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

Abstract

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 (IC50) 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.

Activation of the Nrf2/HO-1 signaling pathway contributes to the protective effects of baicalein against oxidative stress-induced DNA damage and apoptosis in HEI193 Schwann cells

“…After electrophoresis, the slides were rinsed three times with a neutralizing buffer (0.4 M Tris, pH 7.5) for at least 5 min each, dehydrated in absolute ethanol at 4°C, and allowed to dry. The cells were stained with 20 μg/ml of propidium iodide (PI, Sigma-Aldrich Chemical Co.) 34. Images were then captured using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) at ×200 magnification.…”

Shikonin Exerts Cytotoxic Effects in Human Colon Cancers by Inducing Apoptotic Cell Death via the Endoplasmic Reticulum and Mitochondria-Mediated Pathways

“…Cells were seeded onto chamber slides at a density of 1.0×10 5 cells/well and after 20 h, they were treated with shikonin (3 µM) for 48 h. Then, the chamber slides were fixed, and the cells were permeabilized. After washing in PBS, the stained cells were visualized with a fluorescence microscope (Olympus IX 70, Olympus Optical Co., Tokyo, Japan) and quantified (Jeon et al ., 2017).…”

Protective Effects of Nargenicin A1 against Tacrolimus-Induced Oxidative Stress in Hirame Natural Embryo Cells

“…For the cell viability study, 5 × 10 3 HINAE cells were seeded per well in 96-well plates, incubated for 24 h, and then incubated with different concentrations of tacrolimus or nargenicin A1 for 24 h or pre-incubated with nargenicin A1 for 1 h before being treated with tacrolimus for 24 h. Cells were also treated with N -acetyl cysteine (NAC, Sigma-Aldrich Chemical Co.), a known ROS scavenger, for 1 h in the presence or absence of tacrolimus. Subsequently, cell viability was determined using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich Chemical Co.) assay as previously described [28]. The experiments were repeated three times with at least triplicate wells for each concentration, and results are expressed as percentage reductions in absorbance versus non-treated controls.…”