Toxicology International (Formerly Indian Journal of Toxicology)

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


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

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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.

Keywords

Apoptosis, Neuro-2a, Neuropathy, Phosphorylated Neurofilament Heavy Chain, Statin.
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  • A Screening System for Detection of Neurotoxic Potency of Chemicals using in vitro Model

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Authors

Masahiro Ogawa
Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031, Japan
Takahiro Kimura
Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031, Japan
Junya Kitamoto
Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031, Japan
Takeo Takeda
Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031, Japan
Kenta Kitazumi
Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031, Japan
Takahiro Kyoya
Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031, Japan
Megumi Terada
Life Science Research Institute, Kumiai Chemical Industry Co., Ltd. 3360 Kamo, Kikugawa-shi, Shizuoka, 439-0031, Japan

Abstract


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.

Keywords


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

References





DOI: https://doi.org/10.18311/ti%2F2020%2Fv27i1%262%2F25249