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Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse...

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Autores principales: Jeon, Hye Lyun, Yi, Jung-Sun, Kim, Tae Sung, Oh, Youkyung, Lee, Hye Jeong, Lee, Minseong, Bang, Jin Seok, Ko, Kinarm, Ahn, Il Young, Ko, Kyungyuk, Kim, Joohwan, Park, Hye-Kyung, Lee, Jong Kwon, Sohn, Soo Jung
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Korean Society of Toxicology 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402864/
https://www.ncbi.nlm.nih.gov/pubmed/28443181
http://dx.doi.org/10.5487/TR.2017.33.2.107
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author Jeon, Hye Lyun
Yi, Jung-Sun
Kim, Tae Sung
Oh, Youkyung
Lee, Hye Jeong
Lee, Minseong
Bang, Jin Seok
Ko, Kinarm
Ahn, Il Young
Ko, Kyungyuk
Kim, Joohwan
Park, Hye-Kyung
Lee, Jong Kwon
Sohn, Soo Jung
author_facet Jeon, Hye Lyun
Yi, Jung-Sun
Kim, Tae Sung
Oh, Youkyung
Lee, Hye Jeong
Lee, Minseong
Bang, Jin Seok
Ko, Kinarm
Ahn, Il Young
Ko, Kyungyuk
Kim, Joohwan
Park, Hye-Kyung
Lee, Jong Kwon
Sohn, Soo Jung
author_sort Jeon, Hye Lyun
collection PubMed
description Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration (IC(50)) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity.
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spelling pubmed-54028642017-04-25 Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells Jeon, Hye Lyun Yi, Jung-Sun Kim, Tae Sung Oh, Youkyung Lee, Hye Jeong Lee, Minseong Bang, Jin Seok Ko, Kinarm Ahn, Il Young Ko, Kyungyuk Kim, Joohwan Park, Hye-Kyung Lee, Jong Kwon Sohn, Soo Jung Toxicol Res Original Article Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration (IC(50)) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity. Korean Society of Toxicology 2017-04 2017-04-15 /pmc/articles/PMC5402864/ /pubmed/28443181 http://dx.doi.org/10.5487/TR.2017.33.2.107 Text en Copyright © 2017 The Korean Society Of Toxicology http://creativecommons.org/licenses/by-nc/3.0/ This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Article
Jeon, Hye Lyun
Yi, Jung-Sun
Kim, Tae Sung
Oh, Youkyung
Lee, Hye Jeong
Lee, Minseong
Bang, Jin Seok
Ko, Kinarm
Ahn, Il Young
Ko, Kyungyuk
Kim, Joohwan
Park, Hye-Kyung
Lee, Jong Kwon
Sohn, Soo Jung
Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells
title Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells
title_full Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells
title_fullStr Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells
title_full_unstemmed Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells
title_short Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells
title_sort development of a test method for the evaluation of dna damage in mouse spermatogonial stem cells
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402864/
https://www.ncbi.nlm.nih.gov/pubmed/28443181
http://dx.doi.org/10.5487/TR.2017.33.2.107
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