Sadaf Jahan

Differential responses of Trans -Resveratrol on proliferation of neural progenitor cells and aged rat hippocampal neurogenesis

The plethora of literature has supported the potential benefits of Resveratrol (RV) as a life-extending as well as an anticancer compound. However, these two functional discrepancies resulted at different concentration ranges. Likewise, the role of Resveratrol on adult neurogenesis still remains controversial and less understood despite its well documented health benefits. To gather insight into the biological effects of RV on neurogenesis, we evaluated the possible effects of the compound on the proliferation and survival of neural progenitor cells (NPCs) in culture, and in the hippocampus of aged rats. Resveratrol exerted biphasic effects on NPCs; low concentrations (10 μM) stimulated cell proliferation mediated by increased phosphorylation of extracellular signal-regulated kinases (ERKs) and p38 kinases, whereas high concentrations (>20 μM) exhibited inhibitory effects. Administration of Resveratrol (20 mg/kg body weight) to adult rats significantly increased the number of newly generated cells in the hippocampus, with upregulation of p-CREB and SIRT1 proteins implicated in neuronal survival and lifespan extension respectively. We have successfully demonstrated that Resveratrol exhibits dose dependent discrepancies and at a lower concentration can have a positive impact on the proliferation, survival of NPCs and aged rat hippocampal neurogenesis implicating its potential as a candidate for restorative therapies against age related disorders.

Expression and Inducibility of Cytochrome P450s (CYP1A1, 2B6, 2E1, 3A4) in Human Cord Blood CD34+ Stem Cell–Derived Differentiating Neuronal Cells

The status of xenobiotic metabolism in developing human brain cells is not known. The reason is nonavailability of developing human fetal brain. We investigate the applicability of the plasticity potential of human umbilical cord blood stem cells for the purpose. Characterized hematopoietic stem cells are converted into neuronal subtypes in eight days. The expression and substrate-specific catalytic activity of the cytochrome P450s (CYPs) CYP1A1 and 3A4 increased gradually till day 8 of differentiation, whereas CYP2B6 and CYP2E1 showed highest expression and activity at day 4. There was no significant increase in the expression of CYP regulators, namely, aryl hydrocarbon receptor (AHR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and glutathione-S-transferase (GSTP1-1) during differentiation. Differentiating cells showed significant induction in the expression of CYP1A1, 2B6, 2E1, 3A4, AHR, CAR, PXR, and GSTP1-1 when exposed to rifampin, a known universal inducer of CYPs. The xenobiotic-metabolizing capabilities of these differentiating cells were confirmed by exposing them to the organophosphate pesticide monocrotophos (MCP), a known developmental neurotoxicant, in the presence and absence of a universal inhibitor of CYPs-cimetidine. Early-differentiating cells (day 2) were found to be more vulnerable to xenobiotics than mature well-differentiated cells. For the first time, we report significant expression and catalytic activity of selected CYPs in human cord blood hematopoietic stem cell-derived neuronal cells at various stages of maturity. We also confirm significant induction in the expression and catalytic activity of selected CYPs in human cord blood stem cell-derived differentiating neuronal cells exposed to known CYP inducers and MCP.

Short-term exposure of 4-hydroxynonenal induces mitochondria-mediated apoptosis in PC12 cells

4-Hydroxynonenal (4-HNE) is one of the most reactive aldehydic by-products of lipid peroxidation. The role of 4-HNE in the etiology of various neurodegenerative disorders including cerebral ischemia/reperfusion, Alzheimer’s disease, Parkinson’s disease, etc. has been documented. We and others have reported that long-term toxic insults of 4-HNE triggers apoptotic signals and oxidative stress in various cells. However, the status of apoptosis following short-term exposure and underlying mechanisms has not been explored so far. We studied the apoptotic changes in PC12 cells receiving short-term exposure of 4-HNE. A significant dose-dependent induction in reactive oxygen species (ROS) and early response markers (c-Fos, c-Jun, and GAP-43) were observed in cells exposed to 4-HNE (10, 25, and 50 µM) for 1h. Following the exposure of PC12 cells to 4-HNE, the levels of protein and messenger RNA expressions of P53, Bax, and caspase 3 were significantly upregulated, whereas the levels of Bcl2 was downregulated. We could record the apoptotic signals and ROS generation in PC12 cells receiving 4-HNE exposure for such a short period of time. Induction in the expression and activity of caspase 3 has also indicated the mitochondrial mediation in the apoptosis induction.

Differentiating neurons derived from human umbilical cord blood stem cells work as a test system for developmental neurotoxicity

Differentiating neuronal cells derived from human umbilical cord blood stem cells have been used as an in vitro tool for the assessment of developmental neurotoxicity of monocrotophos (MCP), an organophosphate pesticide. The differentiating cells were exposed to MCP during the different stages of maturation, viz., days 2, 4, and 8, and changes in the makers of cell proliferation, neuronal differentiation, neuronal injuries, and receptors were studied. We found significant upregulation in the different MAPKs, apoptosis, and neurogenesis markers and downregulation in the cell proliferation markers during neuronal differentiation. We further identified significant upregulation in the expression of different MAPKs and proteins involved in oxidative stress, apoptosis, and calpain pathways in the mid-differentiating cells exposed to MCP. The upregulated levels of these proteins seem to be the main cause of alteration during the differentiation process towards apoptosis as a fine-tune of pro-apoptotic and anti-apoptotic proteins are desirable for the process of differentiation without apoptosis. The decreased acetylcholinesterase activity, dopaminergic, and cholinergic receptors and increased acetylcholine levels in the differentiating neuronal cells indicate the vulnerability of these cells towards MCP-induced neurotoxicity. Our data confirms that differentiating neuronal cells derived from human umbilical cord stem cells could be used as a powerful tool to assess the developmental neurotoxicity in human beings.

Pkb/Akt1 Mediates Wnt/GSK3β/β-Catenin Signaling-Induced Apoptosis in Human Cord Blood Stem Cells Exposed to Organophosphate Pesticide Monocrotophos

Inhibition mechanisms of protein kinase B (Pkb)/Akt and its consequences on related cell signaling were investigated in human umbilical cord blood stem cells (hUCBSCs) exposed to monocrotophos (MCP, an organophosphate pesticide). In silico data reveal that MCP interacts with kinase and c-terminal regulatory domains of Akt1, resulting into a total docking score of 5.2748 and also forms H-bond between its N-H and Thr-291 residue of Akt1, in addition to possessing several hydrophobic interactions. The main cause of Akt inhibition is considered to be the strong hydrogen bond between N-H and Thr-291, and hydrophobic interactions at Glu-234, and Asp-292 in the vicinity, which is usually occupied by the ribose of ATP, and interaction with residue Phe-161, thus leading to a significant conformational change in that particular portion of the protein. In silico data on Akt inhibition were confirmed by examining the downregulation of phosphorylated (Thr308/Ser493) Akt1 in MCP-exposed hUCBSCs. MCP-mediated altered levels of pAkt downstream targets viz., downregulated pGSK3β (Ser9), unchanged GSK3αβ, and upregulated levels of Bad, P(53), and caspase-9 further confirm the inhibition of pAkt. The cellular fate of such pAkt inhibition was confirmed by increased terminal deoxynucleotide transferase dUTP nick-end labeling positive cells, reduced mitochondrial membrane potential, and the activation of various MAPKs, proapoptotic markers-Bax, and caspases-9/3. Our data demonstrate that Akt1 plays a key role in MCP-induced apoptosis in hUCBSCs. We also identified that such cellular responses of human cord blood stem cells against MCP were due to strong binding and inhibition of kinase and AGC-Kinase-C terminal regulatory domains of Akt1.

Monocrotophos Induces the Expression and Activity of Xenobiotic Metabolizing Enzymes in Pre-Sensitized Cultured Human Brain Cells

The expression and metabolic profile of cytochrome P450s (CYPs) is largely missing in human brain due to non-availability of brain tissue. We attempted to address the issue by using human brain neuronal (SH-SY5Y) and glial (U373-MG) cells. The expression and activity of CYP1A1, 2B6 and 2E1 were carried out in the cells exposed to CYP inducers viz., 3-methylcholanthrene (3-MC), cyclophosphamide (CPA), ethanol and known neurotoxicant- monocrotophos (MCP), a widely used organophosphorous pesticide. Both the cells show significant induction in the expression and CYP-specific activity against classical inducers and MCP. The induction level of CYPs was comparatively lower in MCP exposed cells than cells exposed to classical inducers. Pre-exposure (12 h) of cells to classical inducers significantly added the MCP induced CYPs expression and activity. The findings were concurrent with protein ligand docking studies, which show a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR, PXR and AHR. Similarly, the known CYP inducers- 3-MC, CPA and ethanol have also shown significantly high docking scores with all the three studied CYP regulators. The expression of CYPs in neuronal and glial cells has suggested their possible association with the endogenous physiology of the brain. The findings also suggest the xenobiotic metabolizing capabilities of these cells against MCP, if received a pre-sensitization to trigger the xenobiotic metabolizing machinery. MCP induced CYP-specific activity in neuronal cells could help in explaining its effect on neurotransmission, as these CYPs are known to involve in the synthesis/transport of the neurotransmitters. The induction of CYPs in glial cells is also of significance as these cells are thought to be involved in protecting the neurons from environmental insults and safeguard them from toxicity. The data provide better understanding of the metabolizing capability of the human brain cells against xenobiotics.

Progress toward the development of in vitro model system for chemical-induced developmental neurotoxicity: potential applicability of stem cells

committed toward the neuronal lineage and are very similar to the fetal neural stem cells. In our so far published papers, we have successfully attempted to establish stem cell-based in vitro model systems for the study of the various aspects of DNT and have proven that human umbilical cord blood-derived stem cells (hUCBSCs) when differentiated into neurons serve as one of the classical tools for the same. These were employed to assess the developmental neurotoxicity potential of Monocrotophos (MCP), a known developmental neurotoxin. Differentiating cells were given MCP exposure at different stages of maturation, i.e., day 2, 4 and 8, and significant changes in the markers of differentiation, proliferation, injury and receptors were studied. Our group reported that there was a significant upregulation of the different MAPKs, apoptosis and neurogenesis markers and a down regulation in the various cell proliferation markers during the neuronal differentiation process. Expression analysis was done both at the transcriptional (RT-PCR) as well as translational levels (immunocytochemistry and Western blotting). As differentiation proceeded, there was a significant quantitative reduction in the expression of stems markers, i.e., CD133, MYC, NANOG, SHH, KLF4, SOX2, POU5F1 and FRAP1, whereas the differentiation marker expression was significantly increased except in case of NGF, CHRM2 and NR42A. The highest expression for differentiation markers, i.e., NFM, MBP, NFH, NCM, BDNF and STAT4, was seen on day 8. These results support that hUCBSCs successfully differentiated into the neuronal lineage. Post MCP exposure there was again a significant alteration in the various markers of apoptosis, oxidative stress, metabolism, etc. It was concurred from the results that MCP exposure leads to ROS generation, oxidative stress, depleted glutathione levels and even promoted apoptosis. Receptor studies further demonstrated that the Developmental neurotoxicity (DNT) has been the prime focus of our research group since it is one of the most crucial and significant parts of the neuroscience research arena. The restricted availability of live human fetal brain tissue and major ethical issues makes the field further challenging. So far, it has been best represented via the in vivo model systems; however, the inaccurate extrapolation of data to humans restricts its scope and applications. Therefore, there is a dire need for samples (cells and tissues) of human origin. Based on the OECD guidelines 426, rodent data are present for about 200 compounds, but there is a scarcity of data available for humans. The developing brain is highly vulnerable to even minute doses of substances which are otherwise harmless for a fully developed mature brain. This is primarily because of the poorly developed blood–brain barrier, low oxygen levels and high mitotic rates. The entire process of brain development is quite complex, and in order to generate DNT models in vitro, cells and tissues of human origin are needed. Stem cells with their inherent potential for pluripotency and unique ability for unlimited proliferation serve as one of the best tools. Stem cells can be made to differentiate into the neuronal lineages and can thereby sufficiently mimic cells of the developing brain. Human umbilical cord blood was employed for the isolation of stem cells as it is not only a rich source of stem cells, but also a waste material, therefore does not pose any major ethical dubious. CD34+ hematopoietic stem cells (HSCs)/non-hematopoietic stem cells (non-HSCs) derived from cord blood are greatly

Glycine propionyl l-carnitine attenuates d-Galactosamine induced fulminant hepatic failure in wistar rats.

Glycine propionyl l-carnitine (GPLC) is a propionyl ester of carnitine that includes an additional glycine component. The present study evaluated hepatoprotective effect of GPLC in d-Galactosamine (d-GalN) induced fulminant hepatic failure. Rats were intraperitonially administered d-GalN (700mg/kgBW). GPLC was given as a pre-treatment (35mg/kgBW/day) for 1month followed by a single dose of d-GalN on the 31st day. d-GalN administration resulted in increased mortality and serum ALT and AST activities. These increases were significantly attenuated by GPLC. d-GalN treatment increased hepatic lipid peroxidation and a decrease in reduced glutathione content was observed. GPLC pre-treatment significantly decreased lipid peroxidation and augmented the level of GSH. d-GalN increased the circulating level of TNF-α and ATM-Kinase and MAP-Kinase expression. GPLC supplementation inhibited the increase in serum TNF-α and ATM-Kinase and MAP-Kinase expression. d-GalN treatment increased the level of Bax and Caspase-3 m-RNA while as a decline was observed in Bcl2 m-RNA. GPLC prevented the increase in Caspase-3 and Bax m-RNA and at the same time augmented the expression of Bcl2 m-RNA. Our findings suggest that GPLC alleviates d-GalN induced liver injury by strengthening antioxidative defense system and reducing apoptotic signalling pathways.

Adoptive Autophagy Activation: a Much-Needed Remedy Against Chemical Induced Neurotoxicity/Developmental Neurotoxicity

The profound significance of autophagy as a cell survival mechanism under conditions of metabolic stress is a well-proven fact. Nearly a decade-long research in this area has led scientists to unearth various roles played by autophagy other than just being an auto cell death mechanism. It is implicated as a vital cell survival pathway for clearance of all the aberrant cellular materials in case of cellular injury, metastasis, disease states, cellular stress, neurodegeneration and so on. In this review, we emphasise the critical role of autophagy in the environmental stressors-induced neurotoxicity and its therapeutic implications for the same. We also attempt to shed some light on the possible protective role of autophagy in developmental neurotoxicity (DNT) which is a rapidly growing health issue of the human population at large and hence a point of rising concern amongst researchers. The intimate association between DNT and neurodegenerative disorders strongly indicates towards adopting autophagy activation as a much-needed remedy for DNT.

Differences in the expression and sensitivity of cultured rat brain neuronal and glial cells toward the monocrotophos

Inducible expressions cytochrome P450s (CYPs) against environmental chemicals in brain tissues of experimental animals is well-documented. However, the precise role of specific brain cell type in the metabolism of different class of xenobiotics has not been explored adequately. We study the expression of selected CYPs (1A1/1A2, 2B1/2B2, 2E1) in primary cultures of rat brain neuronal and glial cell exposed to an organophosphate pesticide-monocrotophos (MCP), a known neurotoxicant. The cultured neurons and glial cells express significant expression of CYP1A1, 2B2 and 2E1 isoenzymes, where the levels were comparatively higher in neuronal cells. Neuronal cells exhibited greater induction of CYP2E1 against MCP exposure, while glial cells were having more vulnerability for CYP1A and 2B isoenzymes. Similarly, cells were showing substrate specific responses against the specific inducers of CYPs, that is, ethanol (2E1), cyclophosphamide (2B1/2B2), 3-methylcholanthrene (1A1/1A2). The altered expression and activity of selected CYPs in cultured neuronal and glial cells could be helpful in explaining the association between MCP-induced neurotoxicity/metabolism and synthesis or transport of the neurotransmitters. The induction of CYPs in glial cells may also have significance as these cells are thought to be involved in protecting the neurons from environmental insults and safeguard them from toxicity. The differential expression pattern of CYPs in neuronal and glial cells exposed to MCP also indicate the selective sensitivity of these cells against the xenobiotics, hence suggested their suitability as tool to screen neurotoxicity potential of variety of xenobiotics.