity and mitochondrial membrane potential were attenuated. Apocynin alleviated MB- and/or PQ-induced changes in total ROS, superoxide radicals, expression/catalytic activity of NADPH oxidase and SOD1/2 along with the mitochondrial ROS and membrane potential. CCCP also inhibited ROS and superoxide levels in the PMNs of MB and/or PQ-treated animals. The results demonstrate the involvement of NADPH oxidase and mitochondrial dysfunction in MB and PQ-induced oxidative stress in PMNs and a plausible crosstalk between them.

The manganese-salen compound EUK-134 and N-acetyl cysteine rescue from zinc- and paraquat-induced toxicity in rat polymorphonuclear leukocytes.

Oxidative stress is implicated in toxicant-induced inflammation leading to chronic diseases. Polymorphonuclear leukocytes (PMNs) offer the first line of defense against infection in the mammals and protect against inflammation-mediated pathological anomalies. Conversely, activated PMNs contribute to the oxidative stress-mediated damage and inflammation. The study aimed to investigate the status of oxidative stress and antioxidant defense system in the PMNs of rats treated with/without zinc (Zn) and/or paraquat (PQ) in the presence or absence of a synthetic superoxide dismutase/catalase mimetic, a manganese-salen compound-EUK-134 and/or a glutathione precursor, N-acetyl cysteine (NAC). While Zn and/or PQ elevated the total free radical generation, lipid peroxidation (LPO) and catalytic activity of myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione S-transferase alpha 4-4 (GSTA4-4), a pronounced decrease in reduced glutathione (GSH) and glutathione reductase (GR) activity was also observed. Zn and/or PQ augmented the expression of metallothionein-I and II and GSTA4-4. Pre-treatment of EUK-134 or NAC alone altered the level of total free radical generation, LPO, GSH content and catalytic activity of MPO, SOD, GR and GPx and the expression of metallothionein I and II towards normalcy. The alterations were more pronounced in the PMNs of rats treated with EUK-134 and NAC in combination. Catalytic activity/expression of GSTA4-4 remained unchanged in the PMNs of EUK-134 or NAC treated rats. The results demonstrate that EUK-134 and NAC protect PMNs from the toxic effects of Zn and PQ in rats and also suggest that metallothioneins I/II might contribute to antioxidant defense under GSH depleted conditions.