ABSTRACT
The original version of this article unfortunately contained errors in Fig. 4a. Representative image of b-actin of brain region were copied incorrectly during the preparation of the figures.
ABSTRACT
The original version of this article unfortunately contained an error at Fig. 10.
ABSTRACT
Proteomic studies were carried out in immature (3 week), adult (18 week) and aged (48 week) rats to understand the age dependent vulnerability to lindane induced neurodegeneration. 2-D and western blot analysis of protein extracts of hippocampus and substantia-nigra isolated from lindane treated rats (2.5â¯mg/kg; p.o. X 21 days) revealed marked dysregulation in the expression of proteins related to ubiquitin proteasome pathway, antioxidant activity, chaperones, energy metabolism, calcium homeostasis and proteins involved in neurodegeneration. These alterations were associated with marked increase in reactive oxygen species formation, lipid peroxidation, reduced glutathione content and antioxidant enzyme activities in lindane treated rats. Aged rats, in particular showed higher magnitude of alteration in these proteins when compared to immature or adult rats. Proteins involved in apoptosis and autophagy also showed marked alterations in their expression, particularly in the aged rats. Ultrastructural analysis revealed greater number of autophagic vesicle in hippocampus and substantia-nigra in treated aged rats. The data suggest that proteomic approaches could be used to investigate the vulnerability to lindane induced neurodegeneration in rats.
Subject(s)
Hexachlorocyclohexane/toxicity , Hippocampus/drug effects , Insecticides/toxicity , Proteomics , Substantia Nigra/drug effects , Age Factors , Animals , Antioxidants/metabolism , Apoptosis/drug effects , Autophagy/drug effects , Blotting, Western , Electrophoresis, Gel, Two-Dimensional , Glutathione/metabolism , Hippocampus/metabolism , Hippocampus/ultrastructure , Lipid Peroxidation/drug effects , Male , Microscopy, Electron, Transmission , Molecular Chaperones/metabolism , Nerve Tissue Proteins/metabolism , Oxidative Stress/drug effects , Rats, Wistar , Reactive Oxygen Species/metabolism , Substantia Nigra/metabolism , Substantia Nigra/ultrastructureABSTRACT
Oral administration of low doses of cypermethrin to pregnant Wistar rats led to a dose-dependent differences in the induction of xenobiotic-metabolizing cytochrome P450s (CYPs) messenger RNA (mRNA) and protein in brain regions isolated from the offsprings postnatally at 3 weeks that persisted up to adulthood. Similar alterations were observed in the expression of rate-limiting enzymes of neurotransmitter synthesis in brain regions of rat offsprings. These persistent changes were associated with alterations in circulating levels of growth hormone (GH), cognitive functions, and accumulation of cypermethrin and its metabolites in brain regions of exposed offsprings. Though molecular docking studies failed to identify similarities between the docked conformations of cypermethrin with CYPs and neurotransmitter receptors, in silico analysis identified regulatory sequences of CYPs in the promoter region of rate-limiting enzymes of neurotransmitter synthesis. Further, rechallenge of the prenatally exposed offsprings at adulthood with cypermethrin (p.o. 10 mg/kg × 6 days) led to a greater magnitude of alterations in the expression of CYPs and rate-limiting enzymes of neurotransmitter synthesis in different brain regions. These alterations were associated with a greater magnitude of decrease in the circulating levels of GH and cognitive functions in rechallenged offsprings. Our data has led us to suggest that due to the immaturity of CYPs in fetus or during early development, even the low-level exposure of cypermethrin may be sufficient to interact with the CYPs, which in turn affect the neurotransmission processes and may help in explaining the developmental neurotoxicity of cypermethrin.
Subject(s)
Brain/pathology , Cytochrome P-450 Enzyme System/metabolism , Neurotransmitter Agents/biosynthesis , Prenatal Exposure Delayed Effects/enzymology , Prenatal Exposure Delayed Effects/pathology , Pyrethrins/adverse effects , Xenobiotics/metabolism , Animals , Animals, Newborn , Biocatalysis , Brain/enzymology , Computer Simulation , Female , Gene Expression Regulation , Intercellular Signaling Peptides and Proteins/blood , Learning/drug effects , Male , Metabolome/genetics , Molecular Docking Simulation , Pregnancy , Prenatal Exposure Delayed Effects/blood , Prenatal Exposure Delayed Effects/genetics , Pyrethrins/chemistry , Pyrethrins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats, Wistar , Sequence Analysis, DNA , Spatial Memory/drug effects , Structural Homology, ProteinABSTRACT
RNA interference offers effective control of several economically important insect pests. Bemisia tabaci is an important field crop pest, which causes significant yield loss worldwide. In our earlier study, we have demonstrated successful control of B. tabaci through transgenic plant mediated RNAi. However, selection of target genes without off-target effect(s) has been major concern so far and therefore, a critical exploration for B. tabaci specific targets is frantically required. In this study, we have followed proteomics approach to discover B. tabaci specific targets for RNAi and identified unique nucleotide sequences in functional genes (n=11) of the pest. For this, we have developed proteome profile of B. tabaci extract using two-dimensional electrophoresis. A total of 504 protein spots were analyzed on mass-spectrometer and 453 proteins including 246 non-redundant proteins have been identified successfully. Complementation of the proteome data with available nucleotide database has helped us to interpret the unique nucleotide sequences. These nucleotide stretches may serve as environmentally safe targets for RNAi mediated control of the pest through crop genetic engineering. To the best of our knowledge, it is the most complete proteome of any whitefly species. We have also demonstrated application of proteomics in the identification of functional transcripts for RNAi. BIOLOGICAL SIGNIFICANCE: Insects cause major loss to crop productivity through direct and indirect damages. Among them, hemipteran group of insects are major contributor of global crop yield loss. In current study, gel based proteome profile of B. tabaci (one of the major hemipteran crop insect pest) is developed and characterized, which is a gap area in field of whitefly biology. It is an important data set of future whitefly studies like insect-plant interaction, virulence of whiteflies, their control program and discovery of new pesticides. Out of various control strategies, RNA interference offers a great potential to combat the whitefly successfully. However, the uniqueness of target genes and off target impact of the technology remains a challenge to scientific community. We used our proteome data set for the identification of B. tabaci specific gene targets for RNAi mediated control. The identified genes are critical for the life cycle of B. tabaci hence, could be proven as good molecules for making transgenic crop plant for efficient control of whiteflies in the field.
Subject(s)
Hemiptera/genetics , Hemiptera/metabolism , Insect Proteins/genetics , Insect Proteins/metabolism , Proteome/metabolism , RNA Interference/physiology , Amino Acid Sequence , Animals , Base Sequence , Gene Expression Regulation/genetics , Insect Proteins/chemistry , Molecular Sequence Data , Proteome/chemistry , Proteome/geneticsABSTRACT
Previous studies have reported that lindane, an organochlorine pesticide induces oxidative stress in rat brain that may lead to neurodegeneration. However, as the proteins involved in lindane induced neurodegeneration are yet to be identified, the present study aims to identify the proteins that may regulate lindane induced neurotoxicity. The data showed that repeated exposure of lindane (2.5 mg/kg) for 21 days to adult rats significantly increased the reactive oxygen species and lipid peroxidation in different brain regions. Proteomic study revealed that lindane induces major dysregulation in the ubiquitin proteasome pathway. Alterations in the expression of molecular chaperones in brain regions and an increase in the expression of α-synuclein in substantia-nigra and corpus-striatum and amyloid precursor protein in hippocampus and frontal-cortex suggests the accumulation of proteins in these brain regions. Western blotting also revealed alterations in the dopaminergic and cholinergic pathways in hippocampus and substantia-nigra isolated from lindane treated rats. Neurobehavioural data indicating alterations in learning and working memory, conditioned avoidance response and motor function, supports the proteomic data. The data suggest that repeated exposure of lindane to adult rats induces alterations, which are similar to that seen in neurodegenerative diseases.
Subject(s)
Brain/drug effects , Hexachlorocyclohexane/toxicity , Neurodegenerative Diseases/metabolism , Pesticides/toxicity , Proteome/metabolism , Animals , Brain/metabolism , Catalase/metabolism , Glutathione/metabolism , Lipid Peroxidation/drug effects , Male , Motor Activity/drug effects , Neurodegenerative Diseases/chemically induced , Neurodegenerative Diseases/psychology , Oxidative Stress/drug effects , Proteasome Endopeptidase Complex/metabolism , Rats, Wistar , Reactive Oxygen Species/metabolism , Sensory Receptor Cells/metabolism , Signal Transduction , Superoxide Dismutase/metabolism , Ubiquitin/metabolismABSTRACT
The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A1 increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A1) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cell's compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be established as a biomarker of xenoestrogen exposure.
Subject(s)
Autophagy/drug effects , Benzhydryl Compounds/toxicity , Environmental Pollutants/toxicity , Hippocampus/drug effects , Neurons/drug effects , Phenols/toxicity , Protein Kinases/metabolism , TOR Serine-Threonine Kinases/metabolism , AMP-Activated Protein Kinase Kinases , Androstadienes/pharmacology , Animals , Animals, Newborn , Apoptosis Regulatory Proteins/antagonists & inhibitors , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Autophagy/genetics , Beclin-1 , Benzhydryl Compounds/antagonists & inhibitors , Environmental Pollutants/antagonists & inhibitors , Gene Expression Regulation , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Hippocampus/metabolism , Hippocampus/pathology , Humans , Macrolides/pharmacology , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Neurons/metabolism , Neurons/pathology , Oxidative Stress , Phenols/antagonists & inhibitors , Primary Cell Culture , Protein Kinases/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Sequestosome-1 Protein , Signal Transduction , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/genetics , WortmanninABSTRACT
Oral administration of low doses (1.25, 2.5, or 5 mg/kg) of cypermethrin to pregnant Wistar rats from gestation days 5 to 21 led to dose-dependent differences in the induction of cytochrome P450 2D1 (CYP2D1) and 3A1 messenger RNA (mRNA) and protein in brain regions isolated from the offsprings postnatally at 3 weeks that persisted up to adulthood (12 weeks). Similar alterations were observed in the expression of GABAergic, muscarinic, dopaminergic, and serotonergic neurotransmitter receptors in brain regions of rat offsprings. Rechallenge of the prenatally exposed offsprings at adulthood (12 weeks old) with cypermethrin (p.o., 10 mg/kg for 6 days) led to a greater magnitude of alterations in the expression of CYPs, neurotransmitter receptors, and neurotransmitter receptor binding in the brain regions when compared to the control offsprings treated at adulthood with cypermethrin or prenatally exposed offsprings. A greater magnitude of decrease was also observed in the spontaneous locomotor activity (SLA) in prenatally exposed offsprings rechallenged with cypermethrin. The present data indicating similarities in the alterations in the expression of CYPs (2D1 and 3A1) and neurotransmitter receptors in brain has led us to suggest that endogenous function regulating CYPs is possibly associated with neurotransmission processes. A greater magnitude of alterations in CYP2D1, 3A1, neurotransmitter receptors, and SLA in rechallenged animals has further provided evidence that alterations in CYPs are possibly linked with neurotransmission processes.