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

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

TiO₂-NPs and MWCNTs (50 µg/ml) induced translational changes (HSP27, CYP2E1 and P⁵³) in the absences or presence of DMTU (10 mM)/ NAC (2 mM).

<p><b>a (I)</b> Assessment of altered expressions of proteins involved in the induction of oxidative stress (HSP27 and CYP2E1) and genotoxicity (P⁵³) in A549 cells exposed to TiO₂-NPs (50 µg/ml) for 24 h. GAPDH was used as internal control to normalize the data. Lane (1) unexposed control (2) 50.0 µg/ml TiO₂-NPs (3) 50.0 µg/ml TiO₂-NPs + DMTU (10 mM) (4) 50.0μg/ml TiO₂-NPs + NAC (2 mM). <b>(II)</b> Relative quantification (fold change) of proteins involved in the induction of oxidative stress (HSP27 and CYP2E1) and genotoxicity (P⁵³) in A549 cells exposed to TiO₂-NPs for 24h. GAPDH was used as internal control to normalize the data. Quantification was done in Gel Documentation System (Alpha Innotech, USA) with the help of AlphaEase^TM FC StandAlone V.4.0 software. All values are mean ± S.E. of 3 experiments. ^**P<0.01 (unexposed control Vs TiO₂-NPs exposure). ##P<0.01 (TiO₂-NPs exposure Vs DMTU/NAC treatment). <b>b (I)</b> Assessment of altered expressions of proteins involved in the induction of oxidative stress (HSP27 and CYP2E1) and genotoxicity (P⁵³) in A549 cells exposed to MWCNTs (50 µg/ml) for 24 h. GAPDH was used as internal control to normalize the data. Lane (1) unexposed control (2) 50.0 µg/ml MWCNTs (3) 50.0 µg/ml MWCNTs + DMTU (10 mM) (4) 50.0μg/ml MWCNTs + NAC (2 mM). <b>(III)</b> Relative quantification (fold change) of proteins involved in the induction of oxidative stress (HSP27 and CYP2E1) and genotoxicity (P⁵³) in A549 cells exposed to MWCNTs for 24h. GAPDH was used as internal control to normalize the data. Quantification was done in Gel Documentation System (Alpha Innotech, USA) with the help of AlphaEase^TM FC StandAlone V.4.0 software. All values are mean ± S.E. of 3 experiments. ^**P<0.01 (unexposed control Vs MWCNTs exposure). ##P<0.01 (MWCNTs exposure Vs DMTU/NAC treatment).</p

Transmission Electron Microscopy (TEM) analysis of internalization of TiO₂-NPs in A549 cells.

<p>TEM microphotographs showing internalization of TiO₂-NPs (a, b) in human lung cancer cells-A549. Arrows indicates the nanoparticles were adhered on the cell surface between microvilli and pseudopodes within few minutes of time (a) and subsequently internalized in small vacuoles deep in the cytoplasm, when observed at 24 h (b).</p

Effect of DMTU/NAC on the restoration of ROS generated in A549 cells exposed to TiO₂-NPs/ MWCNTs.

<p>ROS generation was detected using ‘2, 7-dichlorofluorescin diacetate (DCFH-DA) dye. (<b>a</b>) In short term group, cells were pre-treated with either of DMTU or NAC for 30 minutes followed by exposure of TiO₂-NPs (10 & 50 µg/ml) for 24 h. In long term group, cells were receiving a co-exposure (24 h) of DMTU/NAC and TiO₂-NPs (10 & 50 µg/ml). Cells were then analyzed for DMTU/NAC mediated restoration in the levels of intracellular ROS, which was induced by the exposure of TiO₂-NPs. All values are mean ± S.E. of 3 experiments. ^**P<0.01 (unexposed control Vs TiO₂-NPs exposure). ##P<0.01 (TiO₂-NPs exposure Vs DMTU/NAC treatment). The positive control group was consisting of A549 cells pre-treated (1h) with 500 µM of H₂O₂. (<b>b</b>) In short term group, cells were pre-treated with either of DMTU or NAC for 30 minutes followed by exposure of MWCNTs (10 & 50 µg/ml) for 24 h. In long term group, cells were receiving a co-exposure (24 h) of DMTU/NAC and MWCNTs (10 & 50 µg/ml). Cells were then analyzed for DMTU/NAC mediated restoration in the levels of intracellular ROS, which was induced by the exposure of MWCNTs. All values are mean ± S.E. of 3 experiments. ^**P<0.01 (unexposed control Vs MWCNTs exposure). ##P<0.01 (MWCNTs exposure Vs DMTU/NAC treatment). The positive control group was consisting of A549 cells pre-treated (1h) with 500 µM of H₂O₂.</p

Experimental design.

<p>Experimental design showing the exposure protocol of nanoparticles (TiO2-NPs and MWCNTs) and scavenges (DMTU and NAC), study groups and endpoints studied in human lung cancer cells-A549.</p

Apoptosis induction in PC12 cells exposed to MCP.

<p>(a) Apoptosis detection in PC12 cells exposed to MCP using Mitolight™ apoptosis detection kit (catalog no. APT142, Chemicon, USA). (A) Unstained cells; (B) Control cells; (C) PC12 cells exposed to MCP (10⁻⁶ M) for 6 h; (D) PC12 cells exposed to MCP (10⁻⁵ M) for 6 h; (E) PC12 cells exposed to MCP (10⁻⁴ M) for 6 h; (F) Experimental positive control- PC12 cells exposed to campothecin (3 µg/ml) for 6 h; (G) Cells pretreated with 10 µM NAC for 1 h and then exposed with MCP(10⁻⁵ M) for 6 h. (b) Apoptosis detection by Mitolight™ apoptosis detection kit using Upright Phasecontrast Microscope (Nikon 80i, Japan) at 10×100x oil immersion magnification. The images were snapped by Nikon DS-Ri1 (12.7 megapixel) camera. Figure A1- Control cells showing intense red color due to polymerization of Mitolight dye in mitochondria indicative of healthy mitochondria. Figure A2- green color indicates the accumulation of non-polymerized dye in cytoplasm. Figure A3- Nuclei stained with DAPI.Figure A4- Superimposed microphotographs showing healthy mitochondria with intact membrane. Figure B1-B4: PC12 cells exposed to MCP (10⁻⁶ M) for 6 h shows significant dissipation in Mitochondrial membrane potential. Figure C1–C4: PC12 cells exposed to MCP (10⁻⁵ M) for 6 h. C-3: cells showing nuclear condensation and fragmentations (D1 and D2 are magnified view highlighting the same). C-4: Superimposed microphotograph showing apoptotic events.</p

MCP induced alterations in the expression of early response genes.

<p>(7a) Representative microphotographs of immunocytochemical localization of C-fos and C-jun proteins in PC12 cells exposed to MCP (10⁻⁵ and 10⁻⁶ M). Images were taken by Nikon Eclipse 80i equipped with Nikon DS-Ri1 12.7 megapixel camera, Japan. (7b I & II) Relative quantification of fold inductions in the expression of C-fos and C-jun proteins in PC12 cells exposed to MCP (10⁻⁵ and 10⁻⁶ M) for 6 h. Leica Q-Win 500 image analysis software was used to quantify the expression of C-fos and C-jun. Data were calculated as mean ± SE of at least 20 fields from three independent experiments.</p