Mp Kashyap

Multi-walled carbon nanotubes induce oxidative stress and apoptosis in human lung cancer cell line-A549

Abstract Multi-walled carbon-nanotubes (MWCNTs)-induced apoptotic changes were studied in human lung epithelium cell line-A549. Non-cytotoxic doses of MWCNTs were identified using tetrazolium bromide salt (MTT) and lactate dehydrogenase (LDH) release assays. Cells were exposed to MWCNTs (0.5–100 μg/ml) for 6–72 h. Internalization and characterization of CNTs was performed by electron microscopy. Apoptotic changes were estimated by nuclear condensation, DNA laddering, and confirmed by expression of associated markers: p53, p21WAF1/CIP1, Bax, Bcl2 and activated caspase-3. MWCNTs induced the production of reactive oxygen species and malondialdehyde along with significant decrease in the activity of catalase and glutathione. MWCNTs-induced ROS generation was found not to be associated with the mitochondrial activity. In general, the changes were significant at 10 and 50 μg/ml only. Results indicate the involvement of oxidative stress and apoptosis in A549 cells exposed to MWCNTs. Our studies provide insights of the mechanisms involved in MWCNTs-induced apoptosis at cellular level.

Protective potential of trans-resveratrol against 4-hydroxynonenal induced damage in PC12 cells

The role of 4-hydroxynonenal (4-HNE), a byproduct of membrane lipid peroxidation has been suggested in neurodegeneration. However, the underlying mechanisms are poorly understood. The investigations were carried out to study the preventive potential of trans-resveratrol against 4-HNE induced damage in PC12 cells. Trans-resveratrol, a natural compound obtained from grape skin and found in red wine, is reported to have wide pharmacological window. Cells pretreated with trans-resveratrol (5, 10 and 25 microM) for 24 h were exposed to 4-HNE (25 microM) for 2 h. Pre-treatment of trans-resveratrol was found to be significantly effective in countering the cytotoxic responses of 4-HNE. Significant reduction in reactive oxygen species generation, restoration of intracellular glutathione, and lipid peroxidation levels suggest the improved antioxidant defense system in the cells pretreated with trans-resveratrol. Further, 4-HNE induced alterations in the protein expression of mitochondria-mediated apoptosis markers (Bax, Bcl-2 and Caspase-3) were significantly restored by pre-treatment of trans-resveratrol suggesting the protective potential of trans-resveratrol in PC12 cells against 4-HNE induced oxidative damage. Together these data show the prophylactic potential of trans-resveratrol in oxidative stress mediated apoptotic neurodegeneration.

Caspase cascade regulated mitochondria mediated apoptosis in monocrotophos exposed PC12 cells.

Monocrotophos (MCP) is a commonly used organophosphorus (OP) pesticide. We studied apoptotic changes in PC12 cells exposed to MCP. A significant induction in reactive oxygen species (ROS), lipid peroxide (LPO), and the ratio of glutathione disulfide (GSSG)/reduced glutathione (GSH) was observed in cells exposed to selected doses of MCP. Following the exposure of PC12 cells to MCP, the levels of protein and mRNA expressions of Caspase-3, Caspase-9, Bax, p53, P(21), Puma, and cytochrome-c were significantly upregulated, whereas the levels of Bcl(2), Bcl(w), and Mcl1 were downregulated. TUNEL assay, DNA laddering, and micronuclei induction show that long-term exposure of PC12 cells to MCP at higher concentration (10(-5) M) decreases the number of apoptotic events due to an increase in the number of necrotic cells. MCP-induced translocation of Bax and cytochrome-c proteins between the cytoplasm and mitochondria confirmed the role of p53 and Puma in mitochondrial membrane permeability. Mitochondria mediated apoptosis induction was confirmed by the increased activity of caspase cascade. We believe that this is the first report showing MCP-induced apoptosis in PC12 cells, which is mitochondria mediated and regulated through the caspase cascade. Our data demonstrates that MCP induced the apoptotic cell death in neuronal cells and identifies the possible cellular and molecular mechanisms of organophosphate pesticide-induced apoptosis in neuronal cells.

Monocrotophos Induced Apoptosis in PC12 Cells: Role of Xenobiotic Metabolizing Cytochrome P450s

Monocrotophos (MCP) is a widely used organophosphate (OP) pesticide. We studied apoptotic changes and their correlation with expression of selected cytochrome P450s (CYPs) in PC12 cells exposed to MCP. A significant induction in reactive oxygen species (ROS) and decrease in glutathione (GSH) levels were observed in cells exposed to MCP. Following the exposure of PC12 cells to MCP (10−5 M), the levels of protein and mRNA expressions of caspase-3/9, Bax, Bcl2, P53, P21, GSTP1-1 were significantly upregulated, whereas the levels of Bclw, Mcl1 were downregulated. A significant induction in the expression of CYP1A1/1A2, 2B1/2B2, 2E1 was also observed in PC12 cells exposed to MCP (10−5 M), whereas induction of CYPs was insignificant in cells exposed to 10−6 M concentration of MCP. We believe that this is the first report showing altered expressions of selected CYPs in MCP-induced apoptosis in PC12 cells. These apoptotic changes were mitochondria mediated and regulated by caspase cascade. Our data confirm the involvement of specific CYPs in MCP-induced apoptosis in PC12 cells and also identifies possible cellular and molecular mechanisms of organophosphate pesticide-induced apoptosis in neuronal cells.

Ischemic insult induced apoptotic changes in PC12 cells: Protection by trans resveratrol

In this study, we determined the protective potential of trans resveratrol against oxygen-glucose deprivation (OGD) induced reactive oxygen species mediated apoptotic damages in PC12 cells. In vitro model of ischemic cerebral stroke was created by keeping cells in an OGD condition for 6h followed by 24h reoxygenation. Cells received biologically safe doses (5, 10, and 25 μM) of trans resveratrol in the following schedules for 24h prior to OGD; during 6h of OGD; for 24h post OGD and whole treatment group which starts from 24h before OGD and lasted to 24h post OGD. Anti-ischemic potential of trans resveratrol was assessed by measuring the regulation of lipid peroxidation, reactive oxygen species production, glutathione content, and expression (mRNA and protein) of apoptotic markers such as Bax, Bcl(2) and Caspase-3. Hypoxia inducible factor-1α (HIF-1α) was also assessed to correlate the changes with ischemic injuries. Significant (P<0.05) restoration in lipid peroxidation, reactive oxygen species, and glutathione content were observed following the treatment of trans resveratrol in cells receiving OGD and re-oxygenation. Changes induced by trans resveratrol could be correlated well with alterations in the expression of Bax, Bcl(2), Caspase-3 and HIF-1α. These results indicate that trans resveratrol administration attenuates free radical formation and mitochondria mediated apoptosis perhaps by regulating the expressions of Bax, Bcl(2,) and Caspase-3 in PC12 cells receiving OGD and re-oxygenation insult.

trans-Resveratrol protects ischemic PC12 Cells by inhibiting the hypoxia associated transcription factors and increasing the levels of antioxidant defense enzymes.

An in vitro model of ischemic cerebral stroke [oxygen-glucose deprivation (OGD) for 6 h followed by 24 h reoxygenation (R)] with PC12 cells increases Ca(2+) influx by upregulating native L-type Ca(2+) channels and reactive oxygen species (ROS) generation. This reactive oxygen species generation and increase in intracellular Ca(2+) triggers the expression of hypoxic homeostasis transcription factors such as hypoxia induced factor-1 alpha (HIF-1α), Cav-beta 3 (Cav β3), signal transducer and activator of transcription 3 (STAT3), heat shock protein 27 (hsp-27), and cationic channel transient receptor potential melastatin 7 (TRPM7). OGD insulted PC12 cells were subjected to biologically safe doses (5, 10, and 25 μM) of trans-resveratrol in three different treatment groups: 24 h prior to OGD (pre-treatment); 24 h post OGD (post-treatment); and from 24 h before OGD to end of reoxygenation period (whole-treatment). Here, we demonstrated that OGD-R-induced neuronal injury/death is by reactive oxygen species generation, increase in intracellular calcium levels, and decrease in antioxidant defense enzymes. trans-Resveratrol increases the viability of OGD-R insulted PC12 cells, which was assessed by using MTT, NRU, and LDH release assay. In addition, trans-resveratrol significantly decreases reactive oxygen species generation, intracellular Ca(2+) levels, and hypoxia associated transcription factors and also increases the level of antioxidant defense enzymes. Our data shows that the whole-treatment group of trans-resveratrol is most efficient in decreasing hypoxia induced cell death through its antioxidant properties.

Atrazine induces transcriptional changes in marker genes associated with steroidogenesis in primary cultures of rat Leydig cells

Reproductive toxicity of atrazine (ATZ) is well reported in mammals. However, the underlying mechanisms are poorly understood and need to be explored. Thus, we investigate ATZ induced transcriptional changes in selected markers of steroidogenesis in primary cultures of rat interstitial Leydig cells (ILCs). Cytotoxic studies were carried out by exposing the cells to ATZ (0.5-50 μg/mL or 2.32-232 μM) for 24-72 h, whereas; the exposure period of expression studies was for 2 h. ATZ exposure of (25 and 50 μg/ml) for 48 h onwards was found to be cytotoxic in MTT (dimethylthiazol-diphenyl tetrazolium bromide salt) assay, while in NRU (neutral red uptake) assay, cytotoxicity could be recorded at 50 μg/ml exposure of 72 h only. A significant dose dependent induction in the levels of mRNA expression of genes of steroidogenic acute regulatory protein (STAR), cytochrome P45011A1, 3β-hydroxysteroid dehydrogenase (3β-HSD), and other steroidogenic proteins were observed in cells exposed to ATZ. Our data suggest the applicability of these selected marker genes of steroidogenesis as an indicator of short term exposure of ATZ induced testicular toxicity in rats interstitial Leydig cells (ILCs).

Ameliorative Effects of Dimetylthiourea and N-Acetylcysteine on Nanoparticles Induced Cyto-Genotoxicity in Human Lung Cancer Cells-A549

We study the ameliorative potential of dimetylthiourea (DMTU), an OH• radical trapper and N-acetylcysteine (NAC), a glutathione precursor/H2O2 scavenger against titanium dioxide nanoparticles (TiO2-NPs) and multi-walled carbon nanotubes (MWCNTs) induced cyto-genotoxicity in cultured human lung cancer cells-A549. Cytogenotoxicity was induced by exposing the cells to selected concentrations (10 and 50 µg/ml) of either of TiO2-NPs or MWCNTs for 24 h. Anti-cytogenotoxicity effects of DMTU and NAC were studied in two groups, i.e., treatment of 30 minutes prior to toxic insult (short term exposure), while the other group received DMTU and NAC treatment during nanoparticles exposure, i.e., 24 h (long term exposure). Investigations were carried out for cell viability, generation of reactive oxygen species (ROS), micronuclei (MN), and expression of markers of oxidative stress (HSP27, CYP2E1), genotoxicity (P53) and CYP2E1 dependent n- nitrosodimethylamine-demethylase (NDMA-d) activity. In general, the treatment of both DMTU and NAC was found to be effective significantly against TiO2-NPs and MWCNTs induced cytogenotoxicity in A549 cells. Long-term treatment of DMTU and NAC during toxic insults has shown better prevention than short-term pretreatment. Although, cells responded significantly to both DMTU and NAC, but responses were chemical specific. In part, TiO2-NPs induced toxic responses were mediated through OH• radicals generation and reduction in the antioxidant defense system. While in the case of MWCNTs, adverse effects were primarily due to altering/hampering the enzymatic antioxidant system. Data indicate the applicability of human lung cancer cells-A549 as a pre-screening tool to identify the target specific prophylactic and therapeutic potential of drugs candidate molecules against nanoparticles induced cellular damages.

Differential protection of pre-, co- and post-treatment of curcumin against hydrogen peroxide in PC12 cells

Pharmacological potential of curcumin was assessed in PC12 cells against hydrogen peroxide (H2 O2) exposure. In MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and lactate dehydrogenase (LDH) assays, 24-hour exposure of H2O2 (0.5 mM and above) was found to be cytotoxic. A significant (p < 0.001) increase in percentage cell viability was recorded in PC12 cells pretreated with curcumin (25, 50 and 100 µg/mL) for 24 hours prior to H2O2 (0.5 and 1 mM) exposure for 24 hours. Co-exposure to H2O2 and curcumin was also found effective. However, a therapeutic treatment of curcumin for 24 hours after H2O 2 exposure to the cells was found ineffective. Differential response of PC12-H2O2 model to curcumin in MTT and LDH assays suggests the utility of these endpoints to sort the drug candidates to study their antioxidant potential.

Potential of the propolis as storage medium to preserve the viability of cultured human periodontal ligament cells: an in vitro study

AIMnIn vitro experiments were carried out to evaluate the potential of propolis, a natural resin known for its wide therapeutic window, as storage medium to preserve the viability of cultured human periodontal ligament (PDL) cells.nnnMATERIALS AND METHODSnPrimary cultures of human PDL cells were subjected to either independent exposure of propolis (2.5%, 5.0%, 10.0%, and 20.0%), Hanks balanced salt solution (HBSS), milk (0.5%), artificial saliva, Dulbeccos modified Eagles medium (DMEM) or combination of propolis 10% + DMEM, propolis 20% + DMEM for 30 min to 24 h at 37 °C. Cell viability was assessed using standard endpoints i.e., tetrazolium bromide salt (MTT), neutral red uptake, and trypan blue dye exclusion assay.nnnRESULTSnIn general, combinations of propolis 10% + DMEM, propolis 20% + DMEM, and DMEM alone were found to be better than other media used in this study. The difference in the potentials of these media to maintain the cell viability reached to the statistically significant levels by 24 h, when compared with other media used viz., propolis 2.5% (P < 0.01), propolis 5.0% (P < 0.05), propolis 10.0% (P < 0.05), propolis 20.0% (P < 0.001), HBSS (P < 0.001), and milk (P < 0.01). Trypan blue dye exclusion assay could be recorded the most sensitive among all the assays selected to study the cell viability of PDL cells.nnnCONCLUSIONSnStudy indicates that combinations of propolis 10% + DMEM, propolis 20% + DMEM, and DMEM alone are equally good as storage media of choice to keep PDL cells viable during extra-alveolar period up to 24 h. Other more readily available medium such as milk may serve as appropriate alternative storage medium for shorter time periods i.e., up to 12 h.