• Molecular & Cellular Toxicology
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• v.5 no.2
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• pp.126-132
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• 2009

The usage and types of chemicals are advancing, specializing, large-scaled increasing, and new chemical exposed workers are concerning to occupational disease. The generation of reactive oxygen in the body from carcinogen, mutation and DNA damage in cancer is protected by natural antioxidants (phytochemicals) with antimutagenic effect. There were many reports of ginsenoside Rb$_1$, Rg$_1$ grievances of the genetic mutation to suppress the effect confirm the genetic toxicity test with chromosomal aberration test and the Comet (SCGE) assay confirmed the suppression effect occurring chromosomal DNA damage. We had wanted to evaluate the compatibility and sensitivity between the chromosomal aberration (CA) test and the Comet assay. We used the CA test and Comet assay to evaluate the anti-genotoxicity of ginsenoside Rb$_1$ and Rg$_1$, in CHO-K1 (Chinese hamster ovary fibroblast) cell in vitro, composed negative control (solvent), positive control (benzo[a]pyrene), test group (carcinogen+variety concentration of ginsenoside) group. The positive control was benzo[a]pyrene (50 $\mu$M), well-known carcinogen, and the negative control was the 1 % DMSO solvent. The test group was a variety concentration of ginsenoside Rb$_1$, Rg$_1$ with 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1%, 10%. In chromo-somal aberration test, we measured the number of cells with abnormally structured chromosome. In Comet assay, the Olive tail moment (OTM) and Tail length (TL) values were measured. The ratio of cell proliferation was increased 8.3% in 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1%, 10% Rb$_1$ treated groups, and increased 10.4% in 10$^{-10}$%, 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1% Rg$_1$ treated groups. In the CA test, the number of chromosomal aberration was decreased all the Rb$_1$ and Rg$_1$ treated groups. In the Comet assay, the OTM values were decreased in all the Rb$_1$ and Rg$_1$ treated groups. To evaluate the compatibility between CA and Comet assay, we compared the reducing ratio of chromosomal abnormalities with its OTM values, it was identified the antimutagenicity of ginsenoside, but it was more sensitive the CA test than the Comet assay. Ginsenoside Rb$_1$ and Rg$_1$ significantly decrease the number of cells with chromosomal aberration, and decrease the extent of DNA migration. Therefore, ginsenoside Rb$_1$, Rg$_1$ are thought as an antioxidant phytochemicals to protect mutagenicity. The in vitro Comet assay seems to be less sensitive than the in vitro chromosomal aberration test.

• Korean Journal of Environmental Agriculture
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• v.34 no.4
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• pp.350-354
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• 2015

BACKGROUND: Botanical insecticides, especially Azadirachta Indica extract (AIE) and Sophorae radix extract (SRE) are widely used in Agriculture field. In our previous studies on genotoxicity test of AIE and SRE samples, a suspicious clastogenic properties was shown. Herein, we investigated the DNA damage effect of these botanical insecticide samples through the in vitro comet assay. METHODS AND RESULTS: Chinese hamster lung (CHL) fibroblast cell line was used, and methyl methanesulphonate was as positive control. Respective two samples of AIE and SRE were evaluated using Single Cell Gel Electrophoresis (Comet) assay and measured as the Olive tail moment (OTM). Results from this study indicated that all tested AIE and SRE samples did not show DNA damage in comet assay using CHL cells, compared with control. CONCLUSION: AIE and SRE samples used in this study were not cause genetic toxicity and are suitable for use as organic materials.

• Molecular & Cellular Toxicology
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• v.4 no.3
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• pp.211-217
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• 2008

To clarify the DNA damage from reactive oxygen species, we measured the DNA damage through Fpg/Endo III FLARE (Fragment Length Analysis with Repair Enzyme) assay and real time RT-PCR. The 80 SD rats assigned to 4 dose groups exposed to cumene vapor for 90 days. With Fpg/Endo III FLARE assay in hepatocytes, we found the OTM (Olive Tail Moment) and TL (Tail Length) significantly increased in no-enzyme treated and Fpg-treated control and 8 ppm groups with 28 days exposure. In Endo III-treated 8 ppm group, significantly increased the values with 90 days exposure. With lymphocytes, it was founded the values significantly increased in no-enzyme treated 800 ppm group in 28 and 90 days. It was significantly increased in Endo III-treated 80 ppm for 28 days and 800 ppm for 90 days. From the above findings, FLARE assay was suggested as being available as a biological marker for DNA damage induced by cumene exposure in SD rats. And we used real time RT-PCR for the OGG1 mRNA expression, it had dose-dependent biologic effects in 1 day exposure, but decrease the levels of rOGG1 mRNA. Our findings provide evidence that cumene exposure may cause suppression of rOGG1 in the rat hepatocytes or lymphocytes.

• Toxicological Research
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• v.33 no.2
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• pp.107-118
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• 2017

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.