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

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Monocrotophos Induced Apoptosis in PC12 Cells: Role of Xenobiotic Metabolizing Cytochrome P450s
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  Monocrotophos Induced Apoptosis in PC12 Cells: Role of Xenobiotic Metabolizing Cytochrome P450s Mahendra Pratap Kashyap 1,2 , Abhishek Kumar Singh 1,2 , Vivek Kumar 1,2 , Vinay Kumar Tripathi 1,2 , RiteshKumar Srivastava 1,2 , Megha Agrawal 1,2 , Vinay Kumar Khanna 1,2 , Sanjay Yadav 1,2 , Swatantra KumarJain 3 , Aditya Bhushan Pant 1,2 * 1 Indian Institute of Toxicology Research, Lucknow, India,  2 Council of Scientific and Industrial Research, New Delhi, India,  3 Department of Biotechnology, Jamia HamdardUniversity, New Delhi, India Abstract Monocrotophos (MCP) is a widely used organophosphate (OP) pesticide. We studied apoptotic changes and theircorrelation with expression of selected cytochrome P450s (CYPs) in PC12 cells exposed to MCP. A significant induction inreactive oxygen species (ROS) and decrease in glutathione (GSH) levels were observed in cells exposed to MCP. Followingthe exposure of PC12 cells to MCP (10 2 5 M), the levels of protein and mRNA expressions of caspase-3/9, Bax, Bcl 2 , P 53 , P 21 ,GSTP1-1 were significantly upregulated, whereas the levels of Bclw, Mcl1 were downregulated. A significant induction in theexpression of CYP1A1/1A2, 2B1/2B2, 2E1 was also observed in PC12 cells exposed to MCP (10 2 5 M), whereas induction of CYPs was insignificant in cells exposed to 10 2 6 M concentration of MCP. We believe that this is the first report showingaltered expressions of selected CYPs in MCP-induced apoptosis in PC12 cells. These apoptotic changes were mitochondriamediated and regulated by caspase cascade. Our data confirm the involvement of specific CYPs in MCP-induced apoptosisin PC12 cells and also identifies possible cellular and molecular mechanisms of organophosphate pesticide-inducedapoptosis in neuronal cells. Citation:  Kashyap MP, Singh AK, Kumar V, Tripathi VK, Srivastava RK, et al. (2011) Monocrotophos Induced Apoptosis in PC12 Cells: Role of XenobioticMetabolizing Cytochrome P450s. PLoS ONE 6(3): e17757. doi:10.1371/journal.pone.0017757 Editor:  Neeraj Vij, Johns Hopkins School of Medicine, United States of America Received  November 23, 2010;  Accepted  February 9, 2011;  Published  March 21, 2011 Copyright:    2011 Kashyap et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the srcinal author and source are credited. Funding:  Financial support by Council of Scientific and Industrial Research, New Delhi, (SIP-08) is acknowledged. University Grant Commission, New Delhi isacknowledged for providing the fellowship to Mr. M.P. Kashyap (GAP-155). The funders had no role in study design, data collection and analysis, decision topublish, or preparation of the manuscript. Competing Interests:  The authors have declared that no competing interests exist.* E-mail: abpant@rediffmail.com Introduction Organophosphorus (OP) group of pesticides have been usedextensively across the world for more than fifty years [1] resulting annual exposure to 2–3 million people [2]. OPs are known to induceacuteandchronicneurotoxicityinmammaliansprimarilybyinhibiting acetylcholinesterase (AChE) activity [3,4]. However, neurotoxicity of  OPs has also been reported to link with necrosis [5], apoptosis [6,7], and oxidative stress mediated pathways [7,8]. OPs have also been foundtoinduceoxidativestressindevelopingbrain,leadingtoaltertheexpression and functions of antioxidant genes [9]. Most of the OPs donot produce the same pattern of behavioral deficits or toxic responses,in part, because of the involvement of different toxicologicalmechanisms that contribute to the net adverse outcomes [10]. Thetoxic responses of OPs on cellular and molecular level have beenexplored in cultured cells using standard endpoints of cytotoxicity andgenotoxicity [5,11]. However, the knowledge on specific pathway(s) involved forindividual OP-induced toxicityisneeded to be elaborating completely. The involvement of different CYPs has been suggested inthe process of oxidative stress [12], mutagenicity [13], apoptosis [14,15], and behavioural deficits [16]. Significant induction in the expression of different CYPs has been reported in liver exposed tostructurally unrelated chemicals [16]. Although, liver is known to be aprimary site for CYPs-mediated metabolism, but the expression andinducibility of CYPs in extrahepatic systems such as blood and brainhave also been reported [16,17]. Involvements of the several CYPs in the metabolic activation of drugs and chemicals have also beenreported in primary cultures of rat brain neuronal and glial cells [18].CYPs facilitate biotransformation of xenobiotics by oxidizing themresult the formation of number of reactive oxygenated intermediates(ROMs). ROMs are highly unstable in nature, but their presence forshort duration in the cells may lead cellular damages [19,20]. ROMs- induced damages have been suggested to cause abrupt xenobioticmetabolism as well as the formation of more hazards intermediates,which could ultimately lead hyper-mutability, genomic instability,adverse effects on number of proteins related to cell cycle checkpointsand neuronal cell death [21].Thus, we studied apoptotic changes and their correlation withexpression of selected cytochrome P450s (CYPs) in PC12 cellsexposed to MCP. MCP was selected as model pesticide, since ithas been used extensively worldwide and is known for itsneurotoxicity [22,23]. PC12 cells were selected because of known expressions of CYPs [24] and most of the marker associated withneuronal structures, functions, toxicity and repair [9,25] Results Intracellular glutathione levels Data of MCP-induced alterations in the levels of intracellularGSH concentrations are summarized in figure 1. Statistically PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e17757  significant (p , 0.001) decrease in the values were observed at 6, 12,and 24 h exposures, i.e., 31.4 6 1.5 mM, 29.7 6 1.3 mM, and27.8 6 1.1 mM following an exposure of MCP (10 2 6 M) and28.2 6 1.3 mM, 22.3 6 1.1 mM, and 19.9 6 1.4 mM in cells ex-posed to MCP (10 2 5 M) when compared with unexposed controlsi.e., 37.8 6 0.8 mM (6 h), 37.1 6 1.0 mM (12 h) and 36.3 6 0.9 mM(24 h) respectively. ROS generation MCP (10 2 6 M and 10 2 5 M) induces significant ROS produc-tion in PC12 cells at all the incubation periods, i.e., 132 6 11% and116 6 10% (6 h); 155 6 3.6% and 138 6 7.9% (12 h), and144 6 2.7% and 169 6 5.6% (24 h) respectively. ROS productionwas insignificant following 10 2 7 M and 10 2 8 M concentrations of MCP at all the time points. While, MCP (10 2 4 M) exposure for 12and 24 h was significantly cytotoxic (Figure 2b). Fluorescencemicroscopic analysis using DCFH-DA fluorescence dye maintainsthe linearity with the data obtained by spectro-fluorimetric analysis(Figure 2a). Apoptosis detection MCP (10 2 5 M) exposure for 6 h induces significant (3 fold of control) apoptosis in PC12 cells. While the magnitude of apoptosis induction was low (2 fold of control) in cells exposedto MCP (10 2 6 M) for 6 h. Increase in necrosis and decrease inapoptosis was observed in cells exposed to MCP (10 2 4 M) for6 h. System optimization was confirmed by induction of apoptosis in 31.3% cell population following camptothecin(3  m g/ml) exposure for 6 h, and served as positive control(Figure 3a). Fluorescence imaging of the cells kept under identicalexperimental conditions confirms our data obtained by FACSanalysis (Figure 3b). Bis-benzimide Staining Nuclear condensation and DNA fragmentation were studied asmarkers of apoptosis in PC12 cells following the exposure of selected concentrations of MCP. Findings of the assays wereshowing the similar trends as observed in case of MMP andresponded to MCP insult in a dose dependent manner in PC12cells (Figure 4a & b). Transcriptional changes MCP (10 2 5 M) exposure for 2, 6, 12, and 24 h inducessignificant alterations in the expression levels of mRNA of CYP1A1, 1A2, 2B1, 2B2, and 2E1. In a biphasic response, at 2 and6 h, expression was increased and thereafter levels were decreasedat 12 and 24 h. However, at 12 h, values were significantly higherthan unexposed control cells for all the CYPs except for CYP2B1.Interestingly, the peak levels of all the CYPs were observed in cellsexposed for 6 h i.e., 4.79 6 0.56; 17.00 6 1.63; 2.54 6 0.07;3.52 6 0.77; and 3.28 6 0.95 fold of control for CYP 1A1, 1A2,2B1, 2B2, and 2E1 respectively. The elevated levels of mRNAwere almost restored to basal level rather below to that by 24 hexposure (Figure 5a).Since, MCP (10 2 5 M) exposure for 6 h was found to be mosteffective in the induction of mRNA expression of CYPs, themRNA expression study for the genes associated with apoptosiswas restricted for 6 h only. Results show significant up-regulationin the expression of mRNA for Caspase-3 (6.37 6 0.31), Caspase-9(3.51 6 0.21), Bax (1.28 6 0.12), Bcl2 (1.50 6 0.25), Bnip3(1.43 6 0.08), p53 (1.67 6 0.17), and p21 (1.31 6 0.12) fold of control, whereas down regulation was observed in case of Bclw(0.69 6 0.03), and Mcl1 (0.88 6 0.01) fold of control (Figure 5b). Western blot analysis MCP (10 2 5 M) exposure for 6 h shows peak upregulation of protein expression of CYP 1A1 (1.89 6 0.23), 1A2 (1.53 6 0.19), 2B1(1.23 6 0.05), 2B2 (2.06 6 0.23), 2E1 (3.13 6 0.47), p53 (1. 94 6 0.24),GSTP1-1: 23 kda (1.85 6 0.27), GSTP1-1: 42 Kda (1.39 6 0.17),GSTP1-1: 46 kda (1.46 6 0.16), Bax (2.75 6 0.34), Bcl 2  (1.33 6 0.12),activated Caspase-9 (2.43 6 0.14) and activated Caspase-3(3.62 6 0.41) fold of control. Protein expression of CYP1A1/1A2and Bax came to the basal level in cells exposed to MCP (10 2 5 M)for 12, and 24 h. However, the levels of protein expression of CYP2B2, 2E1, p53 and all forms of GSTP1-1 were higher thanunexposed control cells following MCP exposure for 12 and 24 h.In case of Bcl 2  values were observed below the basal level at 12, and24 h exposure. Significant restoration of altered levels wereobserved in recovery group i.e., CYP1A1 (0.74 6 0.09), CYP1A2(1.16 6 0.17),CYP2B1(1.19 6 0.08),CYP2B2(1.72 6 0.26),CYP2E1(1.75 6 0.24), P 53 (1.27 6 0.19), Bax (1.18 6 0.14), Bcl 2  (0.42 6 0.02),and activated Caspase-3 (0.86 6 0.07) [Figure 6 (i) a & b; (ii) a & b;(iii) a & b]. Immunocytochemical analysis MCP (10 2 6 M and 10 2 5 M) exposure for 6 h inducessignificant (p , 0.001) protein expression of C-fos (2.20 6 0.51 fold,2.81 6 0.78), and C-jun (1.93 6 0.51 fold, 3.30 6 0.72) fold of controlrespectively. MCP exposure of 10 2 5 M induces the alteration inthe expression with greater magnitude than MCP 10 2 6 Mconcentration and this magnitude difference was statisticallysignificant (p , 0.001) (Figure 7a & b-I, II). Discussion The high lipid contents, high oxygen consumption, and lowlevels of glutathione contents are suggested reasons for ROS- Figure 1. Glutathione (GSH) levels in PC12 cells exposed toMCP (10 2 4 –10 2 7 M) for 6, 12, and 24 h assessed by usingfluorescence based Glutathione Detection Kit (Catalog no.APT250, Chemicon, USA).  To estimate the GSH levels, the lysedsamples (90  m l/well) were transferred to 96 well black bottom platesand mixed with freshly prepared assay cocktail (10  m l) containingmonochlorobimane (MCB), a dye has high affinity for glutathione incells compared to other thiols. Plates were read at excitationwavelength 380 nm and emission wavelength 460 nm after theincubation for 1–2 h at 37 u C by using Multiwell Microplate Reader(Synergy HT, Bio-Tek, USA). Standard curve was plotted using theglutathione standard supplied in the kit and used to calculate theexperimental values. The data are expressed in intracellular concentra-tions of GSH 6 SEM, n=3. * =P , 0.05, **=p , 0.001.doi:10.1371/journal.pone.0017757.g001MCP-Induced Apoptosis in PC12: Role of CYPsPLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e17757  mediated vulnerability of brain cells against xenobiotics [26]. Inthe present study, we also observed the significant dose and timedependent induction in ROS generation and decrease inglutathione (GSH) levels, which were found to be associated withapoptotic changes. Earlier we reported the increase of LPO inPC12 cells exposed to MCP [7]. Similar kind of associations havealso been reported using cultured cells of neural srcin and ratbrain slices [10], blood mononuclear cells [27], and mouse macrophage cell lines [28,29]. The activation of cytochrome P450s and their interaction withmitochondrial chain complexes have been suggested in chemical-induced apoptosis [20,30]. The involvement of CYPs in organophosphates-induced apoptosis in neuronal cells has alsobeen indicated [31]. However, we are reporting first time thatMCP-induced apoptosis and oxidative stress are associated/regulated by specific isoforms of CYPs in PC12 cells. We observedsignificant induction in the expression of CYPs even at 2 hexposure, which was found to be upstreamed to ROS generationby 6 h in PC12 exposed to MCP. Such induced expression of CYPs in early hours might have played important role in theproduction of reactive oxygenated molecules (ROMs), which areknown to induce ROS generation [32], LPO [33], GSTs [34], and eventually to apoptosis [15,20]. In the present investigations, apoptosis induction and oxidative stress was found to be associatedwith upregulation of CYP1A1. Such increased expression of CYP1A1 has also been reported increase the excretion rate of 8-oxoguanine (oxo8Gua) in human hepatoma cell line, a biomarkerof oxidative DNA damage [35]. CYP1A1 and CYP1B1 have beendemonstrated to catalyze catechol estrogen formations, which playa key role in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin-inducedoxidative damage in cultured human mammary epithelium cells[36]. Induced CYP2E1 was found to cause oxidative stress bydepleting the intracellular GSH levels [37], activation of the p38MAP kinase pathway, and induction of the transcription factorNrf2 [38], in human hepatoma cell line-HepG2. The role of CYP2E1has been suggested in alcohol-induced oxidative DNAdamage in liver of null mice [39].Induction in the expression levels of CYPs (CYP1A1/1A2,2B1/B2 and 2E1) were higher at 6 h, which brought downtowards the basal level by 24 h. Similarly, apoptotic events werealso found to reduce with the passage of time. This could be due toincreased necrosis at 12 and 24 h exposures, as discussed in ourearlier report too [7]. Since, induced expression of CYPs isregarded as defence mechanism to detoxify the effect of xenobiotics [40], thus, initial increase in the expression (mRNAand protein) of CYPs suggest responsiveness of cells against MCP Figure 2. Reactive Oxygen Species (ROS) generation in PC12 cells exposed to MCP.  (a) Representative microphotographs showing MCP-induced reactive oxygen species (ROS) generation in PC12 cells. ROS generation was studied using dichlorofluorescin diacetate (DCFH-DA) dye.Images were captured by Nikon phase contrast cum fluorescence microscope (model 80i) attached with 12.7 Megapixel Nikon DS-Ri1 digital CCDcool camera. (b) Percent change in ROS generation following 6, 12 and 24 h exposure of various concentrations of MCP in PC12 cells assessed byspectrofluorometric analysis. In brief, cells (1 6 10 4 per well) were seeded in poly L-lysine pre-coated 96 well black bottom culture plates and allowedto adhere for 24 h in 5% CO 2 –95% atmosphere at 37 u C. Cells were exposed to MCP (10 2 4 to 10 2 8 M) for 6, 12 and 24 h. Following the exposure, cellswere re-incubated with 2 9 , 7 9  dichlorodihydrofluorescein-diacetate (DCFH-DA) (20  m M) for 30 min at 37 u C and fluorescence intensity was measuredusing multiwall micro plate reader (Synergy HT, Bio-Tek, USA) on excitation wavelength at 485 nm and emission wavelength at 528 nm. The data areexpressed in mean of percent of the unexposed control 6 SEM, n=8. * =P , 0.05, **=p , 0. 001.doi:10.1371/journal.pone.0017757.g002MCP-Induced Apoptosis in PC12: Role of CYPsPLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e17757  MCP-Induced Apoptosis in PC12: Role of CYPsPLoS ONE | www.plosone.org 4 March 2011 | Volume 6 | Issue 3 | e17757  exposure. Whereas, the decreased levels of CYPs in cells exposedto MCP for longer period might be due to significant necrotic celldeath. It has already been demonstrated in case of variousxenobiotics that higher doses for low time periods and lower dosesfor higher time periods can convert apoptosis into necrosis [41].Following MCP exposure, we observed a significant up-regulation in the expression of immediate early response geneproteins, i.e., C-fos and C-jun. Such significant up-regulationmight be due to oxidative stress induced by the massive productionof ROS/ROMs or induction of JNK pathway during CYPs-mediated metabolism of MCP. The association of chemical-induced over expression of the various CYPs, and oxidativedamage is well established [42]. The induced level of GSH is anindicator of strong anti-oxidant status in cell system [28], whereas,reduced GSH levels were found to be associated with impairedanti-oxidant activities [43]. The lower levels of GSH in brain cellshave been reported to facilitate the dissociation of GSTP1-1/JNKcomplex, and activation of JNK pathway [44]. In the presentstudy, increased expression of GSTP1-1 and decreased GSH levelsmay also be correlated with the activation of JNK pathway, andsubsequent cell death. However, upon the longer exposure, theGSTP1-1 levels came down very near to basal, which indicateeither the failure of self defense due to activation of JNK pathwayor necrotic cell death. Such GSTP1-1 dependent activation of  JNK pathway is well documented in Jurkat [45], humanneuroblastoma cell line [46], and in NB4 cell line [44], against variety of chemical exposures.The other possible reason for our findings might be due to thenon-enzymatic direct binding of GSH with CYPs mediatedreactive metabolites of MCP. This phenomenon has already beenreported in case CYPs mediated metabolism of paracetamol,where the levels of GSH were found to be depleted upon theaccumulation of reactive metabolite - N-acetyl-p-benzoquinoneimine (NAPQI) [47].In the present investigation, synchronization was also observedbetween the increased expressions of CYPs (1A1, 1A2, 2B1, 2B2,and 2E1) and altered expressions of caspase 3 and caspase 9, genesinvolved in apoptosis signalling cascade in PC12 cells. The caspasecascade activation has been reported by two different routes, i.e.,binding of procaspase-9 with Apaf-1 to form the apoptosomecomplex following the release of cytochrome- c   from damagedmitochondria [29], while in other route OMI, and SMACsreleased from intra-mitochondrial space is binds with caspaseinhibitors, and thus activates the caspases [48]. But, we arehypothesizing the involvement of CYPs in the activation of caspases as another possible route to trigger the apoptosissignalling in PC12 cells receiving MCP exposure. Since, CYPs-mediated apoptotic changes have already been reported in E47cells [49], and Hepa1c1c7 cells [24,50] exposed to buthionine sulfoximine and Benzo[a] pyrene respectively. Based on thefindings, we propose a schematic flow diagram showing theinvolvement of selected CYPs in the triggering of ROM inducedoxidative stress and apoptosis cascade in PC12 cells exposed toMCP. Apoptosis induction was routed through mitochondrialactivity and by the involvement of caspase 3/9 (Figure 8).In summary, we believe that this is the first report showing altered expressions of selected CYPs in MCP induced apoptosisand oxidative damage in PC12 cells. These apoptotic changeswere mitochondria-mediated and regulated through caspasecascade. Our data confirm the involvement of specific CYPs inMCP induced apoptosis in PC12 cells and also identifies possiblecellular and molecular mechanisms of organophosphate pesticide-induced apoptosis in neuronal cells. Materials and Methods Cell culture PC12 cells were procured from National Centre for CellSciences, Pune, India, and have been maintained at In VitroToxicology Laboratory, Indian Institute of Toxicology Research, Figure 4. DAPI staining for the detection of MCP-inducedapoptosis.  (a) Representative microphotographs showing induction of Apoptosis in PC12 cells exposed to various concentrations of MCP forvariable time periods. (A): Unexposed control cells (B): cells exposed to10 2 6 M MCP showing apoptotic body; (C): Cells exposed to 10 2 5 MMCP Showing more damages. (b) Apoptosis induction in PC12 cellsexposed to various concentrations of MCP for different time periods.Apoptotic Bodies were counted by using Upright phase contrastFluorescent microscope (Nikon 80i, Japan) at 10 6 100x oil immersionmagnification and images were grabbed by Nikon DS-Ri1 (12.7megapixel) camera. Minimum 1000 cells were counted in each slidein triplicate. * p , 0.05, **p , 0.001doi:10.1371/journal.pone.0017757.g004 Figure 3. Apoptosis induction in PC12 cells exposed to MCP.  (a) Apoptosis detection in PC12 cells exposed to MCP using Mitolight TM apoptosis detection kit (catalog no. APT142, Chemicon, USA). (A) Unstained cells; (B) Control cells; (C) PC12 cells exposed to MCP (10 2 6 M) for 6 h; (D)PC12 cells exposed to MCP (10 2 5 M) for 6 h; (E) PC12 cells exposed to MCP (10 2 4 M) for 6 h; (F) Experimental positive control- PC12 cells exposed tocampothecin (3  m g/ml) for 6 h; (G) Cells pretreated with 10  m M NAC for 1 h and then exposed with MCP(10 2 5 M) for 6 h. (b) Apoptosis detection byMitolight TM apoptosis detection kit using Upright Phasecontrast Microscope (Nikon 80i, Japan) at 10 6 100x oil immersion magnification. The imageswere snapped by Nikon DS-Ri1 (12.7 megapixel) camera. Figure A1- Control cells showing intense red color due to polymerization of Mitolight dye inmitochondria 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 cellsexposed to MCP (10 2 6 M) for 6 h shows significant dissipation in Mitochondrial membrane potential. Figure C1–C4: PC12 cells exposed to MCP(10 2 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.doi:10.1371/journal.pone.0017757.g003MCP-Induced Apoptosis in PC12: Role of CYPsPLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e17757
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