Tempol and silymarin rescue from zinc-induced degeneration of dopaminergic neurons through modulation of oxidative stress and inflammation.

Oxidative stress and inflammation are the key players in the toxic manifestation of sporadic Parkinson's disease and zinc (Zn)-induced dopaminergic neurodegeneration. A synthetic superoxide dismutase (SOD) mimetic, tempol, and a naturally occurring antioxidant, silymarin protect against oxidative stress-mediated damage. The study intended to explore the effects of tempol and silymarin against Zn-induced dopaminergic neurodegeneration. Exposure to Zn produced neurobehavioral deficits and striatal dopamine depletion. Zn reduced glutathione content and glutathione-S-transferase activity and increased lipid peroxidation, superoxide dismutase activity, and level of pro-inflammatory mediators [nuclear factor-kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6)]. Zn also attenuated the expression of tyrosine hydoxylase (TH), vesicular monoamine transporter 2 (VMAT-2), mitochondrial B-cell lymphoma-2 (Bcl-2), and procaspase-3 and 9 proteins and number of TH-positive neurons. Conversely, Zn elevated the expression of dopamine transporter (DAT) and mitochondrial Bcl-2-associated X (Bax) protein along with mitochondrial cytochrome c release. Administration of tempol significantly alleviated Zn-induced motor impairments, dopamine depletion, reduction in TH expression, and loss of TH-positive neurons similar to silymarin. Silymarin mitigated Zn-induced oxidative stress and inflammation and restored the expression of dopamine transporters and levels of pro-apoptotic proteins akin to tempol. The results demonstrate that both tempol and silymarin protect against Zn-induced dopaminergic neuronal loss through the suppression of oxidative stress and inflammation.

Cyclooxygenase-2 activates the free radical-mediated apoptosis of polymorphonuclear leukocytes in the maneb- and paraquat-intoxicated rats.

Overproduction of free radicals and inflammation could lead to maneb (MB)- and paraquat (PQ)-induced toxicity in the polymorphonuclear leukocytes (PMNs). Cyclooxygenase-2 (COX-2), an inducible COX, is imperative in the pesticides-induced pathological alterations. However, its role in MB- and PQ-induced toxicity in the PMNs is not yet clearly deciphered. The current study explored the contribution of COX-2 in MB- and PQ-induced toxicity in the PMNs and the mechanism involved therein. Combined MB and PQ augmented the production of free radicals, lipid peroxides and activity of superoxide dismutase (SOD) in the rat PMNs. While combined MB and PQ elevated the expression of COX-2 protein, activation of nuclear factor-kappa B (NF-κB) and phosphorylation of c-Jun N-terminal kinase (JNK), release of mitochondrial cytochrome c and levels of procaspase-3/9 were attenuated in the PMNs. Celecoxib (CXB), a COX-2 inhibitor, ameliorated the combined MB and PQ-induced modulations in the PMNs. MB and PQ augmented the free radical generation, COX-2 protein expression, NF-κB activation and JNK phosphorylation and reduced the cell viability of cultured rat PMNs and human leukemic HL60. MB and PQ elevated mitochondrial cytochrome c release and poly (ADP-ribose) polymerase cleavage whilst procaspase-3/9 levels were attenuated in the cultured PMNs. MB and PQ also increased the levels of phosphorylated c-jun and caspase-3 activity in the HL60 cells. CXB; SP600125, a JNK-inhibitor and pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor, rescued from MB and PQ-induced changes in the PMNs and HL60 cells. However, CXB offered the maximum protection among the three. The results show that COX-2 activates apoptosis in the PMNs following MB and PQ intoxication, which could be linked to NF-κB and JNK signaling.

Mitochondrial Dysfunction Contributes To Zinc-induced Neurodegeneration: a Link with NADPH Oxidase.

Mitochondrial dysfunction and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) are the major sources of augmentation in free radical generation leading to neurodegeneration. Although NADPH oxidase involvement is reported in zinc (Zn)-induced neurodegeneration, contribution of the mitochondrial dysfunction and its association with NADPH oxidase are not known. Therefore, the study was aimed to decipher the role of mitochondrial dysfunction and its link with NADPH oxidase in Zn-induced Parkinsonism. Zn reduced the motor activities, the number of tyrosine hydroxylase (TH)-positive neurons, and level of TH protein. Conversely, Zn increased the mitochondrial reactive oxygen species (ROS) production, lipid peroxidation (LPO), and superoxide dismutase (SOD) activity and reduced the mitochondrial membrane potential and catalytic activities of complex I and III. Zn also attenuated B-cell lymphoma-2 (Bcl-2) and pro-caspase 9/3 levels and augmented the translocation of cytosolic Bcl-2 associated X (Bax) protein to the mitochondria and cytochrome c release into cytosol from the mitochondria. Cyclosporine A, a mitochondrial outer membrane transition pore inhibitor and apocynin, a NADPH oxidase inhibitor, independently, ameliorated the Zn-induced changes. Similarly, Zn reduced cell viability through mitochondrial dysfunction and apoptosis in human neuroblastoma SH-SY5Y cells, which were notably normalized in the presence of cyclosporine or apocynin. The results demonstrate that mitochondrial dysfunction contributes to Zn-induced neurodegeneration, which could be partially aided by the NADPH oxidase.

Autonomous regulation of inducible nitric oxide synthase and cytochrome P450 2E1-mediated oxidative stress in maneb- and paraquat-treated rat polymorphs.

Maneb (MB)- and paraquat (PQ)-induced oxidative stress in rat polymorphonuclear leukocytes (PMNs) is regulated in parallel by cytochrome P450 2E1 (CYP2E1) and inducible nitric oxide synthase (iNOS). However, mechanism underlying their regulation is not yet understood. The study investigated the role of nuclear factor- kappa B (NF-κB) and mitogen-activated protein kinase/extracellular signal regulated kinase/protein kinase C (MEK/ERK/PKC) pathway in the regulation of iNOS- and CYP2E1-induced oxidative stress in PMNs. MB + PQ-induced changes in nitrite content, lipid peroxidation (LPO), iNOS expression/activity and inflammatory mediators were alleviated by aminoguanidine (AG), an iNOS inhibitor, without any change in CYP2E1. Alternatively, diallyl sulphide (DAS), a CYP2E1 inhibitor, rescued from MB + PQ-induced changes in CYP2E1 activity/expression, free radical generation, superoxide dismutase (SOD) activity, LPO and pro-inflammatory cytokines without any alterations in nitrite content and iNOS activity/expression. Pyrrolidine dithiocarbamate (PDTC), NF-κB inhibitor, did not alter CYP2E1 but mitigated free radical generation, SOD activity, LPO, nitrite content, iNOS activity/expression and levels of pro-inflammatory cytokines (tumor necrosis factor-α, interleukine-1ß and interleukine-4). Ex-vivo treatment with MEK inhibitor (PD98059), ERK1/2 inhibitor (AG126) or PKC inhibitor (rottlerin) ameliorated MB + PQ-induced increase in free radical generation and CYP2E1 activity/expression in PMNs. While PD98059 and AG126 abated MB + PQ-induced increase in ERK1/2, PKC-α/δ and CYP2E1 levels, rottlerin restored PKC-α/δ and CYP2E1 towards normalcy without affecting ERK1/2 level in MB + PQ-treated group. The results suggest that iNOS and CYP2E1 contributing to MB + PQ-induced oxidative stress in rat PMNs exhibit differential regulatory mechanisms. The inflammatory mediators regulate iNOS expression while CYP2E1 expression is triggered via MEK-ERK1/2-PKC pathway.

Postnatal zinc or paraquat administration increases paraquat or zinc-induced loss of dopaminergic neurons: insight into augmented neurodegeneration.

Epidemiological evidences have shown an association of exposure to pesticides or heavy metals with increased incidences of Parkinson's disease (PD) in humans. Exposure to pesticides or metals during the decisive period of the brain development increases the susceptibility of dopaminergic neurons upon re-exposure in adult rodents. However, the effect of early life exposure to pesticide on the heavy metal-induced neurodegeneration or heavy metal on pesticide-induced neurodegeneration is not yet explored. The current study explored the effect of developmental exposure to zinc (Zn), a metal or paraquat (PQ), a pesticide on the nigrostriatal dopaminergic neurons of rats challenged to Zn or PQ during adulthood. Exposure of Zn or PQ during adulthood alone exhibited marked reduction in motor activities, striatal dopamine and metabolites, glutathione content and number of dopaminergic neurons. However, the levels of lipid peroxidation, protein carbonyls, superoxide dismutase activity, pro-inflammatory cytokines and 4-hydroxynonenal-protein adducts were increased. While the expression of vesicular monoamine transporter-2 and tyrosine hydroxylase were attenuated, dopamine transporter and microglial marker Iba-1 expression, activated microglia, nuclear factor-kappa B activation, mitochondrial cytochrome c release and caspase-3/9 activation were augmented following Zn or PQ exposure. Albeit postnatal alone exposure did not alter any of the studied parameters, the developmental administration of Zn/PQ in re-challenged adult rats produced more pronounced changes in the aforementioned variables as compared with adulthood Zn or PQ alone intoxicated animals. The results demonstrate that postnatal Zn/PQ intoxication dents the oxidative stress, inflammation, cell death and dopamine metabolism and storage regulating machineries, which speed up the toxicant-induced degeneration during adulthood.

Correction to: CYP2E1-mediated oxidative stress regulates HO-1 and GST expression in maneb- and paraquat-treated rat polymorphonuclear leukocytes.

In the original publication of the article, the wrong ß-actin blots were pasted in Figs. 1b and 2c. The correct versions of Figs. 1b and 2c are given in this correction.

Inhibition of glutathione S-transferase-pi triggers c-jun N-terminal kinase-dependent neuronal death in Zn-induced Parkinsonism.

Oxidative stress is recognized as one of the major wrongdoers in Parkinson's disease (PD) while glutathione S-transferase (GST), an endogenous antioxidant, protects from oxidative stress-induced neurodegeneration. Despite GST-pi (GST-π) encounters the toxic manifestations in PD, its role in zinc (Zn)-induced nigrostriatal dopaminergic neurodegeneration remains elusive. The study aimed to explore the role of GST-π in Zn-induced Parkinsonism and its underlying molecular mechanism. Male Wistar rats were treated intraperitoneally with zinc (zinc sulfate), twice a week, for 2-12 weeks. GST-π inducer, benzyl isothiocyanate (BITC) was also administered in a few sets of experiments along with respective vehicle. Catalytic activity and expression of GST-π protein, total GST activity, neurobehavioral indexes, striatal dopamine and its metabolites, nigral tyrosine hydroxylase (TH)-positive neurons and expression of TH and B-cell lymphoma-2 (Bcl-2) proteins were reduced in Zn-treated rats. Conversely, oxidative stress indicators, c-jun N-terminal kinase (JNK) activation, c-jun phosphorylation, cytochrome c release, Bcl-2-associated X protein (Bax) translocation, and procaspase 3/9 to caspase 3/9 conversion were significantly increased in Zn-exposed rats. BITC ameliorated GST-π activity/expression and normalized Zn-induced changes in neurodegenerative indicators, oxidative stress, JNK activation, c-jun phosphorylation and apoptotic indexes. The results demonstrate that Zn inhibits GST-π expression leading to increased oxidative stress and JNK activation, which induce apoptosis thereby degeneration of the nigrostriatal dopaminergic neurons.

Ionic liquid based ultrasound assisted dispersive liquid-liquid micro-extraction for simultaneous determination of 15 neurotransmitters in rat brain, plasma and cell samples.

Neurotransmitters are signaling molecules which play a key role in the central nervous system allowing signal transmission in the neuronal synapses. The role of these compounds is very crucial in the biological systems. A rapid, sensitive, economical and derivatisation free method has been developed for the analysis of 15 neurotransmitters in a single run on liquid chromatography tandem mass spectrometry. These 15 neurotransmitters are categorized into 5 groups on the basis of their fragmentation pattern. The proposed method "ionic liquid based ultrasound assisted dispersive liquid-liquid microextraction" hyphenated with tandem mass spectrometry is the first report for the analysis of neurotransmitters in cell samples along with other two matrices (rat brain and plasma). All the parameters that influence the extraction efficiency are optimized with aid of response surface methodology and desirability profile. Under these optimized conditions the developed method has been validated. The limit of detection was in the range of (1) 0.021-0.912 µg L-1 for rat brain samples, (2) 0.028-0.978 µg L-1 for plasma samples and (3) 0.025-0.945 µg L-1 for cell samples with good linearity behavior for all analytes in the concentration range of 0.04-200 µg L-1 in all the three matrices. The coefficient of determination for all the neurotransmitters was found in the range of (1) (R2) ≥ 0.996 to 0.999 for rat brain samples and (2) (R2) ≥ 0.991 to 0.999 for plasma and cell samples. The intra-day and inter-day variations were found less than (1) 1.78% and 8.94% for rat brain samples, (2) 1.83% and 8.37% for plasma samples and (3) 1.64% and 8.04% for cell samples respectively. The method has mean recoveries varied between (1) 81-128% for brain samples, (2) 88-107% for plasma samples and (3) 91-104% for cell samples at different spiking levels. The optimized and validated method was found free from matrix interferences and successfully applied for quantitative determination of 15 neurotransmitters in the rat brain, plasma and cell samples.

Cyclooxygenase-2 Directs Microglial Activation-Mediated Inflammation and Oxidative Stress Leading to Intrinsic Apoptosis in Zn-Induced Parkinsonism.

Inflammation is decisive in zinc (Zn)-induced nigrostriatal dopaminergic neurodegeneration; however, the contribution of cyclooxygenase-2 (COX-2) is not yet known. The present study aimed to explore the role of COX-2 in Zn-induced Parkinsonism and its association with the microglial activation. Male Wistar rats were treated intraperitoneally (i.p.) with Zn as zinc sulphate (20 mg/kg) along with respective controls for 2-12 weeks. In a few sets, animals were also treated with/without celecoxcib (CXB, 20 mg/kg, i.p.), a selective COX-2 inhibitor. Indexes of the nigrostriatal neurodegeneration, oxidative stress, inflammation and apoptosis were measured in the animals/nigrostriatal tissue. Zn induced time-dependent increase in the expression of COX-2 while COX-1 expression was unaltered. Zn reduced the neurobehavioral activities, striatal dopamine content, tyrosine hydroxylase (TH) expression and number of dopaminergic neurons. While oxidative stress; microglial activation; expression of microglial cell surface marker-CD11b; cytochrome c release; caspase-9/3 activation; level of pro-inflammatory cytokines, such as TNF-α, IL-1ß and IL-6 and Bcl-2-associated protein x (Bax) translocation from the cytosol to mitochondria were induced in the Zn-treated group, expression of B-cell lymphoma-2 (Bcl-2) was found to be reduced. CXB significantly attenuated Zn-induced increase in COX-2 expression and restored TH-expression, dopamine content, level of inflammatory cytokines and neurobehavioral indexes towards normalcy. Moreover, CXB also attenuated Zn-induced increase in microglial activation, oxidative stress and apoptotic markers towards normal levels. Results of the study thus demonstrate that COX-2 induces microglial activation that provokes the release of inflammatory mediators, which in turn augments oxidative stress and intrinsic apoptosis leading to dopaminergic neurodegeneration in Zn-induced Parkinsonism.

Alpha-synuclein aggregation, Ubiquitin proteasome system impairment, and L-Dopa response in zinc-induced Parkinsonism: resemblance to sporadic Parkinson's disease.

Alpha-synuclein (α-synuclein) aggregation and impairment of the Ubiquitin proteasome system (UPS) are implicated in Parkinson's disease (PD) pathogenesis. While zinc (Zn) induces dopaminergic neurodegeneration resulting in PD phenotype, its effect on protein aggregation and UPS has not yet been deciphered. The current study investigated the role of α-synuclein aggregation and UPS in Zn-induced Parkinsonism. Additionally, levodopa (L-Dopa) response was assessed in Zn-induced Parkinsonian model to establish its closeness with idiopathic PD. Male Wistar rats were treated with zinc sulfate (Zn; 20 mg/kg; i.p.) twice weekly for 12 weeks along with respective controls. In few subsets, animals were subsequently treated with L-Dopa for 21 consecutive days following Zn exposure. A significant increase in total and free Zn content was observed in the substantia nigra of the brain of exposed groups. Zn treatment caused neurobehavioral anomalies, striatal dopamine decline, and dopaminergic neuronal cell loss accompanied with a marked increase in α-synuclein expression/aggregation and Ubiquitin-conjugated protein levels in the exposed groups. Zn exposure substantially reduced UPS-associated trypsin-like, chymotrypsin-like, and caspase-like activities along with the expression of SUG1 and ß-5 subunits of UPS in the nigrostriatal tissues of exposed groups. L-Dopa treatment rescued from Zn-induced neurobehavioral deficits and restored dopamine levels towards normalcy; however, Zn-induced dopaminergic neuronal loss, reduction in tyrosine hydroxylase expression, and increase in oxidative stress were unaffected. The results suggest that Zn caused UPS impairment, resulting in α-synuclein aggregation subsequently leading to dopaminergic neurodegeneration, and that Zn-induced Parkinsonism exhibited positive L-Dopa response similar to sporadic PD.

Implementation and results of an integrated data quality assurance protocol in a randomized controlled trial in Uttar Pradesh, India.

BACKGROUND: There are few published standards or methodological guidelines for integrating Data Quality Assurance (DQA) protocols into large-scale health systems research trials, especially in resource-limited settings. The BetterBirth Trial is a matched-pair, cluster-randomized controlled trial (RCT) of the BetterBirth Program, which seeks to improve quality of facility-based deliveries and reduce 7-day maternal and neonatal mortality and maternal morbidity in Uttar Pradesh, India. In the trial, over 6300 deliveries were observed and over 153,000 mother-baby pairs across 120 study sites were followed to assess health outcomes. We designed and implemented a robust and integrated DQA system to sustain high-quality data throughout the trial. METHODS: We designed the Data Quality Monitoring and Improvement System (DQMIS) to reinforce six dimensions of data quality: accuracy, reliability, timeliness, completeness, precision, and integrity. The DQMIS was comprised of five functional components: 1) a monitoring and evaluation team to support the system; 2) a DQA protocol, including data collection audits and targets, rapid data feedback, and supportive supervision; 3) training; 4) standard operating procedures for data collection; and 5) an electronic data collection and reporting system. Routine audits by supervisors included double data entry, simultaneous delivery observations, and review of recorded calls to patients. Data feedback reports identified errors automatically, facilitating supportive supervision through a continuous quality improvement model. RESULTS: The five functional components of the DQMIS successfully reinforced data reliability, timeliness, completeness, precision, and integrity. The DQMIS also resulted in 98.33% accuracy across all data collection activities in the trial. All data collection activities demonstrated improvement in accuracy throughout implementation. Data collectors demonstrated a statistically significant (p = 0.0004) increase in accuracy throughout consecutive audits. The DQMIS was successful, despite an increase from 20 to 130 data collectors. CONCLUSIONS: In the absence of widely disseminated data quality methods and standards for large RCT interventions in limited-resource settings, we developed an integrated DQA system, combining auditing, rapid data feedback, and supportive supervision, which ensured high-quality data and could serve as a model for future health systems research trials. Future efforts should focus on standardization of DQA processes for health systems research. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT02148952 . Registered on 13 February 2014.

Neuronal Nitric Oxide Synthase Negatively Regulates Zinc-Induced Nigrostriatal Dopaminergic Neurodegeneration.

The study aimed to investigate the role of NO and neuronal NO synthase (nNOS) in Zn-induced neurodegeneration. Animals were treated with zinc sulfate (20 mg/kg), twice a week, for 2-12 weeks along with control. In a few sets, animals were also treated with/without a NO donor, sodium nitroprusside (SNP), or S-nitroso-N-acetyl penicillamine (SNAP) for 12 weeks. Moreover, human neuroblastoma (SH-SY-5Y) cells were also employed to investigate the role of nNOS in Zn-induced toxicity in in vitro in the presence/absence of nNOS inhibitor, 7-nitroindazole (7-NI). Zn caused time-dependent reduction in nitrite content and total/nNOS activity/expression. SNP/SNAP discernibly alleviated Zn-induced neurobehavioral impairments, dopaminergic neurodegeneration, tyrosine hydroxylase (TH) expression, and striatal dopamine depletion. NO donors also salvage from Zn-induced increase in lipid peroxidation (LPO), mitochondrial cytochrome c release, and caspase-3 activation. While Zn elevated LPO content, it attenuated nitrite content, nNOS activity, and glutathione level along with the expression of TH and nNOS in SH-SY-5Y cells. 7-NI further augmented Zn-induced changes in the cell viability, oxidative stress, and expression of TH and nNOS. The results obtained thus demonstrate that Zn inhibits nNOS that partially contributes to an increase in oxidative stress, which subsequently leads to the nigrostriatal dopaminergic neurodegeneration.

IFN-γ regulates xanthine oxidase-mediated iNOS-independent oxidative stress in maneb- and paraquat-treated rat polymorphonuclear leukocytes.

Maneb (MB) and paraquat (PQ) provoke oxidative stress-mediated cell damage. Role of xanthine oxidase (XO) in oxidative stress and its association with nitric oxide (NO)/NO synthase (NOS) have been widely reported. While inducible NOS (iNOS) is implicated in MB+PQ-induced toxicity in rat polymorphonuclear leukocytes (PMNs), role of XO and its alliance with iNOS have not yet been established. The study investigated the role of XO in MB+PQ-induced oxidative stress in rat PMNs and its regulation by iNOS and inflammatory cytokines. MB+PQ-augmented reactive oxygen species (ROS), superoxide, nitro-tyrosine, lipid peroxidation (LPO), and nitrite levels along with the catalytic activity of iNOS, superoxide dismutase (SOD), and XO. XO inhibitor, allopurinol (AP), alleviated MB+PQ-induced changes except nitrite content and iNOS activity. Conversely, an iNOS inhibitor, aminoguanidine, mitigated MB+PQ-induced LPO, nitrite, iNOS, and nitro-tyrosine levels; however, no change was observed in ROS, SOD, and XO. Nuclear factor-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), tumor necrosis factor-alpha (TNF-α) inhibitor, pentoxyfylline, and an anti-inflammatory agent, dexamethasone, attenuated MB+PQ-induced increase in XO, superoxide, and ROS with parallel reduction in the expression of interferon-gamma (IFN-γ), TNF-α, and interleukin-1ß (IL-1ß) in rat PMNs. Exogenous IFN-γ, TNF-α, and IL-1ß enhanced superoxide, ROS, and XO in the PMNs of control and MB+PQ-treated rats; however, IFN- γ was found to be the most potent inducer. Moreover, AP ameliorated cytokine-induced free radical generation and restored XO activity towards normalcy. The results thus demonstrate that XO mediates oxidative stress in MB+PQ-treated rat PMNs via iNOS-independent but cytokine (predominantly IFN-γ)-dependent mechanism.

Inflammation and B-cell Lymphoma-2 Associated X Protein Regulate Zinc-Induced Apoptotic Degeneration of Rat Nigrostriatal Dopaminergic Neurons.

Clinical evidences showing zinc (Zn) accumulation in the post-mortem brain of Parkinson's disease (PD) patients and experimental studies on rodents chronically exposed to Zn suggested its role in PD. While oxidative stress is implicated in Zn-induced neurodegeneration, roles of inflammation and apoptosis in degeneration of the nigrostriatal dopaminergic neurons have yet been elusive. The present study investigated the contribution of the nuclear factor kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), and B-cell lymphoma 2 (Bcl-2) family proteins in Zn-induced Parkinsonism. Male Wistar rats were treated with/without zinc sulfate (Zn; 20 mg/kg, intraperitoneally), twice a week, for 2-12 weeks. In a few sets, animals were treated intraperitoneally with a NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC; 100 mg/kg), a TNF-α inhibitor, pentoxyfylline (PTX; 50 mg/kg), and an anti-inflammatory agent, dexamethasone (DEX; 5 mg/kg), prior to Zn exposure along with respective controls. Zn caused neurobehavioral impairments and reduction in dopamine and its metabolites, tyrosine hydroxylase (TH)-positive neurons, catalase activity, and expression of TH, Bcl-2, and NOXA. On the contrary, Zn augmented lipid peroxidation, activity of superoxide dismutase, expression of TNF-α, IL-1ß, Bcl-xl, and p53-upregulated modulator of apoptosis (PUMA), and translocation of NF-κB and Bax from the cytosol to the nucleus and mitochondria, respectively, with concomitant increase in the mitochondrial cytochrome c release and activation of procaspase-3 and -9. Pre-treatment with PTX, DEX, or PDTC invariably ameliorated Zn-induced changes in behavioral and neurodegenerative indexes, inflammatory mediators, and apoptosis. Results demonstrate that inflammation regulates Bax expression that subsequently contributes to the nigrostriatal dopaminergic neurodegeneration.

Minocycline Rescues from Zinc-Induced Nigrostriatal Dopaminergic Neurodegeneration: Biochemical and Molecular Interventions.

Accumulation of zinc (Zn) in dopaminergic neurons is implicated in Parkinson's disease (PD), and microglial activation plays a critical role in toxin-induced Parkinsonism. Oxidative stress is accused in Zn-induced dopaminergic neurodegeneration; however, its connection with microglial activation is still not known. This study was undertaken to elucidate the role and underlying mechanism of microglial activation in Zn-induced nigrostriatal dopaminergic neurodegeneration. Male Wistar rats were treated intraperitoneally with/without zinc sulphate (20 mg/kg) in the presence/absence of minocycline (30 mg/kg), a microglial activation inhibitor, for 2-12 weeks. While neurobehavioral and biochemical indexes of PD and number of dopaminergic neurons were reduced, the number of microglial cells was increased in the substantia nigra of the Zn-exposed animals. Similarly, Zn elevated lipid peroxidation (LPO) and activities of superoxide dismutase (SOD) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; however, catalase activity was reduced. Besides, Zn increased an association of NADPH oxidase subunit p67(phox) with membrane, cytochrome c release from the mitochondria and cleavage of pro-caspase 3. Zn attenuated the expression of tyrosine hydroxylase (TH) and vesicular monoamine transporter-2 (VMAT-2) while augmented the expression of dopamine transporter (DAT) and heme oxygenase-1 (HO-1). Minocycline alleviated Zn-induced behavioural impairments, loss of TH-positive neurons, activated microglial cells and biochemical indexes and modulated the expression of studied genes/proteins towards normalcy. The results demonstrate that minocycline reduces the number of activated microglial cells and oxidative stress, which rescue from Zn-induced changes in the expression of monoamine transporter and nigrostriatal dopaminergic neurodegeneration.

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

Oxidative stress is a key factor in Parkinson's 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.

CYP2E1-mediated oxidative stress regulates HO-1 and GST expression in maneb- and paraquat-treated rat polymorphonuclear leukocytes.

Cytochrome P4502E1 (CYP2E1), glutathione-S-transferase A4-4 (GSTA4-4), and inducible nitric oxide synthase (iNOS) are implicated in maneb- and paraquat-induced toxicity leading to various pathological conditions. The study aimed to investigate the role of CYP2E1 in maneb- and paraquat-induced oxidative stress in rat polymorphonuclear leukocytes (PMNs) and its crosstalk with iNOS-mediated nitrosative stress and GSTA4-4-linked protective effect, if any and their consequent links with the nuclear factor erythoid 2-related factor 2 (Nrf2) activation and heme oxygenase-1 (HO-1) expression. Rats were treated with/without maneb and/or paraquat for 1, 2, and 3 weeks along with vehicle controls. Subsets of rats were also treated with diallyl sulfide (DAS) or aminoguanidine (AG) along with the respective controls. Maneb and paraquat augmented the reactive oxygen species (ROS), lipid peroxidation (LPO) and 4-hydroxy nonenal (4-HNE) contents, and superoxide dismutase (SOD) activity in the PMNs. However, maneb and paraquat attenuated the reduced glutathione (GSH) level and the expression/activity of total GST and GST-pi. Maneb and paraquat increased the expression/activity of CYP2E1, GSTA4-4, iNOS, Nrf2 and HO-1, and nitrite content. CYP2E1 inhibitor, DAS noticeably alleviated maneb- and paraquat-induced ROS, LPO, 4-HNE, SOD, Nrf2 and HO-1, GST, GSH, and GST-pi while iNOS, nitrite content and GSTA4-4 levels were unchanged. Conversely, AG, an iNOS inhibitor, attenuated maneb- and paraquat-directed changes in nitrite, LPO, iNOS but it did not alter ROS, GSH, SOD, GST, GST-pi, Nrf2, HO-1, CYP2E1, and GSTA4-4. The results demonstrate that CYP2E1 induces iNOS-independent free radical generation and subsequently modulates the Nrf2-dependent HO-1 and 4-HNE-mediated GST expression in maneb- and paraquat-treated PMNs.

Silymarin- and melatonin-mediated changes in the expression of selected genes in pesticides-induced Parkinsonism.

Parkinson's disease (PD) is the second most unconcealed neurodegenerative disorder labelled with motor impairments. Two pesticides, manganese ethylene-1,2-bisdithiocarbamate (maneb) and 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat), together, are reported to increase the incidence of PD in humans and Parkinsonism in mice. Conversely, silymarin and melatonin, two naturally occurring antioxidants, rescue from maneb- and paraquat-induced Parkinsonism. The study examined silymarin- and melatonin-mediated changes in the expression of selected genes in maneb- and paraquat-induced Parkinsonism employing mouse discover chips microarrays. The mice were treated intraperitoneally (i.p.), daily, with silymarin (40 mg/kg) or melatonin (30 mg/kg) for 9 weeks along with vehicles. Subsets of animals were also treated with maneb (30 mg/kg; i.p.) and paraquat (10 mg/kg; i.p.), twice a week, for 9 weeks. Whilst the expression of genes in the striatum was determined by microarray, the expression of randomly selected transcripts was validated by quantitative real-time polymerase chain reaction (qRT-PCR). Combined maneb- and paraquat-treatment altered the expression of several genes associated with apoptosis, inflammation, cell cycle, cell-signalling, etc. pathways. Silymarin and melatonin significantly resisted the changes in the expression of a few genes related to apoptosis, inflammation, cell cycle, cell-signalling, etc. The expression patterns of seven randomly selected genes were analyzed by qRT-PCR, which were found to follow the similar trends, as observed with microarray. The results obtained from the study thus demonstrate that despite resemblances, silymarin and melatonin differentially offset maneb- and paraquat-induced changes in transcriptome.

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-1ß (IL-1ß) 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-1ß 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.