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.
With ultraviolet and visible light exposure, some pharmaceutical substances applied systemically or topically may cause phototoxic skin irritation. The major factor in phototoxicity is the generation of reactive oxygen species (ROS) such as singlet oxygen and superoxide anion that cause oxidative damage to DNA, lipids and proteins. Thus, measuring the generation of ROS can predict the phototoxic potential of a given substance indirectly. For this reason, a standard ROS assay (ROS assay) was developed and validated and provides an alternative method for phototoxicity evaluation. However, negative substances are over-predicted by the assay. Except for ultraviolet A (UVA), other UV ranges are not a major factor in causing phototoxicity and may lead to incorrect labeling of some non-phototoxic substances as being phototoxic in the ROS assay when using a solar simulator. A UVA stimulator is also widely used to evaluate phototoxicity in various test substances. Consequently, we identified the applicability of a UVA simulator to the ROS assay for photoreactivity. In this study, we tested 60 pharmaceutical substances including 50 phototoxins and 10 non-phototoxins to predict their phototoxic potential via the ROS assay with a UVA simulator. Following the ROS protocol, all test substances were dissolved in dimethyl sulfoxide or sodium phosphate buffer. The final concentration of the test solutions in the reaction mixture was 20 to 200 μM. The exposure was with 2.0~2.2 mW/cm2 irradiance and optimization for a relevant dose of UVA was performed. The generation of ROS was compared before and after UVA exposure and was measured by a microplate spectrophotometer. Sensitivity and specificity values were 85.7% and 100.0% respectively, and the accuracy was 88.1%. From this analysis, the ROS assay with a UVA simulator is suitable for testing the photoreactivity and estimating the phototoxic potential of various test pharmaceutical substances.
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