Jai Shankar

Green Synthesis of Silver and Titanium Dioxide Nanoparticles Using Euphorbia prostrata Extract Shows Shift from Apoptosis to G0/G1 Arrest followed by Necrotic Cell Death in Leishmania donovani

ABSTRACT The aim of the present study was to synthesize silver (Ag) and titanium dioxide (TiO2) nanoparticles (NPs) using green synthesis from aqueous leaf extract of Euphorbia prostrata as antileishmanial agents and to explore the underlying molecular mechanism of induced cell death. In vitro antileishmanial activity of synthesized NPs was tested against promastigotes of Leishmania donovani by alamarBlue and propidium iodide uptake assays. Antileishmanial activity of synthesized NPs on intracellular amastigotes was assessed by Giemsa staining. The leishmanicidal effect of synthesized Ag NPs was further confirmed by DNA fragmentation assay and by cell cycle progression and transmission electron microscopy (TEM) of the treated parasites. TEM analysis of the synthesized Ag NPs showed a spherical shape with an average size of 12.82 ± 2.50 nm, and in comparison to synthesized TiO2 NPs, synthesized Ag NPs were found to be most active against Leishmania parasites after 24 h exposure, with 50% inhibitory concentrations (IC50) of 14.94 μg/ml and 3.89 μg/ml in promastigotes and intracellular amastigotes, respectively. A significant increase in G0/G1 phase of the cell cycle with a subsequent decrease in S (synthesis) and G2/M phases compared to controls was observed. The growth-inhibitory effect of synthesized Ag NPs was attributed to increased length of S phase. A decreased reactive oxygen species level was also observed, which could be responsible for the caspase-independent shift from apoptosis (G0/G1 arrest) to massive necrosis. High-molecular-weight DNA fragmentation as a positive consequence of necrotic cell death was also visualized. We also report that the unique trypanothione/trypanothione reductase (TR) system of Leishmania cells was significantly inhibited by synthesized Ag NPs. The green-synthesized Ag NPs may provide promising leads for the development of cost-effective and safer alternative treatment against visceral leishmaniasis.

A comprehensive toxicity study of zinc oxide nanoparticles versus their bulk in Wistar rats Toxicity study of zinc oxide nanoparticles

The purpose of this study was to characterize the zinc oxide nanoparticles (ZnO-NPs) and their bulk counterpart in suspensions and to access the impact of their acute oral toxicity at doses of 300 and 2000 mg/kg in healthy female Wistar rats. The hematological, biochemical, and urine parameters were accessed at 24 and 48 h and 14 days posttreatment. The histopathological evaluations of tissues were also performed. The distribution of zinc content in liver, kidney, spleen, plasma, and excretory materials (feces and urine) at 24 and 48 h and 14 days posttreatment were accessed after a single exposure at dose of 2000 mg/kg body weight. The elevated level of alanine amino transferase, alkaline phosphatase, lactate dehydrogenase, and creatinine were observed in ZnO-NPs at a dose of 2000 mg/kg at all time points. There was a decrease in iron levels in all the treated groups at 24 h posttreatment as compared to control groups but returned to their normal level at 14 days posttreatment. The hematological parameters red blood cells, hemoglobin, hematocrit, platelets, and haptoglobin were reduced at 48 h posttreatment at a dose of 2000 mg/kg ZnO-NPs and showed hemolytic condition. All the treated groups were comparable to control group at the end of 14 days posttreatment. The zinc concentration in the kidney, liver, plasma, feces, and urine showed a significant increase in both groups as compared to control. This study explained that ZnO-NPs produced more toxicological effect as compared to their bulk particles as evidenced through alteration in some hemato-biochemical parameters and with few histopathological lesions in liver and kidney tissues.

Endophytic Fungi Piriformospora indica Mediated Protection of Host from Arsenic Toxicity

Complex intercellular interaction is a common theme in plant-pathogen/symbiont relationship. Cellular physiology of both the partners is affected by abiotic stress. However, little is known about the degree of protection each offers to the other from different types of environmental stress. Our current study focused on the changes in response to toxic arsenic in the presence of an endophytic fungus Piriformospora indica that colonizes the paddy roots. The primary impact of arsenic was observed in the form of hyper-colonization of fungus in the host root and resulted in the recovery of its overall biomass, root damage, and chlorophyll due to arsenic toxicity. Further, fungal colonization leads to balance the redox status of the cell by adjusting the antioxidative enzyme system which in turn protects photosynthetic machinery of the plant from arsenic stress. We observed that fungus has ability to immobilize soluble arsenic and interestingly, it was also observed that fungal colonization restricts most of arsenic in the colonized root while a small fraction of it translocated to shoot of colonized plants. Our study suggests that P. indica protects the paddy (Oryza sativa) from arsenic toxicity by three different mechanisms viz. reducing the availability of free arsenic in the plant environment, bio-transformation of the toxic arsenic salts into insoluble particulate matter and modulating the antioxidative system of the host cell.

Impaired lysosomal activity mediated autophagic flux disruption by graphite carbon nanofibers induce apoptosis in human lung epithelial cells through oxidative stress and energetic impairment

BackgroundGraphite carbon nanofibers (GCNF) have emerged as a potential alternative of carbon nanotubes (CNT) for various biomedical applications due to their superior physico-chemical properties. Therefore in-depth understanding of the GCNF induced toxic effects and underlying mechanisms in biological systems is of great interest. Currently, autophagy activation by nanomaterials is recognized as an emerging toxicity mechanism. However, the association of GCNF induced toxicity with this form of cell death is largely unknown. In this study, we have assessed the possible mechanism; especially the role of autophagy, underlying the GCNF induced toxicity.MethodsHuman lung adenocarcinoma (A549) cells were exposed to a range of GCNF concentrations and various cellular parameters were analyzed (up to 48 h). Transmission electron microscopy, immunofluorescent staining, western blot and quantitative real time PCR were performed to detect apoptosis, autophagy induction, lysosomal destabilization and cytoskeleton disruption in GCNF exposed cells. DCFDA assay was used to evaluate the reactive oxygen species (ROS) production. Experiments with N-acetyl-L-cysteine (NAC), 3-methyladenine (3-MA) and LC3 siRNA was carried out to confirm the involvement of oxidative stress and autophagy in GCNF induced cell death. Comet assay and micronucleus (MN) assay was performed to assess the genotoxicity potential.ResultsIn the present study, GCNF was found to induce nanotoxicity in human lung cells through autophagosomes accumulation followed by apoptosis via intracellular ROS generation. Mechanistically, impaired lysosomal function and cytoskeleton disruption mediated autophagic flux blockade was found to be the major cause of accumulation rather than autophagy induction which further activates apoptosis. The whole process was in line with the increased ROS level and their pharmacological inhibition leads to mitigation of GCNF induced cell death. Moreover the inhibition of autophagy attenuates apoptosis indicating the role of autophagy as cell death process. GCNF was also found to induce genomic instability.ConclusionOur present study demonstrates that GCNF perturbs various interrelated signaling pathway and unveils the potential nanotoxicity mechanism of GCNF through targeting ROS-autophagy-apoptosis axis. The current study is significant to evaluate the safety and risk assessment of fibrous carbon nanomaterials prior to their potential use and suggests caution on their utilization for biomedical research.

Oral subchronic exposure to silver nanoparticles causes renal damage through apoptotic impairment and necrotic cell death

Abstract Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials. Following oral exposure, AgNPs can accumulate in various organs including kidneys where they show gender specific accumulation. There is limited information on their effect on renal system following long-term animal exposure especially at the ultramicroscopic and molecular level. In this study, we have assessed the effect of 60 days oral AgNPs treatment on kidneys of female Wistar rats at doses of 50 ppm and 200 ppm that are below previously reported lowest observed adverse effect level (LOAEL). AgNPs treatment led to decrease in kidney weight and some loss of renal function as seen by increased levels of serum creatinine and early toxicity markers such as KIM-1, clusterin and osteopontin. We also observed significant mitochondrial damage, loss of brush border membranes, pronounced swelling of podocytes and degeneration of their foot processes using transmission electron microscopy (TEM). These symptoms are similar to those seen in nephrotic syndrome and ‘Minimal change disease’ of kidney where few changes are visible under light microscopy but significant ultrastructural damage is observed. Prolonged treatment of AgNPs also led to the activation of cell proliferative, survival and proinflammatory factors (Akt/mTOR, JNK/Stat and Erk/NF-κB pathways and IL1β, MIP2, IFN-γ, TNF-α and RANTES) and dysfunction of normal apoptotic pathway. Our study shows how long term AgNPs exposure may promote ultrastructural damage to kidney causing inflammation and expression of cell survival factors. These changes, in the long term, could lead to inhibition of the beneficial apoptotic pathway and promotion of necrotic cell death in kidneys.

Synthesis of Nanoparticles Using Euphorbia prostrata Extract Reveals a Shift from Apoptosis to G0/G1 Arrest in Leishmania donovani

The aim of the present investigation was to synthesize silver (Ag) and titanium dioxide (TiO2) nanoparticles (NPs) using the aqueous leaves extract of Euphorbia prostrata as antileishmanial agents and to explore the mechanism of induced cell death. In vitro antileishmanial activity of synthesized NPs was tested against promastigotes of Leishmania donovani by alamar Blue® cell viability reagent and propidium iodide uptake assay. The effective leishmanicidal activity of synthesized Ag NPs was further confirmed by cell cycle progression, externalized phosphatidylserine, DNA fragmentation assay, reactive oxygen species (ROS) level, intracellular non-protein thiols and transmission electron microscopy (TEM) of the treated parasites. TEM analysis of the synthesized Ag NPs and TiO2 NPs showed spherical shape with an average size of 12.82 ± 2.50 and 83.22 ± 1.50 nm, respectively. Ag NPs was found to be the most active agent against Leishmania parasites after 24 h exposure with IC50 value of 14.94 μg/mL. A significant increase in G0/ G1 phase of the cell cycle with subsequent decrease in S and G2/M phases was observed when compared to control and thus confirming the growth inhibitory effect of synthesized Ag NPs. Decreased ROS level was also observed which could be responsible for caspase independent shift from apoptosis (G0/G1 arrest) to massive necrosis. High molecular weight DNA fragmentation as a positive consequence of necrotic cell death was also visualized. In the present study, the unique trypanothione/trypanothione reductase (TR) system of Leishmania cells was significantly inhibited by synthesized Ag NPs was reported. The green synthesized Ag NPs may provide promising leads for the development of cost effective and safer alternative treatment against visceral leishmaniasis.

Chemoprevention of Leishmaniasis: In-vitro antiparasitic activity of dibenzalacetone, a synthetic curcumin analog leads to apoptotic cell death in Leishmania donovani

Curcumin is the major phenolic compound found in turmeric, a dry powder of rhizomes and roots of the plant, Curcuma longa L., which is widely used as spice and food colorant around the world, and in herbal medicinal practice in Asian countries. The present study reports the leishmanicidal activity of trans-dibenzalacetone (DBA), a synthetic monoketone analog of curcumin, against Leishmania donovani parasites. We for the first time report the antiproliferative effect of a curcumin analog (DBA) on the intracellular amastigotes of L. donovani, the clinically more relevant stage of the parasite than its promastigotes stage. The leishmanicidal effect of DBA was further confirmed by scanning and transmission electron microscopies. Cell growth was arrested in G0/G1 phase with increased concentration of cytosolic calcium and dissipation of mitochondrial membrane potential. Further, the unique trypanothione/trypanothione reductase (TR) system of Leishmania cells was significantly inhibited by DBA. This economically synthesizable simple monoketone analog of curcumin has the potential for field use against visceral leishmaniasis which is currently widespread in tropical and subtropical developing countries of the world. In conclusion, we have identified an analog of curcumin for potential applications against leishmaniasis, based on its strong antiparasitic activity and low toxicity. This curcumin analog compares favorably, at least in vitro, with the existing medication miltefosine.

MIR155 Regulation of Ubiquilin1 and Ubiquilin2: Implications in Cellular Protection and Tumorigenesis

Ubiquilin (UBQLN) proteins are adaptors thought to link ubiquitinated proteins to the proteasome. However, our lab has recently reported a previously unappreciated role for loss of UBQLN in lung cancer progression. In fact, UBQLN genes are lost in over 50% of lung cancer samples examined. However, a reason for the loss of UBQLN has not been proposed, nor has a selective pressure that could lead to deletion of UBQLN been reported. Diesel Exhaust Particles (DEP) are a major concern in the large cities of developing nations and DEP exposed populations are at an increased risk of developing a number of illnesses, including lung cancer. A connection between DEP and UBQLN has never been examined. In the present study, we determined the effect of DEP on lung cell lines and were interested to determine if UBQLN proteins could potentially play a protective role following treatment with DEP. Interestingly, we found that DEP treated cells have increased expression of UBQLN proteins. In fact, over-expression of UBQLN was capable of protecting cells from DEP toxicity. To investigate the mechanism by which DEP leads to increased UBQLN protein levels, we identified and interrogated microRNAs that were predicted to regulate UBQLN mRNA. We found that DEP decreases the oncogenic microRNA, MIR155. Further, we showed that MIR155 regulates the mRNA of UBQLN1 and UBQLN2 in cells, such that increased MIR155 expression increased cell invasion, migration, wound formation and clonogenicity in UBQLN-loss dependent manner. This is the first report of an environmental carcinogen regulating expression of UBQLN proteins. We show that exposure of cells to DEP causes an increase in UBQLN levels and that MIR155 regulates mRNA of UBQLN. Thus, we propose that DEP-induced repression of MIR155 leads to increased UBQLN levels, which in turn may be a selective pressure on lung cells to lose UBQLN1.

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.

Genotoxicity evaluation of zinc oxide nanoparticles in Swiss mice after oral administration using chromosomal aberration, micronuclei, semen analysis, and RAPD profile

The expanded uses of zinc oxide nanoparticles (ZnO NPs) have grown rapidly in the field of nanotechnology. Thus, rising production of nanoparticles (NPs) increases the possible risks to the environment and occupationally exposed humans. Hence, it is indispensable to appraise the safety toxicity including genotoxicity for these NPs. In the present study, we have evaluated the genotoxic effect of ZnO NPs after oral administration to Swiss mice at dose levels of 300 and 2000 mg/kg body weight. These doses were administered for 2 days at 24 h apart. Chromosomal aberration (CA) and micronucleus tests were conducted following Organization for Economic Co-operation and Development guidelines. DNA damage was evaluated at 0, 24, 48, and 72 h posttreatment using a randomly amplified polymorphic DNA (RAPD) assay; additionally, semen analyses were also performed at 34.5 days post oral exposure. The reactive oxygen species (ROS), 8-oxo-2′-deoxyguanosine and CAs were increased (p < 0.05) at the highest dosage (2000 mg/kg) of ZnO NPs compared to controls. Aberrant sperm morphology with reduced sperm count and motility were also present (p < 0.05) in the high-dose group. Based on the RAPD assay, the genomic template stability within the high-dose group (<90%) was less than the controls (100%). The results suggested that ZnO NPs are mildly genotoxic in a dose-related manner and this toxicity were induced by generation of ROS.