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KMID : 1177720100040010019
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Journal of Alternatives to Animal Experiments
2010 Volume.4 No. 1 p.19 ~ p.29
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Application of ECVAM proposal for in vitro target organ toxicity using Liver and kidney cell Lines
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Lee Jin-Young
Kang Mi-Sun
Kim Ji-Myoung
Kwack Seung-Jun
Han Eui-Sik
Han Bum-Seok
Kang Tae-Seok
Han Soon-Young
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Abstract
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Repeated dose toxicity testing in rodents is used to have information on potential target organs in terms of toxicity, and the NOAEL (no-observed-adverse-effect-level). ECVAM and ICCVAM have not validated any non-animal methods for assessing chronic toxicity endpoints or repreated exposure target organ toxicity because in vivo systems often poorly resemble in vitro cell culture systems. However, recently ECVAM recommended a proposed approach for the assessment of repeated dose toxicity in vitro. We applied ECVAM’s proposal to assess target organ toxicity using liver and kidney cell lines. In order to predict liver and kidney target organ toxicities, we used three kinds of kidney toxicants(HgCl2, CH3HgCl, CdCl2) and two kinds of liver toxicants(acetaminophen, CuCl2) in human hepatoma(HepG2) cells, human renal tubular epithelial(HK-2) cells and human embryonic kidney(HEK293) cells. We performed cytotoxicity assays, mitochondria damage and calcium influx tests to predict target organ toxicity. Cell viability and cell proliferation were assessed by NRU assay and MTT assays, respectively. Mitochondria potential and cell calcium concentration([Ca2+]i) were estimated by a
fluorescence microscopy using JC-1 dye and a fluorometer using Fluo-4/AM dye, respectively. The cytotoxicity of nephrotoxicants(HgCl2, CH3HgCl, CdCl2) from the result of NRU and MTT assay was higher in the kidney cells than in the liver cells. Also, the cytotoxicity of hepatotoxicants(acetaminophen, CuCl2) was higher in the liver cells than in the kidney cells. Mitochondria potential and [Ca2+]i show compatability with cytotoxicity results in the kidney cells but not in the liver cells. The results demonstrated that nephrotoxicants can predict target organ toxicity by in vitro tests like NRU, MTT assay, mitochondria potential and [Ca2+]i. However, hepatotoxicants can predict target organ toxicity by cytotoxicity assays like NRU and MTT assays but not by mitochondria potential and [Ca2+]i.
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KEYWORD
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Kidney, Liver, ECVAM, in vitro target organ toxicity
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