Aditya Bhushan Pant

Influence of cytotoxic doses of 4-hydroxynonenal on selected neurotransmitter receptors in PC-12 cells.

Effect of 4-hydroxynonenal (HNE), a long-chain alpha, beta unsaturated aldehyde product, generated by the oxidation of omega-6 polyunsaturated fatty acids on the sensitivity of selected neurotransmitter receptors was studied in PC-12 cells. Cytotoxicity profiling was carried out at varying concentrations of HNE (0.1-50microM) for 30min to 24h. Trypan blue dye exclusion, MTT, LDH release and neutral red uptake (NRU) assays were carried out to assess the cytotoxicity of HNE. Cytotoxic response was found to be significant at 2h of exposure. Cytotoxicity of HNE at 50microM was exerted even at 90min. HNE 10-50microM was found to be cytotoxic, whereas, 2-5microM causes physiological stress only and 1-0.1microM non-cytotoxic. Effect on dopamine, cholinergic, serotonin and benzodiazepine receptors was studied at varying concentrations of HNE (1, 10, 25 and 50microM for 1-8h). A significant decrease in binding of 3H-QNB, 3H-Fluinitrazepam and 3H-Ketanserin, known to label cholinergic (muscarinic), benzodiazepine and serotonin (5HT(2A)) receptors respectively was observed at 1h exposure of PC-12 cells to HNE at 25 and 50microM concentrations. The decrease in the binding of (3)H-Spiperone, known to label dopamine (DA-D2) receptors was evident at 4h of exposure of PC-12 cells to HNE. The decrease in the binding with DA-D2 receptors continued till 8h. Effect on the binding of (3)H-Fluinitrazepam and 3H-Ketanserin appeared to be maximum at 25 and 50microM concentrations of HNE for 4h and 8h. The PC-12 cells appear to be vulnerable to cytotoxic concentrations of HNE. Experimental HNE exposure provides an intriguing model of toxicant-cell interactions involving neurotransmitter receptors in HNE neurotoxicity.

Photosensitizing Potential of Ciprofloxacin at Ambient Level of UV Radiation

Ciprofloxacin is a widely used fluoroquinolone drug with broad spectrum antibacterial activities. Clinical experience has shown incidences of adverse effects related to skin, hepatic, central nervous system, gastrointestinal and phototoxicity. India is a tropical country and sunlight is abundant throughout the day. In this scenario exposure to ambient levels of ultraviolet radiation (UV‐R) in sunlight may lead to harmful effects in ciprofloxacin users. Phototoxicity assessment of ciprofloxacin was studied by two mouse fibroblast cell lines L‐929 and NIH‐3T3. Generation of reactive oxygen species (ROS) like singlet oxygen (1O2), superoxide anion radical (O2ḃ−) and hydroxyl radical (ḃOH) was studied under the exposure of ambient intensities of UV‐A (1.14, 1.6 and 2.2 mW cm−2), UV‐B (0.6, 0.9 and 1.2 mW cm−2) and sunlight (60 min). The drug was generating 1O2, O2ḃ− and ḃOH in a concentration and dose‐dependent manner. Sodium azide (NaN3) and 1,4‐diazabicyclo 2‐2‐2‐octane (DABCO) inhibited the generation of 1O2. Superoxide dismutase (SOD) inhibited 90–95% O2ḃ− generation. The drug (5–40 μg mL−1) was responsible for linoleic acid peroxidation. Quenching study of linoleic acid peroxidation with SOD (25 and 50 U mL−1) confirms the involvement of ROS in drug‐induced lipid peroxidation. The generation of ḃOH radical was further confirmed by using specific quenchers of ḃOH such as mannitol (0.5 m) and sodium benzoate (0.5 m). 2′‐deoxyguanosine (2′‐dGuO) assay and linoleic acid peroxidation showed that ROS were mainly responsible for ciprofloxacin‐sensitized photo‐degradation of guanine base. L‐929 cell line showed 29%, 34% and 54% reduced cell viability at higher drug concentration (300 μg mL−1) under UV‐A, UV‐B and sunlight, respectively. 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay in NIH‐3T3 cell line at higher drug concentration (300 μg mL−1) showed a decrease in cell viability by 54%, 56% and 59% under UV‐A, UV‐B and sunlight, respectively. Results of neutral red uptake assay (NRU) in L‐929 cell line were in accordance with MTT assay. The NIH‐3T3 cell line showed a higher photosensitizing potential than L‐929. The phototoxicity end point shows a time‐ and concentration‐dependent statistically significant (P < 0.001) damage. Ciprofloxacin produced ROS by Type I and Type II photodynamic reactions, interacted with nucleic acid moiety and inhibited cell viability. Further, UV‐induced photo‐peroxidation of linoleic acid accorded the involvement of ROS in the manifestation of drug phototoxicity. Appearance of ciprofloxacin‐induced phototoxicity at the ambient level of sunlight is a real risk for the people of India and for those of other tropical countries. We suggest that sunlight exposure should be avoided (especially peak hours) during ciprofloxacin treatment.

Environmental and experimental exposure of phthalate esters: The toxicological consequence on human sperm

Rapid industrialization and urbanization release several chemicals such as phthalates into the environment and cause adverse effects on reproductive system, mainly endocrine disruption, testicular injury and decline in semen quality in humans. There are no reports in extrapolating of the epidemiological data with in vitro findings. Our study show the correlations between in vivo studies and in vitro data for the effect of phthalate esters. Healthy human males, in the age group 21 to 40 years, visiting Chhatrapati Sahuji Maharaj Medical University (CSMMU), Lucknow, as part of infertility investigation, were recruited as volunteers. Semen analysis was performed according to the WHO guidelines. Phthalate esters were analyzed by high-performance liquid chromatography (HPLC) and cell viability by MTT assay. In the in vitro studies, sperms were exposed to highest concentration in semen samples (5—10 times higher) for a period ranging between 30 min and 96 hours. An inverse relationship with sperm motility in epidemiological studies was concurrent by significant dose-and time-dependent decrease in the sperm motility under in vitro environment after 12-hour exposure. Cytotoxicity was observed only with the highest concentration after 96 hours of exposure. There are a significant correlation between phthalate ester diethylhexyl phthalate, di-n-butyl phthalate (DEHP and DBP) and sperm motility both in vitro and in vivo conditions. Additionally, in vitro experiments conducted not only adjunct to the existing in vivo data but also specify the effect of specific toxicants (DEHP and DBP) on sperm motility and viability. Results show the decrease in motility of sperms under in vitro conditions at the maximum range of in vivo measured levels and 5- or 10-folds higher to that found in human semen samples.

Cyto-genotoxicity of amphibole asbestos fibers in cultured human lung epithelial cell line: role of surface iron.

The present investigations correlate the potentials of the reactive oxygen species (ROS) generation and the cyto-genotoxicity of amphibole asbestos fibers (amosite, crocidolite and tremolite) with their surface iron, under in vitro controlled conditions, using A549 cells (human lung epithelial cell line). The mobilizable surface iron was measured by Atomic Absorption Spectroscopy; the production of ROS was investigated using 2, 7 dichloro-dihydrofluorescein-diacetate (DCFH-DA) dye; for cytotoxicity assessment, the intracellular organelles specific damages were measured, using 3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide salt (MTT) assay; and, the genotoxic potential of amphibole fibers was determined by cytokinesis block micronucleus (CBMN) assay. In the study, highest amount of ROS was generated by crocidolite followed by tremolite and minimum with amosite. In MTT assay, the time- and concentration-dependent decrease in percent cell viability was recorded with all the three amphibole fibers, tremolite being most cytotoxic, followed by crocidolite, and then amosite. In genotoxicity assay, an increase in the frequency of micronuclei (MNi) in binucleated (BN) cells was observed, where crocidolite was most genotoxic, followed by tremolite, and amosite the least.The comparison of results depicts a clear trend of cyto-genotoxic potential paralleling the ROS generation, suggesting a definite role of oxidative stress in fiber-induced toxicity. However, amosite contains maximum surface iron (28%), followed by crocidolite (27%), and tremolite carrying least (as contaminant) or no iron, the mobilizable surface iron is maximum in crocidolite followed by amosite and is minimum in tremolite. The mobilizable iron somewhat corresponds with the ROS generation capacity of these fibers. This shows that the surface iron could be mainly responsible for amphibole asbestos-induced ROS toxicity; though it may not be the only factor responsible, other factors like shape and size etc., also play role in amphibole asbestos-induced toxicity.

Oxygen Glucose Deprivation Model of Cerebral Stroke in PC-12 Cells: Glucose as a Limiting Factor

Optimum time points for oxygen-glucose deprivation (OGD) and re-oxygenation have been identified to suggest the suitability of PC-12 cells as rapid and sensitive in vitro model of cerebral stroke. Further, the precise role of glucose as one of the limiting factors was ascertained. PC-12 cells were subjected to receive OGD of 1–8 h followed by re-oxygenation for 6 to 96 h in medium having glucose 0–10 mg/ml. Loss of cell viability was assessed using trypan blue dye exclusion and MTT assays. The significant (p < 0.05) reduction in percent viable cell count was started at 2 h of OGD (80.7 ± 2.0) and continued in further OGD periods (3, 4, 5, 6, 7, and 8 h), i.e. 65.7 ± 3.5, 59.7 ± 4.6, 54.3 ± 3.2, 44.7 ± 2.9, 20.3 ± 4.3, 5.7 ± 2.0 of counted cells, respectively. Cells growing in glucose-free medium have shown a gradual (p < 0.001) decrease in cell viability throughout the re-oxygenation. Re-oxygenation of 24 h was found to be first statistically significant time point for all the glucose concentrations. Glucose concentration during re-oxygenation was found to be one of the key factors involved in the growth and proliferation in PC-12 cells. The OGD of 6 h followed by a re-oxygenation period of 24 h with 4–6 mg/ml glucose concentration could be recorded as optimum conditions under our experimental conditions.

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.

The Anti-Inflammatory and Antibacterial Basis of Human Omental Defense: Selective Expression of Cytokines and Antimicrobial Peptides

Background The wound healing properties of the human omentum are well known and have extensively been exploited clinically. However, the underlying mechanisms of these effects are not well understood. We hypothesize that the omentum tissue promotes wound healing via modulation of anti-inflammatory pathways, and because the omentum is rich in adipocytes, the adipocytes may modulate the anti-inflammatory response. Factors released by human omentum may affect healing, inflammation and immune defense. Methodology Six human omentum tissues (non obese, free from malignancy, and any other systemic disorder) were obtained during diagnostic laparoscopies having a negative outcome. Healthy oral mucosa (obtained from routine oral biopsies) was used as control. Cultured adipocytes derived from human omentum were exposed to lipopolysaccharide (LPS) (1–50 ng/mL) for 12–72 hours to identify the non-cytotoxic doses. Levels of expression (mRNA and protein) were carried out for genes associated with pro- and anti-inflammatory cytokine responses and antibacterial/antimicrobial activity using qRT-PCR, western blotting, and cell-based ELISA assays. Results The study shows significant higher levels of expression (mRNA and protein) of several specific cytokines, and antibacterial peptides in the omentum tissues when compared to oral sub-mucosal tissues. In the validation studies, primary cultures of adipocytes, derived from human omentum were exposed to LPS (5 and 10 ng/mL) for 24 and 48 h. The altered expressions were more pronounced in cultured adipocytes cells when exposed to LPS as compared to the omentum tissue. Conclusions/Significance Perhaps, this is the first report that provides evidence of expressional changes in pro- and anti-inflammatory cytokines and antibacterial peptides in the normal human omentum tissue as well as adipocytes cultured from this tissue. The study provides new insights on the molecular and cellular mechanisms of healing and defense by the omentum, and suggests the potential applicability of cultured adipocytes derived from the omentum for future therapeutic applications.

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.

Protective effects of l-pGlu-(2-propyl)-l-His-l-ProNH2, a newer thyrotropin releasing hormone analog in in vitro and in vivo models of cerebral ischemia

In the present study, the newly synthesized TRH analog (L-pGlu-(2-propyl)-L-His-l-ProNH(2); NP-647) was evaluated for its effects in in vitro (oxygen glucose deprivation (OGD)-, glutamate- and H(2)O(2)-induced injury in PC-12 cells) and in vivo (transient global ischemia) models of cerebral ischemic injury. PC-12 cells were subjected to oxygen and glucose deprivation for 6h. Exposure of NP-647 was given before and during OGD. In glutamate and H(2)O(2) induced injury, exposure of NP-647 was given 1, 6 and 24h prior to exposure of glutamate and H(2)O(2) exposure. NP-647, per se found to be non-toxic in 1-100μM concentrations. NP-647 showed protection against OGD at the 1 and 10μM. The concentration-dependent protection was observed in H(2)O(2)- and glutamate-induced cellular injury. In in vivo studies, NP-647 treatment showed protection of hippocampal (CA1) neuronal damage in transient global ischemia in mice and subsequent improvement in memory retention was observed using passive avoidance retention test. Moreover, administration of NP-647 resulted in decrease in inflammatory cytokines TNF-α and IL-6 as well as lipid peroxidation. These results suggest potential of NP-647 in the treatment of cerebral ischemia and its neuroprotective effect may be attributed to reduction of excitotoxicity, oxidative stress and inflammation.

Galactomannan-PEI based non-viral vectors for targeted delivery of plasmid to macrophages and hepatocytes

Intracellular nature and diversified locations of infectious and parasitic diseases such as leishmaniasis, trypanosomiasis, tuberculosis and hepatitis B and C pose a significant global burden and challenge to the scientists working in the area of drug discovery and drug delivery. The macrophages and hepatocytes are considered as potential target sites as they together play an important role in various infectious diseases. The present study scrutinizes the applicability of a natural biopolymer-based chemical vectors, capable of targeting both macrophages and hepatocytes, that can form a complex with plasmid and administer it into cells to produce a desired protein. The investigations were made to develop a novel series of gene carriers by conjugating depolymerized galactomannan (guar gum), a biocompatible polysaccharide with low molecular weight branched PEI (LMWP). A series of conjugates were developed and characterized using physicochemical techniques. All the GP/pDNA complexes showed significantly higher transfection efficiency with GP-3/pDNA, one of the best formulations, showed ~2.0-7.7-folds higher transfection efficacy when compared with the standard transfection reagents. Further, GP-3/pDNA displayed significantly higher target specific transfection efficiency under both in vitro and in vivo conditions. The data demonstrate the potential of GP vectors to deliver nucleic acids simultaneously to macrophages and hepatocytes in gene delivery applications.