Effects of Chlorpyrifos on Dopaminergic Neuronal Viability with Activation of Microglia_Journal of Biomedical Engineering

Authors：

 LOUYue , LIYaguo

Department of Neurology, Zhejiang Hospital, Hangzhou 310000, China;

Corresponding author：

LOUYue, Email: louyue87@126.com

Keywords：

chlorpyrifos; microglia; dopaminergic neurons; Parkinson's disease

DOI：

10.7507/1001-5515.20160085

Video：

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Excessive microglial activation and subsequent neuroinflammation lead to neuronal cell death, which are involved in the pathogenesis and progression of several neurodegenerative diseases such as Parkinson's disease. The objective of this study was to determine the involvement of chlorpyrifos (CPF) in the activation of microglia and production of inflammatory factors in response to CPF stimulation and the influence on the viability of dopaminergic (DA) neurons. We detected the change of BV-2 cells morphology and expression of inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2) mRNA and protein level upon CPF stimulation (0, 1, 3, 6, 12, 24 h) in BV-2 (mouse brain microglia) cells by reverse transcription polymerase chain reaction (RT-PCR) or Western blot. We randomly assigned BV-2 cells into CPF, menstruum dimethysulfoxide (DMSO) and normal saline (NS) groups. We stimulated The BV-2 cells in the CPF group with CPF, and we stimulated the two control groups with DMSO or NS for 12 hours, respectively. We then collected the used culture media from the culture dishes and centrifuged it to remove the detached cells. Then, we used the supernatants as microglial conditioned media. We treated SH-SY5Y neurons with various groups of microglial conditioned media for 24 hours. We observed the effect of conditioned media collected from BV-2 cell on the viability of dopaminergic cell lines SH-SY5Y using MTT assay. We found that inflammatory factors iNOS, COX-2 mRNA and protein levels were up-regulated upon CPF stimulation. Conditioned media from BV-2 upon CPF stimulation is toxic to SH-SY5Y. It might be concluded that the exposure to CPF may induce dopaminergic neuronal damage by the activation of inflammatory response, and a mechanism may be involved in Parkinson's disease pathogenesis.

Citation： LOUYue, LIYaguo. Effects of Chlorpyrifos on Dopaminergic Neuronal Viability with Activation of Microglia. Journal of Biomedical Engineering, 2016, 33(3): 506-511. doi: 10.7507/1001-5515.20160085 Copy

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2.	LAI B C, MARION S A, TESCHKE K, et al. Occupational and environmental risk factors for Parkinson's disease[J]. Parkinsonism Relat Disord, 2002, 8(5):297-309.
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9.	KREUTZBERG G W. Microglia:a sensor for pathological events in the CNS[J]. Trends Neurosci, 1996, 19(8):312-318.
10.	DAI Hongmei, DENG Yuanying, ZHANG Jie, et al. PINK1/parkin-mediated mitophagy alleviates chlorpyrifos-induced apoptosis in SH-SY5Y cells[J]. Toxicology, 2015, 334:72-80.
11.	TERRY A V Jr. Functional consequences of repeated organophosphate exposure:potential non-cholinergic mechanisms[J]. Pharmacol Ther, 2012, 134(3):355-365.
12.	SLOTKIN T A, TATE C A, COUSINS M M, et al. Functional alterations in CNS catecholamine systems in adolescence and adulthood after neonatal chlorpyrifos exposure[J]. Brain Res Dev Brain Res, 2002, 133(2):163-173.
13.	BLOCK M L, ZECCA L, HONG J S. Microglia-mediated neurotoxicity:uncovering the molecular mechanisms[J]. Nat Rev Neurosci, 2007, 8(1):57-69.
14.	LIBERATORE G T, JACKSON-LEWIS V, VUKOSAVIC S, et al. Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease[J]. Nat Med, 1999, 5(12):1403-1409.
15.	HUNOT S, BOISSIÈRE F, FAUCHEUX B, et al. Nitric oxide synthase and neuronal vulnerability in Parkinson's disease[J]. Neuroscience, 1996, 72(2):355-363.
16.	QURESHI G A, BAIG S, BEDNAR I, et al. Increased cerebrospinal fluid concentration of nitrite in Parkinson's disease[J]. Neuroreport, 1995, 6(12):1642-1644.
17.	DRAPIER J C, HIBBS J B. Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells[J]. J Immunol, 1988, 140(8):2829-2838.
18.	KNOTT C, STERN G, WILKIN G P. Inflammatory regulators in Parkinson's disease:iNOS, lipocortin-1, and cyclooxygenases-1 and -2[J]. Mol Cell Neurosci, 2000, 16(6):724-739.
19.	VIJITRUTH R, LIU Mei, CHOI D Y, et al. Cyclooxygenase-2 mediates microglial activation and secondary dopaminergic cell death in the mouse MPTP model of Parkinson's disease[J]. J Neuroinflammation, 2006, 3:6.
20.	SÁNCHEZ-PERNAUTE R, FERREE A, COOPER O, et al. Selective COX-2 inhibition prevents progressive dopamine neuron degeneration in a rat model of Parkinson's disease[J]. J Neuroinflammation, 2004, 1(1):6.
21.	REALE M, IARLORI C, THOMAS A, et al. Peripheral cytokines profile in Parkinson's disease[J]. Brain Behav Immun, 2009, 23(1):55-63.

1. SHEHADEH L, MITSI G, ADI N, et al. Expression of Lewy body protein septin 4 in postmortem brain of Parkinson's disease and control subjects[J]. Mov Disord, 2009, 24(2):204-210.
2. LAI B C, MARION S A, TESCHKE K, et al. Occupational and environmental risk factors for Parkinson's disease[J]. Parkinsonism Relat Disord, 2002, 8(5):297-309.
3. CHIN M H, QIAN Weijun, WANG Haixing, et al. Mitochondrial dysfunction, oxidative stress, and apoptosis revealed by proteomic and transcriptomic analyses of the striata in two mouse models of Parkinson's disease[J]. J Proteome Res, 2008, 7(2):666-677.
4. BENSINGER S J, TONTONOZ P. A Nurr1 pathway for neuroprotection[J]. Cell, 2009, 137(1):26-28.
5. LI A A, LEVINE T E, BURNS C J, et al. Integration of epidemiology and animal neurotoxicity data for risk assessment[J]. Neurotoxicology, 2012, 33(4):823-832.
6. 张洁,赵玲玲,胡治平,等.低剂量毒死蜱暴露对新生大鼠黑质多巴胺能神经元发育及行为的影响[J].中国当代儿科杂志,2011,13(12):989-994.
7. SLOTKIN T A, SEIDLER F J. Prenatal chlorpyrifos exposure elicits presynaptic serotonergic and dopaminergic hyperactivity at adolescence:critical periods for regional and sex-selective effects[J]. Reprod Toxicol, 2007, 23(3):421-427.
8. ZHANG Jie, DAI Hongmei, DENG Yuanying, et al. Neonatal chlorpyrifos exposure induces loss of dopaminergic neurons in young adult rats[J]. Toxicology, 2015, 336:17-25.
9. KREUTZBERG G W. Microglia:a sensor for pathological events in the CNS[J]. Trends Neurosci, 1996, 19(8):312-318.
10. DAI Hongmei, DENG Yuanying, ZHANG Jie, et al. PINK1/parkin-mediated mitophagy alleviates chlorpyrifos-induced apoptosis in SH-SY5Y cells[J]. Toxicology, 2015, 334:72-80.
11. TERRY A V Jr. Functional consequences of repeated organophosphate exposure:potential non-cholinergic mechanisms[J]. Pharmacol Ther, 2012, 134(3):355-365.
12. SLOTKIN T A, TATE C A, COUSINS M M, et al. Functional alterations in CNS catecholamine systems in adolescence and adulthood after neonatal chlorpyrifos exposure[J]. Brain Res Dev Brain Res, 2002, 133(2):163-173.
13. BLOCK M L, ZECCA L, HONG J S. Microglia-mediated neurotoxicity:uncovering the molecular mechanisms[J]. Nat Rev Neurosci, 2007, 8(1):57-69.
14. LIBERATORE G T, JACKSON-LEWIS V, VUKOSAVIC S, et al. Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease[J]. Nat Med, 1999, 5(12):1403-1409.
15. HUNOT S, BOISSIÈRE F, FAUCHEUX B, et al. Nitric oxide synthase and neuronal vulnerability in Parkinson's disease[J]. Neuroscience, 1996, 72(2):355-363.
16. QURESHI G A, BAIG S, BEDNAR I, et al. Increased cerebrospinal fluid concentration of nitrite in Parkinson's disease[J]. Neuroreport, 1995, 6(12):1642-1644.
17. DRAPIER J C, HIBBS J B. Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells[J]. J Immunol, 1988, 140(8):2829-2838.
18. KNOTT C, STERN G, WILKIN G P. Inflammatory regulators in Parkinson's disease:iNOS, lipocortin-1, and cyclooxygenases-1 and -2[J]. Mol Cell Neurosci, 2000, 16(6):724-739.
19. VIJITRUTH R, LIU Mei, CHOI D Y, et al. Cyclooxygenase-2 mediates microglial activation and secondary dopaminergic cell death in the mouse MPTP model of Parkinson's disease[J]. J Neuroinflammation, 2006, 3:6.
20. SÁNCHEZ-PERNAUTE R, FERREE A, COOPER O, et al. Selective COX-2 inhibition prevents progressive dopamine neuron degeneration in a rat model of Parkinson's disease[J]. J Neuroinflammation, 2004, 1(1):6.
21. REALE M, IARLORI C, THOMAS A, et al. Peripheral cytokines profile in Parkinson's disease[J]. Brain Behav Immun, 2009, 23(1):55-63.

Journal of Biomedical Engineering

Effects of Chlorpyrifos on Dopaminergic Neuronal Viability with Activation of Microglia

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