CASPASE INHIBITION AUGMENTS DICHLORVOS-INDUCED DOPAMINERGIC NEURONAL CELL DEATH BY INCREASING ROS PRODUCTION AND PARP1 ACTIVATION W. Y. WANI, a A. SUNKARIA, a D. R. SHARMA, a R. J. L. KANDIMALLA, a A. KAUSHAL, a E. GERACE, b A. CHIARUGI b AND K. D. GILL a * a Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India b Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy Abstract—Numerous epidemiological studies have shown an association between pesticide exposure and the increased risk of developing Parkinson’s disease. Previ- ously we have reported that Dichlorvos exposure can induce oxidative stress, resulting in over-expression of pro-apoptotic genes and finally caspase-dependent nigro- striatal dopaminergic neuronal cell death in rat brain. Here, we examined the effect of caspase inhibition on PC12 cell death induced by Dichlorvos (30 lM). Reactive oxygen spe- cies (ROS) generation followed by protein carbonylation, lipid peroxidation, decreased antioxidant defenses (decreased Mn-superoxide dismutase (MnSOD) activity and decreased glutathione levels) and subsequent caspase acti- vation mediated the apoptosis. Inhibition of caspase cas- cade with Boc-aspartyl(OMe)-fluoromethylketone (BAF) enhanced the Dichlorvos-induced PC12 cell death, as assessed by the increased cellular efflux of lactate dehydro- genase (LDH). This increase in cell death was accompanied by a marked increase in poly(ADP-ribose) polymerase-1 (PARP1) activity, increased oxidative stress, a reduction in the mitochondrial membrane potential and reduced cellular NAD and ATP levels. Pretreatment of cells with PJ34, a PARP1 inhibitor prevented the cells from undergoing cell death and preserved intracellular NAD and ATP levels. Sub- sequent release of the apoptosis-inducing factor (AIF) from mitochondria and its translocation into the nucleus was also prevented by PJ34 pretreatment. In conclusion, the results of the present study show that caspase inhibition without concurrent inhibition of PARP1 is unlikely to be effective in preventing cell death because in the presence of the caspase inhibitor, caspase-independent cell death predomi- nates due to PARP activation. These results suggest that combined therapeutic strategies directed at multiple cell death pathways may provide superior neuroprotec- tion than those directed at a single mechanism. Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: organophosphate, Dichlorvos, oxidative stress, caspase inhibition, PARP1 activation. INTRODUCTION Parkinson’s disease (PD) is one of the most common neurodegenerative disorders and is characterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and subsequent decrease of dopamine levels in the striatum (Dawson et al., 1996). Epidemiological studies have repeatedly indicated that pesticide exposure is a significant risk factor for PD. For example, case-control surveys from several countries including the United States, Canada, Australia, Hong Kong, and Taiwan have shown statistically significant associations between pesticide exposures and PD (Chan et al., 1991, 1998; Gorell et al., 1998; Golbe, 1999). Dichlorvos is a synthetic insecticide that belongs to the family of chemically related organophosphate (OP) pesticides. India is the world’s largest OP producer, and companies make many hazardous products, including Dichlorvos (Agrow, 1996). Dichlorvos can be released into the environment as a major degradation product of other OP insecticides, such as trichlorfon, naled and metrifonate (Hofer, 1981; Pettigrew et al., 1998). OPs, including Dichlorvos have been reported to exert their primary pharmacological and toxicological effects through the inhibition of acetylcholinesterase (AChE) (Balali-Mood and Shariat, 1998). Studies from our lab have also reported that Dichlorvos exposure is toxic to rat dopaminergic (DA) neurons and this could lead to parkinson-like symptoms. Dichlorvos exposure caused mitochondrial abnormalities, nigrostriatal dopaminergic neurodegeneration, decreased dopamine levels, a-synuclein inclusions, neuroinflammation along with impairments in neurobehavioral indices (Binukumar et al., 2010, 2011a,b). These findings taken together indicate that chronic dichlorvos exposure reproduces neurochemical, neuropathological and behavioral 0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2013.11.004 * Corresponding author. Tel: +91-1722755177. E-mail address: kdgill2002@yahoo.co.in (K. D. Gill). Abbreviations: AChE, acetylcholinesterase; AIF, apoptosis-inducing factor; BAF, Boc-aspartyl(OMe)-fluoromethylketone; DAPI, 4 0 ,6- diamidino-2-phenylindole; DCFH-DA, dichloro-dihydro-fluorescein diacetate; DNPH, 2,4-dinitrophenyl hydrazine; EDTA, ethylene- diaminetetraacetic acid; EGTA, ethylene glycol tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; H2DCF-DA, dihydro- dichlorofluorescein diacetate; HEPES, 4-(2-hydroxyethyl)piperazine-1- ethanesulfonic acid; IC50, inhibitory concentration 50; LDH, lactate dehydrogenase; MDA, malondialdehyde; MMP, mitochondrial membrane potential; MnSOD, Mn-superoxide dismutase; MPT, mitochondrial permeability transition; MTT, 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide; OP, organophosphate; PARP1, poly(ADP-ribose) polymerase-1; PBS, phosphate-buffered saline; ROS, reactive oxygen species; RT, room temperature. Neuroscience 258 (2014) 1–15 1