A Screening System for Detection of Neurotoxic Potency of Chemicals using in vitro Model

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  • Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031 ,JP
  • Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031 ,JP
  • Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031 ,JP
  • Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031 ,JP
  • Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031 ,JP
  • Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031 ,JP
  • Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031 ,JP




Apoptosis, Neuro-2a, Neuropathy, Phosphorylated Neurofilament Heavy Chain, Statin


Background: The major categories of peripheral nerve injury (neuropathy) are generally neuronopathy, axonopathy and myelinopathy. Numerous chemicals are known to cause neuropathy, and the exact mechanism of neuronal toxicity is not known for the majority of chemicals. It is expected that the elucidation of the underlying mechanism of chemicals-induced peripheral neuropathy may lead to its prevention and treatment. Objective: The aim of this study is whether the type of neurotoxicity induced by pravastatin, colchicine, amiodarone, cisplatin, and CoCl2 can be identified by using the in vitro model. Materials and Methods: The cytotoxicity was performed in Neuro-2a neural cells. The levels of DNA fragmentation and the mRNA and protein levels of Bax and Bcl2 were used as apoptosis markers. To evaluate the axon damage, the extracellular phosphorylated neurofilament heavy chain (pNF-H) was investigated by Western blotting. Results: The half of cytotoxic concentration of pravastatin, colchicine, amiodarone, cisplatin and CoCl2 were 19.69 μM, 109.2 nM, 11.89 μM, 5.54 μM and 337.3 μM, respectively. To investigate morphological changes of Neuro-2a exposed to neurotoxic chemicals, Neuro-2a neural cells were incubated with 10 μM pravastatin, 100 nM colchicine, 10 μM amiodarone, 1 μM cisplatin, or 300 μM CoCl2, and evaluated by immunofluorescence assay. Pravastatin, colchicine and cisplatin significantly decreased the axon length. DNA fragmentation and apoptosis-related genes were investigated. Cisplatin significantly increased the degree of fragmentation and the ratio of Bax/Bcl-2. The protein levels of pNF-H in both supernatants and cell lysates were evaluated. The levels of pNF-H in the supernatant of pravastatin-treated, colchicine-treated, and CoCl2-treated cells were higher than in that of vehicle-treated cells. Conclusion: The neuronopathy and axonopathy induced by chemicals could be determined by the evaluation of the levels of apoptosis marker and extracellular pNF-H in Neuro-2a neural cells, respectively.


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How to Cite

Ogawa, M., Kimura, T., Kitamoto, J., Takeda, T., Kitazumi, K., Kyoya, T., & Terada, M. (2020). A Screening System for Detection of Neurotoxic Potency of Chemicals using <i>in vitro</i> Model. Toxicology International, 27(1&amp;2), 44–53. https://doi.org/10.18311/ti/2020/v27i1&2/25249



Research Articles
Received 2020-04-27
Accepted 2020-06-05
Published 2020-10-01



Jortner BS. Mechanisms of toxic injury in the peripheral nervous system: neuropathologic considerations. Toxicologic Pathology. 2000; 28:54–69. https://doi. org/10.1177/019262330002800108. PMid:10668991

Peltier AC, Russell JW. Recent advances in drug-induced neuropathies. Current Opinion in Neurology. 2002; 15:633–8. https://doi.org/10.1097/00019052-200210000- 00015. PMid:12352008

Peltier AC, Russell JW. Advances in understanding druginduced neuropathies. Drug Safety. 2006; 29:23–30. https://doi.org/10.2165/00002018-200629010-00002. PMid:16454532

Valentine WM. Toxic peripheral neuropathies: Agents and mechanisms. Toxicologic Pathology. 2020; 48:152–73. https://doi.org/10.1177/0192623319854326. PMid:31181992

Poncelet AN. An Algorithm for the evaluation of peripheral neuropathy. American Family Physician. 1998; 57:755–64.

Grogan PM, Katz JS. Toxic neuropathies. Neurologic Clinics. 2005; 23:377–96. https://doi.org/10.1016/j. ncl.2004.12.003. PMid:15757790

Jeppesen U, Gaist D, Smith T, Sindrup SH. Statins and peripheral neuropathy. European Journal of Clinical Pharmacology. 1999; 54:835–8. https://doi.org/10.1007/ s002280050562. PMid:10027656

Kuncl RW, Duncan G, Watson D, Alderson K, Rogawski MA, Peper M. Colchicine myopathy and neuropathy. New England Journal of Medicine. 1987; 316:1562–8. https:// doi.org/10.1056/NEJM198706183162502. PMid:3035372

De Deyn PP, Ceuterick C, Saxena V, Crols R, Chappel R, Martin JJ. Chronic colchicine-induced myopathy and neuropathy. Acta Neurologica Belgica. 1995; 95:29–32.

Niimi N, Takaku S, Yako H, Sango K. Drug-induced demyelinating neuropathies. Advances in Experimental Medicine and Biology. 2019; 1190:357–69. https://doi. org/10.1007/978-981-32-9636-7_23. PMid:31760656

Tredici G, Tredici S, Fabbrica D, Minoia C, Cavaletti G. Experimental cisplatin neuronopathy in rats and the effect of retinoic acid administration. Journals of Neurooncology. 1998; 36:31–40. https://doi.org/10.1023/A:1005756023082. PMid:9525823

Kirchmair R, Walter DH, Ii M, Rittig K, Tietz AB, Murayama T, et al. Antiangiogenesis mediates cisplatin-induced peripheral neuropathy: Attenuation or reversal by local vascular endothelial growth factor gene therapy without augmenting tumor growth. Circulation. 2005; 111:2662–70. https://doi.org/10.1161/CIRCULATIONAHA.104.470849. PMid:15897348

Kikuchi S, Ninomiya T, Kohno T, Kojima T, Tatsumi H. Cobalt inhibits motility of axonal mitochondria and induces axonal degeneration in cultured dorsal root ganglion cells of rat. Cell Biology and Toxicology. 2018; 34:93–107. https://doi.org/10.1007/s10565-017-9402-0. PMid:28656345

Gotoh M, Sano-Maeda K, Murofushi H, Murakami- Murofushi K. Protection of neuroblastoma Neuro2A cells from hypoxia-induced apoptosis by cyclic Phosphatidic Acid (cPA). PLoS One. 2012; 7. https://doi.org/10.1371/journal. pone.0051093. PMid:23251428. PMCid:PMC3521017

Hershman DL, Lacchetti C, Loprinzi CL. Prevention and management of chemotherapy-induced peripheral neuropathy in survivors of adult cancers: American society of clinical oncology clinical practice guideline summary. Journal of Oncology Practice. 2014; 10:e421–4. https://doi.org/10.1200/JOP.2014.001776. PMid:29424607

Zajć…czkowska R, Kocot-Kć™pska M, Leppert W, Wrzosek A, Mika J, Wordliczek J. Mechanisms of chemotherapyinduced peripheral neuropathy. International Journal of Molecular Sciences. 2019; 20. https://doi.org/10.3390/ ijms20061451. PMid:30909387. PMCid:PMC6471666

Gill JS, Windebank AJ. Cisplatin-induced apoptosis in rat dorsal root ganglion neurons is associated with attempted entry into the cell cycle. Journal of Clinical Investigation. 1998; 101:2842–50. https://doi.org/10.1172/JCI1130. PMid:9637718. PMCid:PMC508875

Reed JC. Bcl-2 and the regulation of programmed cell death. Journal of Cell Biology. 1994; 124:1–6. https://doi.org/10.1083/jcb.124.1.1. PMid:8294493. PMCid:PMC2119888

Brady HJ, Gil-Gómez G. Bax. The pro-apoptotic Bcl-2 family member, Bax. International Journal of Biochemistry & Cell Biology. 1998; 30:647–50. https://doi.org/10.1016/ S1357-2725(98)00006-5

Perlman H, Zhang X, Chen MW, Walsh K, Buttyan R. An elevated bax/bcl-2 ratio corresponds with the onset of prostate epithelial cell apoptosis. Cell Death & Differentiation. 1999; 6:48–54. https://doi.org/10.1038/ sj.cdd.4400453. PMid:10200547

Raisova M, Hossini AM, Eberle J, Riebeling C, Wieder T, et al. The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. Journal of Investigative Dermatology. 2001; 117:333– 40. https://doi.org/10.1046/j.0022-202x.2001.01409.x. PMid:11511312

Salakou S, Kardamakis D, Tsamandas AC, Zolota V, Apostolakis E, Tzelepi V. Increased Bax/Bcl-2 ratio up-regulates caspase-3 and increases apoptosis in the thymus of patients with myasthenia gravis. Papathanasopoulos P, Bonikos DS, Papapetropoulos T, Petsas T, Dougenis D. In Vivo. 2007; 21:123–32.

Khodapasand E, Jafarzadeh N, Farrokhi F, Kamalidehghan B, Houshmand M. Is Bax/Bcl-2 ratio considered as a prognostic marker with age and tumor location in colorectal cancer? Iranian Biomedical Journal. 2015; 19:69–75.

Collins JA, Schandi CA, Young KK, Vesely J, Willingham MC. Major DNA fragmentation is a late event in apoptosis. Journal of Histochemistry & Cytochemistry. 1997; 45:923–34. https://doi.org/10.1177/002215549704500702. PMid:9212818

Horie H, Kim SU, Takenaka T. Immunofluorescence demonstration of neurofilament polypeptide expression in fetal human neurons in culture. Neuroscience Research. 1989; 6:463–9. https://doi.org/10.1016/0168- 0102(89)90008-4

Natori A, Ogata T, Sumitani M, Kogure T, Yamauchi T, Yamauchi H. Potential role of pNF-H, a biomarker of axonal damage in the central nervous system, as a predictive marker of chemotherapy-induced cognitive impairment. Clinical Cancer Research. 2015; 21:1348–52. https://doi. org/10.1158/1078-0432.CCR-14-2775. PMid:25589615

Hayakawa K, Okazaki R, Ishii K, Ueno T, Izawa N, Tanaka Y, et al. Phosphorylated neurofilament subunit NF-H as a biomarker for evaluating the severity of spinal cord injury patients, a pilot study. Spinal Cord. 2012; 50:493–6. https:// doi.org/10.1038/sc.2011.184. PMid:22270191

Lee Y, Lee BH, Yip W, Chou P, Yip BS. Neurofilament proteins as prognostic biomarkers in neurological disorders. Current Pharmaceutical Design. 2020; 25:456029. https://doi.org/10.2174/1381612825666191210 154535. PMid:31820696

Qiao X Zhang S, Zhao W, Ye H, Yang Y, Zhang Z, et al. Serum phosphorylated neurofilament-heavy chain, a potential biomarker, is associated with peripheral neuropathy in patients with Type 2 diabetes. Medicine (Baltimore). 2015; 94. https://doi.org/10.1097/MD.0000000000001908. PMid:26554790. PMCid:PMC4915891

Ogawa M, Kyoya T, Kimura T, Terada M. Effects of estrogen on fatty-acid-induced cytotoxicity in mouse Neuro-2a neural cells. Fundamental Toxicological Sciences. 2020; 7:115–21. https://doi.org/10.2131/fts.7.115

Ogawa M, Yamaji R, Higashimura Y, Harada N, Ashida H, Nakano Y, et al. 17β-estradiol represses myogenic differentiation by increasing ubiquitin-specific peptidase 19 through estrogen receptor α. Journal of Biological Chemistry. 2011; 286:41455–65. https://doi.org/10.1074/ jbc.M111.276824. PMid:21971047. PMCid:PMC3308857

Harada N, Katsuki T, Takahashi Y, Masuda T, Yoshinaga M, Adachi T, et al. Androgen receptor silences thioredoxininteracting protein and competitively inhibits glucocorticoid receptor-mediated apoptosis in pancreatic β-Cells. Journal of Cellular Biochemistry. 2015; 116:998–1006. https://doi.org/10.1002/jcb.25054. PMid:25639671

Ogawa M, Kimura T, Tanetani Y, Hori M, Kyoya T, Terada M. Sex differences in the effects of high-fat diet on mouse sciatic nerves. Fundam Toxicol Sci 2020; 7:133-9. https:// doi.org/10.2131/fts.7.133.

Anderson KJ, Scheff SW, Miller KM, Roberts KN, Gilmer LK, Yang C, et al. The phosphorylated axonal form of the neurofilament subunit NF-H (pNF-H) as a blood biomarker of traumatic brain injury. Journal of Neurotrauma. 2008; 25:1079–85.https://doi.org/10.1089/neu.2007.0488. PMid:18729720. PMCid:PMC2820728

Sumitani M, Ogata T, Natori A, Hozumi J, Shimojo N, Kida K, et al. Poor efficacy of the phosphorylated highmolecular- weight neurofilament heavy subunit serum level, a biomarker of axonal damage, as a marker of chemotherapy-induced peripheral neuropathy. Biomedical Reports. 2016; 4:758–60. https://doi.org/10.3892/ br.2016.648. PMid:27284419. PMCid:PMC4887842

Al-Kuraishy HM, Al-Gareeb AI, Hussien NR, Al-Naimi MS, Rasheed HA. Statins an oft-prescribed drug is implicated in peripheral neuropathy: The time to know more. Journal of Pakistan Medical Association. 2019; 69(Suppl 3):S108–12.

í–zdemir IH, Copkiran í–, Tıkız H, Tıkız C. Peripheral polyneuropathy in patients receiving long-term statin therapy. Uzun dönem statin kullanan hastalarda periferik polinöropati gelişimi. Turk Kardiyol Dern Ars. 2019; 47:554–63. https://doi.org/10.5543/tkda.2019.78379. PMid:31582682

Pergolizzi JV Jr, Magnusson P, LeQuang JA, Razmi R, Zampogna G, Taylor R Jr. Statins and neuropathic pain: A narrative review. Pain and Therapy. 2020. https:// doi.org/10.1007/s40122-020-00153-9. PMid:32020545. PMCid:PMC7203325

Emad M, Arjmand H, Farpour HR, Kardeh B. Lipidlowering drugs (statins) and peripheral neuropathy. Electron Physician. 2018; 10:6527–33. https://doi. org/10.19082/6527. PMid:29765578. PMCid:PMC5942574

de Chaves EI, Rusiñol AE, Vance DE, Campenot RB, Vance JE. Role of lipoproteins in the delivery of lipids to axons during axonal regeneration. Journal of Biological Chemistry. 1997; 272:30766–73. https://doi.org/10.1074/ jbc.272.49.30766. PMid:9388216

Rundek T, Naini A, Sacco R, Coates K, DiMauro S. Atorvastatin decreases the coenzyme Q10 level in the blood of patients at risk for cardiovascular disease and stroke. Archives of Neurology. 2004; 61:889–92. https://doi. org/10.1001/archneur.61.6.889. PMid:15210526

Ukena TE, Borysenko JZ, Karnovsky MJ, Berlin RD. Effects of colchicine, cytochalasin B, and 2-deoxyglucose on the topographical organization of surface-bound concanavalin A in normal and transformed fibroblasts. Journal of Cell Biology. 1974; 61:70–82. https://doi.org/10.1083/ jcb.61.1.70. PMid:4132067. PMCid:PMC2109267

Schmitz Y, Kohler K. Spinule formulation in the fish retina: Is there an involvement of actin and tubulin? An electronmicroscopic immunogold study. Journal of Neurocytology. 1993; 22:205–14. https://doi.org/10.1007/ BF01246359. PMid:8478642

Niggli V. Microtubule-disruption-induced and chemotactic-peptide-induced migration of human neutrophils: Implications for differential sets of signalling pathways. Journal of Cell Science. 2003; 116:813–22. https://doi.org/10.1242/jcs.00306. PMid:12571279

James KA, Bray JJ, Morgan IG, Austin L. The effect of colchicine on the transport of axonal protein in the chicken. Biochemical Journal. 1970; 117:767–71. https://doi.org/10.1042/bj1170767. PMid:4194591. PMCid:PMC1179029

Choi BY, Hong DK, Suh SW. ZnT3 gene deletion reduces colchicine-induced dentate granule cell degeneration. International Journal of Molecular Sciences. 2017; 18. https://doi.org/10.3390/ijms18102189. PMid:29048371 PMCid:PMC5666870

Diez H, Garrido JJ, Wandosell F. Specific roles of Akt iso forms in apoptosis and axon growth regulation in neurons. PLoS One. 2012; 7. https://doi.org/10.1371/journal. pone.0032715. PMid:22509246. PMCid:PMC3324480

Qian W, Nishikawa M, Haque AM, Hirose M, Mashimo M, Sato E, et al. Mitochondrial density determines the cellular sensitivity to cisplatin-induced cell death. American Journal of Physiology-Cell Physiology. 2005; 289:C1466–75. https://doi.org/10.1152/ajpcell.00265.2005. PMid:16107504

Li J, Wuliji O, Li W, Jiang Z-G, Ghanbari HA. Oxidative stress and neurodegenerative disorders. International Journal of Molecular Sciences. 2013; 14:24438–75. https://doi.org/10.3390/ijms141224438. PMid:24351827. PMCid:PMC3876121

Argyriou AA, Chroni E, Koutras A, Ellul J, Papapetropoulos S, Katsoulas G, et al. Vitamin E for prophylaxis against chemotherapy-induced neuropathy: A randomized controlled trial. Neurology. 2005; 64:26–31. https:// doi.org/10.1212/01.WNL.0000148609.35718.7D. PMid:15642899

Fouladi M, Chintagumpala M, Ashley D, Kellie S, Gururangan S, Hassall T, et al. Amifostine protects against cisplatin-induced ototoxicity in children with averagerisk medulloblastoma. Journal of Clinical Oncology. 2008; 26:3749–55. https://doi.org/10.1200/JCO.2007.14.3974. PMid:18669462. PMCid:PMC2504739

Khademi E, Mahabadi VP, Ahmadvand H, Akbari E, Khalatbary AR. Anti-inflammatory and anti-apoptotic effects of hyperbaric oxygen preconditioning in a rat model of cisplatin-induced peripheral neuropathy. Iranian Journal of Basic Medical Sciences. 2020; 23:321–8.

Ho VT, Bunn HF. Effects of transition metals on the expression of the erythropoietin gene: Further evidence that the oxygen sensor is a heme protein. Biochemical and Biophysical Research Communications. 1996; 223:175–80. https://doi.org/10.1006/bbrc.1996.0865. PMid:8660366

Zhong X, Lin R, Li Z, Mao J, Chen L. Effects of Salidroside on cobalt chloride-induced hypoxia damage and mTOR signaling repression in PC12 cells. Biological and Pharmaceutical Bulletin. 2014; 37:1199–206. https://doi. org/10.1248/bpb.b14-00100. PMid:24989011

Sekhon MS, Ainslie PN, Griesdale DE. Clinical pathophysiology of hypoxic ischemic brain injury after cardiac arrest: a "two-hit” model. Critical Care. 2017; 21:90. https://doi.org/10.1186/s13054-017-1670-9. PMid:28403909. PMCid:PMC5390465

Oncel C, Baser S, Cam M, Akdağ B, Taspinar B, Evyapan F. Peripheral neuropathy in chronic obstructive pulmonary disease. COPD. 2010; 7:11–6. https://doi. org/10.3109/15412550903499480. PMid:20214459

Park HW, Lim MJ, Jung H, Lee SP, Paik KS, Chang MS. Human mesenchymal stem cell-derived Schwann celllike cells exhibit neurotrophic effects, via distinct growth factor production, in a model of spinal cord injury. Glia. 2010; 58:1118–32. https://doi.org/10.1002/glia.20992. PMid:20468053

Hyung S, Lee BY, Park JC, Kim J, Hur EM, Suh JKF. Coculture of primary motor neurons and Schwann cells as a model for in vitro myelination. Scientific Reports. 2015; 5. https://doi.org/10.1038/srep15122. PMid:26456300. PMCid:PMC4601011

Ishii T, Kawakami E, Endo K, Misawa H, Watabe K. Myelinating cocultures of rodent stem cell line-derived neurons and immortalized Schwann cells. Neuropathology. 2017; 37:475–81. https://doi.org/10.1111/neup.12397. PMid:28707715