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KMID : 1177720150090010005
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Journal of Alternatives to Animal Experiments
2015 Volume.9 No. 1 p.5 ~ p.22
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Development of an Alternative Test Method for the Evaluation of Reproductive Toxicity Using Male Germline Stem Cells
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Jeon Hye-Lyun
Kim Tae-Sung
Yi Jung-Sun
Ahn Il-Young
Koh Kyoung-Yuk
Lee Jin-Ha
Kim Joo-Hwan
Shon Soo-Jung
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Abstract
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Currently, alternative test methods are actively being developed as a replacement for animal testing, based on the 3Rs (Replacement, Refinement, Reduction). However, the development of alternative test methods for the evaluation of reproductive and developmental toxicity is in its early stage, and no established test methods exist. This study is aimed at developing an alternative test method to evaluate reproductive toxicity using male germline stem cells (GSC). We selected a negative toxic substance (Dimethyl sufloxide (DMSO)) and two positive toxic substances (NEthyl- N-Nitrosourea (ENU), methyl methanesulfonate (MMS) characterized in OECD TG 489. We also used seven test substances (2,4-diaminotoluene (2,4-DAT), cyclophosphamide (CP), benzo[α]pyrene (BP), cadmium chloride (CdCl2), D-mannitol (MA), n-butyl chloride (NBC) and trimethyl ammonium chloride (TAC)) suggested in a scientific paper published by ECVAM. The endpoints of toxicological evaluation were cell viability (MTT assay) and comet assay which is a method to measure DNA damage. As a result of our study with a 50% inhibitory concentration (IC50) determined using the MTT assay, IC50 values of ENU and MMS were 1.7 mM and 0.4 mM, respectively. Also, IC50 values of 2,4-DAT, CP, BP andCdCl2 were 10.3 mM, 5.5 mM, 0.4 mM and 0.18 mM, respectively. As cell viability wasn’t significantly different from that of the control, IC50 values of MA, NBC and TAC could not be calculated. In the comet assay, Tail DNA%, Tail Length (TL) and Olive Tail Moment (OTM) of the two positive toxic substances (ENU and MMS) and the four test substances (2,4-DAT, CP, BP and CdCl2) significantly grew in comparison with the control. However, Tail DNA%, TL and OTM of the negative toxic substance (DMSO) and the three positive toxic substances (MA, NBC, TAC) were similar to those of the control. In conclusion, this study demonstrated that the comet assay using GSC could be a candidate test method in predicting male reproductive toxicity.
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KEYWORD
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Alternative test method, Male germline stem cell, Reproductive toxicity, Comet assay, DNA damage
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