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박철범 ( Cheol Beom Park ) 한국동물실험대체법학회 2008 한국동물실험대체법학회 학술대회집 Vol.2008 No.1
Reconstructed skin provides advantages to single cell culture testing and leads to promising results as shown by different validation studies. In this study we investigated the efficacy of "KeraskinTM" compared with "EPI-200" and in vivo test. We conducted endpoint analysis including cell viability (MTT reduction, CCK-8) and IL-1 alpha release. Twenty chemical were investigated using keraskinTM model and compared with Epiderm model. Results of MTT assay and CCK-8 assay were not different between EPI-200 and KeraSkinTM, but IL-lα levels of some test items different between EPI-200 and KeraSkinTM. We concluded that this KeraSkinTM model is useful for alternative skin irritation test, although other tests should be conducted for many other chemicals in international validation study.
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Use of CAM-TBS assay as the alternative methods to Draize eye irritation test
( Chae Wook Kim ),( Kyoung Mi Jung ),( Byung Fhy Shu ),( Bae Hwan Kim ) 한국동물실험대체법학회 2007 한국동물실험대체법학회 학술대회집 Vol.2007 No.1
The hen`s egg test-chorioallantoic membrane (HET-CAM) assay, which employs the vascular membrane of a fertile hen`s egg, has been actively studied by several countries as an alternative method to the Draize rabbit eye irritation test. However, the scoring system of the original HET-CAM test is not satisfactory because of its lack of objectivity and quantitativeness. Moreover, the test required skill when the effects of test substance are to be regarded. To overcome these disadvantages, Hagino et al. developed and objective evaluation technique using the amount of trypan blue absorbed at the site of treatment as an indicator of injury to the CAM (Chorioallantoic membrane – trypan blue staining, CAM-TBS). In this study, we evaluate the CAM-TBS methods and used three materials (1%, 2%, 5% SLS, tween 20 and PBS) to evaluate the correlation of CAM-TBS with HET-CAM test. The amounts of trypan blue adsorbed with the CAM showed a good correlation with the HET-CAM test score(r = 0.958). According to these results, we suppose that the trypan blue staining assay using CAM-TBS may be a good method to replace the HET-CAM test as an alternative to Draize rabbit eye irritation test. Despite our results, additional studies with more materials are necessary to validate this method.
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( Jung Hun Ju ),( Young Soo Kim ),( Kyung Rak Min ),( Jong Kwon Lee ),( Seung Hee Kim ),( Kui Lea Park ) 한국동물실험대체법학회 2007 한국동물실험대체법학회 학술대회집 Vol.2007 No.1
Interleukin (IL)-5 is mainly produced from Th2 cells activated by allergen. The cytokine acts preferentially on eosinophils. IL-5 promotes growth and differentiation of eosinophils from their progenitor cells, and activates the cells to release chemical mediators for the inflammatory response. Sophoricoside, a natural isoflavone glycoside from S. japonica, showed an IC50 value of 11.3 uM on IL-5 mediated proliferation of Y16 cells. To develop novel IL-5 inhibitor with sophoricoside as a lead compound, 100 kinds of synthetic isoflavone analogs have been prepared. Among them, synthetic isoflavone(7-cyclohexylmethoxy-3-(4-hydroxyphenyl) chromen-4-one) substituted with cyclohexylmethyl group showed an IC50 value of 5.2 uM on the IL-5 bioassay. Pharmacophore of the isoflavone analogs to inhibit IL-5 bioactivity seems to require of isoflavone 1) introduction of cyclohexylmethoxy group at 5 or 7 position of A ring of isoflavone, which may act as a bulky group for interacting with hydrophobic pocket in putative target, 2) existence of phenolic hydroxy group at 4` position of B ring, and 3) planarity between A and C rings. Furthermore, 7-cyclohexylmethoxy-3-(4-hydroxyphenyl) chromen-4-one compound inhibited IL-3 or GM-CSF mediated proliferations of Y16 cells with IC50 values of 6.8 uM or 5.9 uM, respectively. As the mechanism of action, 7-cyclohexylmethoxy-3-(4-hydroxyphenyl) chromen-4-one suppressed IL-5 induced c-mycexpression through inhibitory action on activation of JAK-2 and MAP kinases (p38 and ERK1/2). However, sophoricoside inhibited IL-5 induced phosphorylation of JAK-2 and ERK1/2 but it did not suppress p38 MAP kinase and c-myc expression.
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( Sue Nie Park ) 한국동물실험대체법학회 2007 한국동물실험대체법학회 학술대회집 Vol.2007 No.1
Recently, the understanding of the mechanism of genotoxicity and / or the prediction of carcinogenicity of chemicals have been attempted by toxicogenomics. However, the procurement of enough amount data set for prediction analysis is usually not so easy for the costs. Therefore, it would be useful to improve the prediction power by applying proper analytical methods on the data set obtained for small number of chemicals. Thus, in this study, we attempted to apply and compare the classification approaches such as k-NN(k-nearest Neithborhood), SVM(Support Vector Machine), PLS(Partial Least Square regression), PCR(Principle Component regression), and ridge regression discriminant classification and compared to ensemble method with bootstrap resampling. Four chemicals each representing genotoxic carcinogen(glycidol), genotoxic noncarcinogen(8-hydroxyquinoline), nongenotoxic carcinogens(o-nitrotoluene), and nongenotoxic noncarcinogen(1,2-dichlorobenzene) were selected to evaluate the classification methods using microarray chip data. Each minimal error rate was 0.13, 0.0, 0.0, 0.0, and 0.0 for training step while 0.8, 0.4, 0.8, 0.8, and 0.8 was obtained in test data, respectively. The application of LogitBoost resulted in minimum error of 0.13 in training data and 0.3 in test data with improvement of prediction power. Our study may offer the more effective method in evaluating toxicity of chemicals related to genotoxicity and / or predicition of carcinogenecity.
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KSAAE vision on the development of the relationship among Asian societies
( Heo Yong ) 한국동물실험대체법학회 2008 한국동물실험대체법학회 학술대회집 Vol.2008 No.1
World-wide restrictions in animal use for research has driven efforts to develop alternative methods. Considering international 3R trends, Korean Society for Alternatives to Animal Experiments (KSAAE) was organized at early 2007. Thereafter KSAAE has been doing various aspects of academic activities, such as hosting winter and summer conference, publishing society`s journal, and exchanging information on the alternatives with foreign countries including Japan and China. National Institute for Toxicological Research has contributed to vitalize alternatives in Korea through sponsoring KSAAE, preparing so called KoCVAM (Korean Center for Validation of Alternative Methods), and performing research activities for development of alternative methods. Various activities will be flourishing through organizing Asian Coalition for Alternatives. Alternative methods under development can be internationally validated among Asian countries belonging to the Coalition, which will facilitate adoption of the methods into world-wide. Elevation and expansion of scientific knowledge or basis on the alternatives can be obtained through joint conference or publication of scientific journal. In addition, development of new alternative methods will be efficiently proceeded through mutual research funding among Asian countries. Furthermore, Fostering predecessor young scientists will be achieved through continuing education at the time of regular conferences or dispatching students to advanced institutes.
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C. elegans as a model organism to study animal Behavior
안주홍 ( Joo Hong Ahnn ) 한국동물실험대체법학회 2007 한국동물실험대체법학회 학술대회집 Vol.2007 No.1
Ca2+ / calmodulin-dependent calcineurin has been shown to have important roles in various Ca2+ signaling pathways. We have previously reported that cnb-1 mutants, null mutants of a regulatory B subunit, displayed pleiotropic defects including uncoordinated movement and delayed egg laying in C. elegans. Interestingly, gain-of-function mutants of a catalytic A subunit showed exactly opposite phenotypes to those of cnb-1 mutants providing an excellent genetic model to define calcium-mediated signaling pathway at the organism level. Furthermore, calcineurin is also important for normal cuticle formation, whichis required for maintenance of normal body size in C. elegans. Genetic interactions between tax-6 and several mutants including egl-30 and egl-10 which are known to be involved in Gprotein signaling pathway suggest that calcineurin indeed regulates locomotion and serotonin-mediated egg laying through goa-1(Goα) and egl-30(Gqα). Our results indicate that, along with CaMKII, calcineurin regulates G-protein-coupled phosphorylation signaling pathways in C. elegans. The enteric muscle contraction (EMC) is the last step of the defecation behavior which occurs every 50 seconds in C. elegans. This EMC is regulated by intestinal and anal depressor muscles, which are innervatedby GABA motor neurons. Our data show that calcineurin (tax-6) is expressed in intestinal muscle and anal depressor muscle, and the gain-of-function mutant of calcineurin, tax-6(jh107), shows defects in enteric muscle contractions. In addition, exogenous GABA failed to rescue the EMC defects of tax-6(jh107) indicating that calcineurin functions in postsynaptic muscles rather than presynaptic neurons regulating EMC, which could be rescued by exogenous GABA treatment. Genetic epistasis with exp -1 mutant, which encodes an excitatory GABA receptor, suggests that calcineurin functions as a negative regulator of exp-1 in excitatory GABA signaling.
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Comparison of bovine corneal opacity and porcine corneal opacity assay for eye irritation test
석승혁 ( Seung Hyeok Seok ),( Ju Hee Han ),( Min Won Baek ),( Hui Young Lee ),( Dong Jae Kim ),( Yi Rang Na ),( Hyun Kyoung Lee ),( Sung Hoon Park ),( Bae Hwan Kim ),( Kui Lea Park ),( Jong Kwon Lee ),( 한국동물실험대체법학회 2008 한국동물실험대체법학회 학술대회집 Vol.2008 No.1
The Draize eye irritation test has been changed to alternative methods based on 3 Rs (Replacement, Reduction, Refinement) of animal experiments. The bovine corneal opacity and permeability (BCOP) assay is well established as one of the alternative methods to the Draize test but the supply of bovine eyes from a slaughterhouse is not sufficient in actuality. The porcine corneal opacity and permeability (PCOP) assay have been proposed as a reliable alternative to the BCOP assay and the Draize test because porcine corneas have numerous advantages including structural similarities with human corneas and the stable capacity for a corneal supply. In this study, three cosmetics and seven chemicals that are already known as irritants at Draize score and the bovine corneal opacity (BCO) assay were tested by the PCOP assay for the first time in Korea. The porcine corneal opacity was measured by quantifying the ability of light to pass through a cornea using an opacitometer. The permeability was measured at 10 and 30 min time points after chemical exposure. The PCOP assay showed the high correlation with the Draize score and BCO assay. These results showed that the PCOP assay can be used as an alternative method for the BCO assay and the Draize test although it needs further studies about insoluble chemicals and others to increase reliability.
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