• Toxicological Research
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• v.35 no.1
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• pp.37-44
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• 2019

A major predictor of the efficacy of natural or synthetic cannabinoids is their binding affinity to the cannabinoid type I receptor ($CB_1$) in the central nervous system, as the main psychological effects of cannabinoids are achieved via binding to this receptor. Conventionally, receptor binding assays have been performed using isotopes, which are inconvenient owing to the effects of radioactivity. In the present study, the binding affinities of five cannabinoids for purified $CB_1$ were measured using a surface plasmon resonance (SPR) technique as a putative non-isotopic receptor binding assay. Results were compared with those of a radio-isotope-labeled receptor binding assay. The representative natural cannabinoid ${\Delta}^9$-tetrahydrocannabinol and four synthetic cannabinoids, JWH-015, JWH-210, RCS-4, and JWH-250, were assessed using both the SPR biosensor assay and the conventional isotopic receptor binding assay. The binding affinities of the test substances to $CB_1$ were determined to be (from highest to lowest) $9.52{\times}10^{-3}M$ (JWH-210), $6.54{\times}10^{-12}M$ (JWH-250), $1.56{\times}10^{-11}M$ (${\Delta}^9$-tetrahydrocannabinol), $2.75{\times}10^{-11}M$ (RCS-4), and $6.80{\times}10^{-11}M$ (JWH-015) using the non-isotopic method. Using the conventional isotopic receptor binding assay, the same order of affinities was observed. In conclusion, our results support the use of kinetic analysis via SPR in place of the isotopic receptor binding assay. To replace the receptor binding affinity assay with SPR techniques in routine assays, further studies for method validation will be needed in the future.

• Biomolecules & Therapeutics
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• v.23 no.5
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• pp.486-492
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• 2015

Drug metabolism mostly occurs in the liver. Cytochrome P450 (CYP) is a drug-metabolizing enzyme that is responsible for many important drug metabolism reactions. Recently, the US FDA and EU EMA have suggested that CYP enzyme induction can be measured by both enzymatic activity and mRNA expression. However, these experiments are time-consuming and their interassay variability can lead to misinterpretations of the results. To resolve these problems and establish a more powerful method to measure CYP induction, we determined CYP induction by using luminescent assay. Luminescent CYP assays link CYP enzyme activity to firefly luciferase luminescence technology. In this study, we measured the induction of CYP isozymes (1A2, 2B6, 2C9, and 3A4) in cryopreserved human hepatocytes (HMC424, 478, and 493) using a luminometer. We then examined the potential induction abilities (unknown so far) of mesalazine, a drug for colitis, and mosapride citrate, which is used as an antispasmodic drug. The results showed that mesalazine promotes CYP2B6 and 3A4 activities, while mosapride citrate promotes CYP1A2, 2B6, and 3A4 activities. Luminescent CYP assays offer rapid and safe advantages over LC-MS/MS and qRT-PCR methods. Furthermore, luminescent CYP assays decrease the interference between the optical properties of the test compound and the CYP substrates. Therefore, luminescent CYP assays are less labor intensive, rapid, and can be used as robust tools for high-throughput CYP screening during early drug discovery.

• Proceedings of the PSK Conference
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• 2000.10a
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• pp.169.2-170
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• 2000

• 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.

• Proceedings of the Korea Environmental Mutagen Society Conference
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• 2003.05a
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• pp.80-80
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• 2003

• Proceedings of the Korean Society of Toxicology Conference
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• 2001.05a
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• pp.147-147
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• 2001

To establish a test protocol for the rodent 20-day thyroid/pubertal assay, we dosed flutamide(fl), a non-steroidal androgen antagonist to intact male SD rats from postnatal day 33 for 20 days, and examined several reproductive endpoints for assessing the sensitivity of a list of parameters for detecting endocrine-related effects of endocrine-disrupting chemicals.(omitted)

• Proceedings of the PSK Conference
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• 2001.04a
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• pp.155-155
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• 2001

• Proceedings of the Korea Environmental Mutagen Society Conference
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• 2004.11a
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• pp.150-150
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• 2004

• Proceedings of the Korean Society of Toxicology Conference
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• 2003.05a
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• pp.80-80
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• 2003

The use of skin whitening agents have been recently increased in various kinds of cosmetic products, although there were reports that whitening agents might cause allergic contact dermatitis. A murine local lymph node assay (LLNA) has been developed as an alternative to guinea pigs for contact sensitization potential. This study was carried out to investigate the skin sensitization potential of three whitening agents, kojic acid, arbutin, azelaic acid, by LLNA using a non-radioistopic endpoint.(omitted)

• Proceedings of the Korean Society of Toxicology Conference
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• 2005.05a
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• pp.130-130
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• 2005