Anshuman Singh

Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways

Background: The effects of xenoestrogen bisphenol-A on autophagy, and association with oxidative stress and apoptosis are still elusive. Results: Transient activation of autophagy protects against bisphenol-A-induced neurodegeneration via AMPK activation and mTOR down-regulation. Conclusion: Autophagy induction against bisphenol-A is an early cells tolerance response. Significance: Autophagy provides an imperative biological marker for evaluation of neurotoxicity by xenoestrogen. 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 cells 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.

Reversibility of changes in brain cholinergic receptors and acetylcholinesterase activity in rats following early life arsenic exposure.

In view of the increasing incidences of arsenic induced health effects and the vulnerability of the developing brain to its toxic effects, studies have been carried out to investigate the mechanism of arsenic induced cholinergic alterations and understand if such changes are persistent or transient on withdrawal of arsenic exposure. Male rats were exposed to arsenic (2 mg/kg or 4 mg/kg body weight, p.o) from post‐lactational day (PD)22 to PD59, and the effect on selected behavioral and neurochemical end points associated with cholinergic functions was assessed on PD60 and PD90. Decrease in the binding of muscarinic‐cholinergic receptors in frontal cortex (26%, 43%) and hippocampus (21%, 34%) associated with reduced CHRM2 mRNA levels, acetylcholinesterase activity and expression of ChAT and PKC β‐1 was observed in arsenic exposed rats on PD60 as compared to controls. Spatial learning and memory and muscle strength were affected following arsenic exposure in rats on PD60 and associated with arsenic induced cholinergic alterations. Enhanced oxidative stress associated with increased expression of pro‐apoptotic proteins and decreased expression of anti‐apoptotic proteins was distinct in both frontal cortex and hippocampus following arsenic exposure in rats on PD60. The cholinergic alterations and other neurochemical modifications were found to be linked with increased arsenic levels in frontal cortex (1.39, 3.90‐fold) and hippocampus (3.23, 5.48‐fold) on PD60. Although a trend of recovery was observed both in behavioral and neurochemical endpoints on withdrawal of arsenic exposure on PD90, the results indicate that continuous arsenic exposure may have detrimental effects.

Identification of Drosophila-Based Endpoints for the Assessment and Understanding of Xenobiotic-Mediated Male Reproductive Adversities

Men are at risk of becoming completely infertile due to innumerable environmental chemicals and pollutants. These xenobiotics, hence, should be tested for their potential adverse effects on male fertility. However, the testing load, a monumental challenge for employing conventional animal models, compels the pursuit of alternative models. Towards this direction, we show here that Drosophila melanogaster, an invertebrate, with its well characterized/conserved male reproductive processes/proteome, recapitulates male reproductive toxicity phenotypes observed in mammals when exposed to a known reproductive toxicant, dibutyl phthalate (DBP). Analogous to mammals, exposure to DBP reduced fertility, sperm counts, seminal proteins, increased oxidative modification/damage in reproductive tract proteins and altered the activity of a hormone receptor (estrogen related receptor) in Drosophila males. In addition, we show here that DBP is metabolized to monobutyl phthalate (MBP) in exposed Drosophila males and that MBP is more toxic than DBP, as observed in higher organisms. These findings suggest Drosophila as a potential alternative to traditional animal models for the prescreening of chemicals for their reproductive adversities and also to gain mechanistic insights into chemical-mediated endocrine disruption and male infertility.

Brain cholinergic alterations in rats subjected to repeated immobilization or forced swim stress on lambda-cyhalothrin exposure

Role of immobilization stress (IMS), a psychological stressor and forced swim stress (FSS), a physical stressor was investigated on the neurobehavioral toxicity of lambda-cyhalothrin (LCT), a new generation type-II synthetic pyrethroid. Pre-exposure of rats to IMS (15 min/day) or FSS (3 min/day) for 28 days on LCT (3.0 mg/kg body weight, p.o.) treatment for 3 days resulted to decrease spatial learning and memory and muscle strength associated with cholinergic-muscarinic receptors in frontal cortex and hippocampus as compared to those exposed to IMS or FSS or LCT alone. Decrease in acetylcholinesterase activity, protein expression of ChAT and PKC-β1 associated with decreased mRNA expression of CHRM2, AChE and ChAT in frontal cortex and hippocampus was also evident in rats pre-exposed to IMS or FSS on LCT treatment, compared to rats exposed to IMS or FSS or LCT alone. Interestingly, changes both in behavioral and neurochemical endpoints were marginal in rats subjected to IMS or FSS for 28 days or those exposed to LCT for 3 days alone, compared to controls. The results suggest that stress is an important contributor in LCT induced cholinergic deficits.

Effect of prenatal exposure of lindane on alterations in the expression of cerebral cytochrome P450s and neurotransmitter receptors in brain regions

Prenatal exposure to low doses (0.0625- or 0.125- or 0.25 mg/kg b. wt., orally) of lindane, an organochlorine insecticide, from gestation day (GD) 5-21 was found to produce a dose-dependent increase in the mRNA expression of cytochrome P450s (CYPs) and associated transcription factors in frontal cortex, cerebellum and corpus striatum isolated from the offsprings. Though the increase in the expression persisted up to postnatal day 60, the increase was significant at postnatal days 21-, and 45- in the offsprings exposed prenatally to relatively higher doses (0.125- or 0.25 mg/kg) of lindane and even up to postnatal day 60 in the offsprings exposed prenatally to the highest dose of lindane. A similar increase in the expression of dopamine D2, 5HT2A and GABAA receptors and associated neurotransmitter receptor binding was observed in the brain regions of the exposed offsprings. Scatchard analysis also suggested an increase in the levels of these neurotransmitter receptors in offsprings prenatally exposed to lindane. The data indicating similarities in the alterations of neurotransmitter receptors and CYPs in brain regions in prenatally exposed offsprings have suggested that neurotransmission processes and CYPs are closely linked that will eventually help in understanding the developmental neurotoxicity of lindane.

Functional male accessory glands and fertility in Drosophila require novel ecdysone receptor

In many insects, the accessory gland, a secretory tissue of the male reproductive system, is essential for male fertility. Male accessory gland is the major source of proteinaceous secretions, collectively called as seminal proteins (or accessory gland proteins), which upon transfer, manipulate the physiology and behavior of mated females. Insect hormones such as ecdysteroids and juvenoids play a key role in accessory gland development and protein synthesis but little is known about underlying molecular players and their mechanism of action. Therefore, in the present study, we examined the roles of hormone-dependent transcription factors (Nuclear Receptors), in accessory gland development, function and male fertility of a genetically tractable insect model, Drosophila melanogaster. First, we carried out an RNAi screen involving 19 hormone receptors, individually and specifically, in a male reproductive tissue (accessory gland) for their requirement in Drosophila male fertility. Subsequently, by using independent RNAi/ dominant negative forms, we show that Ecdysone Receptor (EcR) is essential for male fertility due to its requirement in the normal development of accessory glands in Drosophila: EcR depleted glands fail to make seminal proteins and have dying cells. Further, our data point to a novel ecdysone receptor that does not include Ultraspiracle but is probably comprised of EcR isoforms in Drosophila male accessory glands. Our data suggest that this novel ecdysone receptor might act downstream of homeodomain transcription factor paired (prd) in the male accessory gland. Overall, the study suggests novel ecdysone receptor as an important player in the hormonal regulation of seminal protein production and insect male fertility.

Similarities in lindane induced alterations in protein expression profiling in different brain regions with neurodegenerative diseases

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.

Proteomic approaches to investigate age related vulnerability to lindane induced neurodegenerative effects in rats

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 (2.5 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.

Correction: Functional male accessory glands and fertility in Drosophila require novel ecdysone receptor

[This corrects the article DOI: 10.1371/journal.pgen.1006788.].