Rotenone-induced oxidative stress and apoptosis in human liver HepG2 cells.

Rotenone, a commonly used pesticide, is well documented to induce selective degeneration in dopaminergic neurons and motor dysfunction. Such rotenone-induced neurodegenration has been primarily suggested through mitochondria-mediated apoptosis and reactive oxygen species (ROS) generation. But the status of rotenone induced changes in liver, the major metabolic site is poorly investigated. Thus, the present investigation was aimed to study the oxidative stress-induced cytotoxicity and apoptotic cell death in human liver cells-HepG2 receiving experimental exposure of rotenone (12.5-250 µM) for 24 h. Rotenone depicted a dose-dependent cytotoxic response in HepG2 cells. These cytotoxic responses were in concurrence with the markers associated with oxidative stress such as an increase in ROS generation and lipid peroxidation as well as a decrease in the glutathione, catalase, and superoxide dismutase levels. The decrease in mitochondrial membrane potential also confirms the impaired mitochondrial activity. The events of cytotoxicity and oxidative stress were found to be associated with up-regulation in the expressions (mRNA and protein) of pro-apoptotic markers viz., p53, Bax, and caspase-3, and down-regulation of anti-apoptotic marker Bcl-2. The data obtain in this study indicate that rotenone-induced cytotoxicity in HepG2 cells via ROS-induced oxidative stress and mitochondria-mediated apoptosis involving p53, Bax/Bcl-2, and caspase-3.

Nano-titanium dioxide induces genotoxicity and apoptosis in human lung cancer cell line, A549.

The increased inhaled application of titanium dioxide nanoparticles (TiO(2) NPs) increases the potential pulmonary health risks. The present investigations were carried out to study the TiO(2) NPs-induced apoptosis, oxidative stress and genotoxicity in the human lung cancer cell line, A549, a widely used cell system for pulmonary toxicity studies. Tetrazolium bromide salt and lactate dehydrogenase release assays were used to study the cytotoxicity. The genotoxicity studies were carried out using cytokinesis block micronucleus assay. Apoptosis was confirmed by the formation of apoptotic bodies and altered expression (messenger RNA (mRNA) and protein) of markers such as P(53), P(21), Bax, Bcl(2) and cleaved caspase-3. Cells exposed to TiO(2) NPs (10 and 50 µg/ml) for 6-24 h shows significant induction in oxidative stress, that is, the production of reactive oxygen species and malondialdehyde and decrease in the activity of catalase and glutathione. TiO(2) NPs exposure also induces the formation of apoptotic bodies and micronucleus as marker of genotoxicity. A significant up-regulation in the expression of apoptosis markers such as P(53), P(21) and cleaved caspase-3 was observed, while the levels were down-regulated for Bcl(2) at both mRNA and protein levels. TiO(2) NPs exposure could not pose significant effects on Bax expression. Data indicate that nano-TiO(2) induces oxidative stress, genotoxicity and apoptosis in human lung cancer cell line, A549. Our result also identifies the mechanisms involved in TiO(2) NP-induced changes in A549 cells. Perhaps, reporting for the first time, the association of TiO(2) NPs-induced genotoxicity and apoptosis at transcriptional and translational level in the human lung cancer cell line, A549 cells.

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 P(53), Bax, and caspase 3 were significantly upregulated, whereas the levels of Bcl(2) 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.

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, 3ß-hydroxysteroid dehydrogenase (3ß-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).

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 (H(2) O(2)) exposure. In MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and lactate dehydrogenase (LDH) assays, 24-hour exposure of H(2)O(2) (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 H(2)O(2) (0.5 and 1 mM) exposure for 24 hours. Co-exposure to H(2)O(2) and curcumin was also found effective. However, a therapeutic treatment of curcumin for 24 hours after H(2)O(2) exposure to the cells was found ineffective. Differential response of PC12-H(2)O(2) model to curcumin in MTT and LDH assays suggests the utility of these endpoints to sort the drug candidates to study their antioxidant potential.

Protective potential of 17ß-estradiol against co-exposure of 4-hydroxynonenal and 6-hydroxydopamine in PC12 cells.

4-hydroxynonenal (4-HNE) and 6-hydroxydopamine (6-OHDA)-mediated damage in dopaminergic neurons is well documented. Protective potential of steroidal hormone (17ß-estradiol) has also been suggested. However, therapeutic potential of such promising hormone is hampered due to complex brain anatomy and physiology. Thus, the present investigations were studied to suggest the applicability of dopamine expressing PC12 cells as in vitro tool to screen the pharmacological potential of 17ß-estradiol against 4-HNE and 6-OHDA. MTT assay was conducted for cytotoxicity assessment of both 4-HNE (1 µM to 50 µM) and 6-OHDA (10(-4) to 10(-7) M). Non-cytotoxic concentrations, that is, 4-HNE (1 µM) and 6-OHDA (10(-6) M) were selected to study the synergetic/additive responses. PC12 cells were found to be more vulnerable towards co-exposure of individual exposure of 4-HNE and 6-OHDA, even at non-cytotoxic concentrations. Then, cells were subjected to pre-treatment (24 hours) of 17ß-estradiol (1 µM), followed by a permutation of combinations of both 4-HNE and 6-OHDA. Pretreatment of 17ß-estradiol was found to be significantly effective against the cytotoxic responses of 4-HNE and 6-OHDA, when the damage was at lower level. However, 17ß-estradiol was found to be ineffective against higher concentrations. Physiological-specific responses of PC12 cells against 4-HNE/6-OHDA and 17ß-estradiol suggest its applicability as first tier of screening tool.

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.

Association of dopamine DA-D2 receptor in rotenone-induced cytotoxicity in PC12 cells.

The investigations were aimed to study the possible association of dopamine DA-D(2) receptor in rotenone-induced cytotoxicity in PC12 cells, one among the most studied cell line in neurotoxicity studies. PC12 cells were subjected to receive an exposure of rotenone (10(-6) to 10(-4) M) for 24 and 48 hours. Cytotoxicity studies were carried out using standard end points including, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), lactate dehydrogenase (LDH) release and neutral red uptake (NRU). Cells were found to be vulnerable to rotenone in dose-dependent manner. In general, 10(-4) and 10(-5) M concentrations were found to be cytotoxic, whereas 10(-6) M and lower concentrations used have shown nonsignificant effect on cell viability. Further, studies were extended to study the rotenone-induced alterations in cellular glutathione (GSH) level and dopamine DA-D(2) receptor expression. Significant (p < 0.001) chronological depletion in GSH levels were recorded following rotenone exposure. Expression of dopamine DA-D(2) receptor was also found to be effected significantly (p < 0.001) at 24 hours of rotenone exposure (10(-4) and 10(-5)). However, no further depletion in the expression of dopamine DA-D(2) receptor could be recorded with extended exposure period, that is, 48 hours. Rotenone at 10(-6) M and lower concentrations was found to be ineffective in PC12 cells. Data suggest the vulnerability of PC12 cells against experimental exposure of rotenone, which possibly routed through dopamine DA-D(2) receptor and oxidative stress machinery.

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.

NGF induced differentiated PC12 cells as in vitro tool to study 4-hydroxynonenal induced cellular damage.

Investigations were carried out to examine the suitability of PC12 cells as an in vitro tool to examine 4-hydroxynonenal (4-HNE)-induced toxicity in nervous tissue. On day 8 of differentiation, markers of neural effects and oxidative stress were measured following exposure of PC12 cells to 1-50 microM 4-HNE for 1-8h. Endpoints included dopamine DA-D(2) receptor and glutathione S-transferase (GSTP1-1) protein levels, 4-HNE-protein binding, glutathione (GSH) concentrations and intracellular calcium levels. GSH levels were maximally depleted after 4h. 4-HNE also induced depletion of GSTP1-1 and increased intracellular Ca(++), with the latter seen as early as 1h after exposure. Responses at 8h were not greater than responses at earlier times. The experiments suggest that PC12 cells could be an in vitro tool for understanding toxicant-cell interactions, especially those that result in oxidative stress.

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.

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.