연구논문 : Balb/c 마우스를 이용한 비방사선 BrdU:ELISA 국소림프절 시험법의 연구

정경미 ( Kyoung Mi Jung ),장원희 ( Won Hee Jang ),박영호 ( Young Ho Park ),임경민 ( Kyung Min Lim ) 한국동물실험대체법학회 2010 동물실험대체법학회지 Vol.4 No.1

To avoid the use of radioisotopes, many efforts have been made to develop non-radioisotopic modifications of the LLNA. Recently, OECD presented a guideline for the nonradioisotopic murine local lymph node assay employing BrdU incorporation and ELISA. This method was a non-radioisotopic modification of the LLNA using 5-bromo-2′-deoxyuridine (BrdU) incorporation. However, this method recommends CBA strain mice which is not common in Korea. Here we evaluated the sensitivity of the LLNA:BrdU-ELISA using the Balb/c mice strain, which is a readily available strain in Korea. According to OECD draft guideline, female Balb/c mice were treated for 3 days with a test material or vehicle to each side of both ears and BrdU was given as a intraperitoneal administration at 5 mg/mouse. We found that Balb/c mice is sensitive to BrdU and some premature deaths were found in the treatment groups. Balb/c mice must be older than 11 weeks to tolerate BrdU. BrdU incorporation into draining lymph node cells was measured using ELISA. In this study, isoeugenol, hexylcinnamic aldehyde(HCA), eugenol and isopropanol(IS) were used as reference chemicals. When the 1.6 or greater SI values were defined as sensitizer according to OECD draft guideline, isoeugenol, HCA and eugenol which are classified as moderate and weak sensitizers in the traditional LLNA, were found to evaluate positive results. LLNA:BrdU-ELISA using Balb/c mice, sensitivity and specificity were 100%. The EC1.6 values for isoeugenol, HCA and eugenol were calculated to be 1.8, 19.3 and 15.5% in 5 mg BrdU/mouse. A positive correlation (R2=0.813) between traditional LLNA and LLNA:BrdU-ELISA strongly suggests that the LLNA:BrdU- ELISA could be used as an alternative method to screen allergen.

우리나라 농민의 Chlorpyrifos에 대한 피부 위해성 평가

정경미(Kyoung Mi Jung),이효민(Hyo Min Lee),이은희(Eun Hee Lee),이선희(Sun Hee Lee),김진화(Jin Haw Kim),심영용(Young Yong Sim),홍진태(Jin Tae Hong),이용욱(Yong Wook Lee) 한국환경성돌연변이발암원학회 2002 한국환경성돌연변이·발암원학회지 Vol.22 No.3

Chlorpyrifos is an organophosphate insecticide and one of the most commonly and widely used insecticide. However, a little known about the dermal risk of chlorpyrifos on human being. Therefore, this study was conducted for the dermal risk assessment after exposure to chlorpyrifos in Korean farmers. First, skin irritation by chlorpyrifos (10 mg/㎠, 50 mg/㎠, 100 mg/㎠, 250 mg/㎠ in acetone) was determined in rabbits for 5 days considering the usage of chlorpyrifos short term highly exposure. The index of skin irritation by chlorpyrifos was increased in each dose and length of exposure dependent manners. Next, using benchmark dose (BMD5) approach, the dose-response relationship was assessed to calculate the reference dose (RfD). The value of RfD was 2.84 ㎍/kg/day from 142.16 ㎍/kg/day BMD5 value divided uncertainty factor 50. Finally, we assessed human dermal risk of chlorpyrifos with exposure level and RfD. Skin absorbed levels were assumed with several exposure scenarios encounting the circumstances of exposure that application method, protection equipment and cloth, exposure time and exposure frequency during chlorpyrifos spraying. By the comparison of skin absorbed dose with the reference dose, it was identified that risk values (risk index) to skin chlorpyrifos exposure were 0.958 from the point of above results and it was recommended that the occurrence of hazard effect (skin irritation toxicity) of chlorpyrifos would not be expected. Risk index was smaller than 1 in the case of spraying vehicle mounted application, 1hour exposure time and wearing protective cloth exposure. Whereas, risk index was above 1 in the case of hand-held application, 2hour exposure time and wearing common cloth. Comparing two kinds of application method, total risk index of the hand held application (1.67) was higher than vehicle mounted (0.27). Therefore, chlorpyrifos skin exposure was mainly affected by application equipment and applied form. The results of risk assessment on the human dermal toxicity of chlorpyrifos should be required to control in keeping safety<br/> rules, skin surface area available for contact, spraying time ,and spraying frequency.

Balb/c 마우스를 이용한 비방사선 BrdU:ELISA 국소림프절 시험법의 연구

정경미 ( Kyoung Mi Jung ),장원희 ( Won Hee Jang ),박영호 ( Young Ho Park ),임경민 ( Kyung Min Lim ) 한국동물실험대체법학회 2010 동물실험대체법학회지 Vol.4 No.1

To avoid the use of radioisotopes, many efforts have been made to develop non-radioisotopic modifications of the LLNA. Recently, OECD presented a guideline for the nonradioisotopic murine local lymph node assay employing BrdU incorporation and ELISA. This method was a non-radioisotopic modification of the LLNA using 5-bromo-2′-deoxyuridine (BrdU) incorporation. However, this method recommends CBA strain mice which is not common in Korea. Here we evaluated the sensitivity of the LLNA:BrdU-ELISA using the Balb/c mice strain, which is a readily available strain in Korea. According to OECD draft guideline, female Balb/c mice were treated for 3 days with a test material or vehicle to each side of both ears and BrdU was given as a intraperitoneal administration at 5 mg/mouse. We found that Balb/c mice is sensitive to BrdU and some premature deaths were found in the treatment groups. Balb/c mice must be older than 11 weeks to tolerate BrdU. BrdU incorporation into draining lymph node cells was measured using ELISA. In this study, isoeugenol, hexylcinnamic aldehyde(HCA), eugenol and isopropanol(IS) were used as reference chemicals. When the 1.6 or greater SI values were defined as sensitizer according to OECD draft guideline, isoeugenol, HCA and eugenol which are classified as moderate and weak sensitizers in the traditional LLNA, were found to evaluate positive results. LLNA:BrdU-ELISA using Balb/c mice, sensitivity and specificity were 100%. The EC1.6 values for isoeugenol, HCA and eugenol were calculated to be 1.8, 19.3 and 15.5% in 5 mg BrdU/mouse. A positive correlation (R2=0.813) between traditional LLNA and LLNA:BrdU-ELISA strongly suggests that the LLNA:BrdU- ELISA could be used as an alternative method to screen allergen.

BrdU Elisa 측정법을 이용한 비방사선 국소림프절 시험법의 소개

정경미 ( Kyoung Mi Jung ),김배환 ( Bae Hwan Kim ),박영호 ( Young Ho Park ),임경민 ( Kyung Min Lim ) 한국동물실험대체법학회 2009 동물실험대체법학회지 Vol.3 No.1

The murine local Iymph node assay(LLNA) using H3-thymidine has been developed to detect chemical allergens, replacing the traditional guinea pig maximization test. Recently, non-radioisotopic local lymph node assay employing 5-bromo-2`-deoxyuridine(BrdU) incorporation has been developed to extend its utility further. This method adopts an alternative non-radioisotopic endpoint for the LLNA by using BrdU in place of radioisotopes, 3H-thymidine, employed in the traditional method. International validation studies have been conducted to evaluate the performance of the LLNA by BrdU incorporation using ELISA and OECD presented a guideline. In this study, we evaluated the performance of the guideline with 7 reference chemicals. BrdU was given as a administration at 2 mg/mouse after 2 days following three consecutive day applications of material. BrdU incorporation into draining lymph node cells was measured using ELISA. In this study, 2,4-Dinitrochiorobenzene(DNCB), 4-Phenylenediamine(PPD), Isoeugenol, Hexylcinnamic aldehyde(HCA), Eugenol, Isopropanol(IS) and Sodium lauryl sulfate were used as reference chemicals. When the 3 or greater SI values were defined as sensitizer, DNCB, PPD and Isoeugenol, which are classified as extreme to moderate sensitizers in the traditional LLNA, were also found positive. In contrast, HCA, Eugenol and IS were concluded to be negative. At this SI threshold values(3 or greater), for LLNA:BrdU-ELISA, sensitivity and specificity were determined to be 60% and 100%, respectively. When the 1.5 or greater SI values were defined as sensitizer according to ICCVAM guideline, the all chemicals were evaluated sensitizer suggesting the low specificity. These results suggest that the non-radioactive LLNA by BrdU incorporation using ELISA is a simple and useful method for evaluating sensitivity although the issue of sensitivity and specificity should be resolved in the future.

BrdU Elisa 측정법을 이용한 비방사선 국소림프절 시험법의 소개

정경미 ( Kyoung Mi Jung ),김배환 ( Bae Hwan Kim ),박영호 ( Young Ho Park ),임경민 ( Kyung Min Lim ) 한국동물실험대체법학회 2009 동물실험대체법학회지 Vol.3 No.1

The murine local Iymph node assay(LLNA) using H3-thymidine has been developed to detect chemical allergens, replacing the traditional guinea pig maximization test. Recently, non-radioisotopic local lymph node assay employing 5-bromo-2`-deoxyuridine(BrdU) incorporation has been developed to extend its utility further. This method adopts an alternative non-radioisotopic endpoint for the LLNA by using BrdU in place of radioisotopes, 3H-thymidine, employed in the traditional method. International validation studies have been conducted to evaluate the performance of the LLNA by BrdU incorporation using ELISA and OECD presented a guideline. In this study, we evaluated the performance of the guideline with 7 reference chemicals. BrdU was given as a administration at 2 mg/mouse after 2 days following three consecutive day applications of material. BrdU incorporation into draining lymph node cells was measured using ELISA. In this study, 2,4-Dinitrochiorobenzene(DNCB), 4-Phenylenediamine(PPD), Isoeugenol, Hexylcinnamic aldehyde(HCA), Eugenol, Isopropanol(IS) and Sodium lauryl sulfate were used as reference chemicals. When the 3 or greater SI values were defined as sensitizer, DNCB, PPD and Isoeugenol, which are classified as extreme to moderate sensitizers in the traditional LLNA, were also found positive. In contrast, HCA, Eugenol and IS were concluded to be negative. At this SI threshold values(3 or greater), for LLNA:BrdU-ELISA, sensitivity and specificity were determined to be 60% and 100%, respectively. When the 1.5 or greater SI values were defined as sensitizer according to ICCVAM guideline, the all chemicals were evaluated sensitizer suggesting the low specificity. These results suggest that the non-radioactive LLNA by BrdU incorporation using ELISA is a simple and useful method for evaluating sensitivity although the issue of sensitivity and specificity should be resolved in the future.

인공피부를 이용한 피부자극 대체시험 밸리데이션 연구

정경미 ( Kyoung Mi Jung ),문지영 ( Ji Young Moon ),이수현 ( Su Hyon Lee ),김채욱 ( Chae Wook Kim ),박철범 ( Cheol Beom Park ),김배환 ( Bae Hwan Kim ) 한국동물실험대체법학회 2008 동물실험대체법학회지 Vol.2 No.1

Several alternative in vitro methods for identifying skin irritants have been developed recently, the most promising of which use reconstituted human skin model. In this study, the two reconstituted human skin models, KeraskinTM and EPI-200, showed promising results in terms of interlaboratory variability and predictive capacity. The main endpoint measured for both KeraskinTM and EPI-200 was cell viability via MTT. In samples from chemicals which gave MTT assay results above the threshold of 50% viability, IL-1α release was also measured, to determine whether the additional endpoint would improve the predictive ability of the tests. In the interlaboratory validation study for KeraskinTM, sensitivity, specificity and accuracy of the KeraskinTM were 90%, 44% and 68%(MTT assay only), respectively. The KeraskinTM model was overly sensitive, resulting in the prediction of the non-irritant chemicals as irritant. The EPI-200 model had an accuracy of 63%, with a sensitivity rate of 70% and a specificity rate of 56%. Based on these results, although predictive capacity of KeraSkinTM model was considered insufficient, due to a low specificity, we concluded that this KeraSkinTM model is useful potential alternative skin irritation method. Further studies are in progress to improve the test protocols and prediction models for KeraSkinTM.

연구논문 : 토끼 안점막자극 대체시험법으로서의 HET-CAM assay 소개

김채욱 ( Chae Wook Kim ),정경미 ( Kyoung Mi Jung ),이지해 ( Ji Hae Lee ),윤준원 ( Jun Won Yun ),서병휘 ( Byung Fhy Suh ),김배환 ( Bae Hwan Kim ) 한국동물실험대체법학회 2007 동물실험대체법학회지 Vol.1 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. There are two methods to evaluate the eye irritation potential, reaction time method(RTM) and end point assessment(EPA), between which can be chosen as the apt method depending on the characteristics of test material. In this study, we employed the EPA method to compare with the Draize rabbit eye irritation test method and then calculated the Pearson correlation coefficient value. The Result of Pearson correlation was high(0.986, p=0.002) with 5 test materials: 3 raw materials(IPMS 03, VCP-IS-2Na, Seletinoid: Amorepacific R&D center, Korea), 1 commercial cleansing form(Amorepacific R&D center, Korea) and 1 surfactant (Amisoft LS-11, Ajinomoto, Japan). According to these results, we suppose that the HET-CAM assay is a good method to replace the Draize eye irritation test as the tests of 5 materials have proven. Despite our results, additional studies with more materials are necessary to validate this method as an alternative to the Draize rabbit eye irritation test method.

안점막자극시험의 대체시험법으로서의 CAM-TBS assay 소개

김채욱 ( Chae Wook Kim ),정경미 ( Kyoung Mi Jung ),서병휘 ( Byung Fhy Suh ),김배환 ( Bae Hwan Kim ) 한국동물실험대체법학회 2007 동물실험대체법학회지 Vol.1 No.2

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 CAMTBS 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.

Slim813의 지방산 분해 촉진을 통한 슬리밍 효능 연구

박미영 ( Miyoung Park ),정경미 ( Kyoung-mi Jung ),주경미 ( Kyoung-mi Joo ),김연준 ( Yeon-joon Kim ),박영호 ( Young-ho Park ),임경민 ( Kyung-min Lim ) 대한화장품학회 2012 대한화장품학회지 Vol.38 No.2

Slim813은 2-사이클로펜텐-1-온 옥심 유도체로서 다양한 염증성 자극원에 대한 TNF 생성을 억제하는 항 자극 및 UV에 의한 MMP1 억제를 통한 항 노화 효능을 갖는 물질이다. 본 연구를 통하여 이와 같은 효능 외에도 완전히 분화된 지방 세포에서 cAMP (cyclic adenosine monophosphate) 생성 촉진을 통한 HSL (hormone sensitive lipase) 활성을 증대시킴으로써 지방분해를 증가시킴을 확인하였다. 또한 Slim813을 사람의 팔과 허벅지 부위에 2주간 도포하였을 때 지방의 두께 감소를 유발하는 것으로 나타나 체내 피하 지방을 분해하는 효능을 갖는 것으로 사료된다. Slim813 is a 2-cyclopentene-1-one oxime derivative with potent anti-inflammatory and anti-photoaging effects. Slim813 inhibited LPS-induced TNF-α production and attenuated UVB-induced MMP1 expression. Here in an attempt to find an unrevealed efficacy of Slim813, we found that Slim813 stimulates lipolysis in a dose-dependent manner by increasing intracellular cAMP level through the elevation of HSL activity in fully differentiated adipocytes. Moreover, topical application of Slim813 for two weeks in human reduced the thickness of subcutaneous fat in arm and thigh regions, implying it could be effectively used in the reduction of unwanted local fat accumulation.

인공각막모델을 이용한 안점막자극 대체시험법 연구

정행선 ( Haeng Sun Jung ),이수현 ( Su Hyon Lee ),류양환 ( Yang Hwan Ryu ),정경미 ( Kyoung Mi Jung ),김채욱 ( Chae Wook Kim ),임경민 ( Kyung Min Lim ),손영숙 ( Young Sook Son ) 한국동물실험대체법학회 2009 동물실험대체법학회지 Vol.3 No.1

Alternative methods to the Draize eye irritation test, such as the BCOP, HET-CAM, ICE, and IRE, are used to evaluate the ocular irritation potential of cosmetic, livelihood articles or industrial chemicals. In order to improve the sensitivity and specificity of alternative eye irritation test, we developed a three-dimensional human corneal model that uses normal human corneal epithelial cells. Our corneal tissue model consists of normal human corneal epithelial cells on cell culture inserts at the air liquid interface, differentiating to form a stratified, squamous epithelium similar to that found in the human cornea, exhibiting in vivo like morphological and histological characteristics. In this study, two laboratories have tested 20 reference chemicals using the same study protocol. The results were compared to in vivo data as well as previously published data obtained in the other three-dimensional corneal model test. Although an overall accuracy of 85% was obtained (sensitivity=100% and specificity=67%), further experiments are required to confirm and validate these preliminary results.