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KMID : 1177720140080010005
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Journal of Alternatives to Animal Experiments
2014 Volume.8 No. 1 p.5 ~ p.14
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Establishment of Flow Cytometry-based High-throughput Genotoxicity Testing Using Peripheral Blood
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Ahn Il-Young
Kim Joo-Hwan
Lim Dae-Hyun
Yang Jun-Young
Kim So-Young
Yi Jung-Sun
Yum Young-Na
Lim Chae-Hyung
Lee Jin-Ha
Choi Ki-Hwan
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Abstract
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To identify mutagenic potential of test substances, in vitro Ames tests are commonly used. Recently revised ICH S2(R1) guideline requires in vivo genotoxicity test if the result of the in vitro test is positive. In addition, a method testing multiple endpoints is required for animal welfare. Therefore we established a flow cytometry-based analysis such as Pig-a gene mutation assay and the micronuclei assay for detection of in vivo genotoxic potential using peripheral blood collected from repeated dose toxicity study. To evaluate these new methods, male Sprague Dawley rats were treated for 3, 14 or 28 days with N-nitro-N-ethylurea (ENU). ENU induced mutations in both reticulocytes (RET) and red blood cells of rats dose-dependently from the Pig-a gene mutation assay. ENU also increased micronucleated reticulocytes frequencies in flow cytometry based micronuclei assay, implying chromosomal damage to hematopoetic cells. These data show that both assays were well established. We additionally evaluated urethane and glycidol for applicability of Pig-a gene mutation assay and in vivo micronuclei assay by flow cytometry. Urethane, compared with vehicle control, did not increase Pig-a gene mutation and micronuclei frequency. Glycidol, compared with vehicle control, did not increase in micronuclei frequency, but Pig-a gene mutation significantly increased in the highest concentration for 28 days. Pig-a gene mutation assay for genotoxicity has many advantages: It can detect mutation in various species including humans, primates and rodents; and is integrated with repeated dose toxicity test without additional usage of animals; and has low spontaneous mutation frequency.
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KEYWORD
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Flow cytometry, Pig-a assay, In vivo micronuclei assay, peripheral blood
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