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MC / 9 마우스 비만세포주에서 생성되는 interleukin-4 및 tumor necrosis factor-alpha의 피부감작능 대체시험을 위한 활용도 평가에 대한 연구
임연미 ( Yeon Mi Lim ),허용 ( Yong Heo ) 한국동물실험대체법학회 2008 동물실험대체법학회지 Vol.2 No.1
We have attempted to develop an in vitro assay using MC / 9 murine mast cell line to classify chemicals into contact sensitizers or irritants. MC / 9 murine mast cell line was in vitro sensitized with anti-dinitrophenyl antibody overnight followed by DNP-albumin challenge for 1 hour. Four chemicals judged to be sensitizing (1-chloro-2,4-dinitrobenzene, hexyl cinnamic aldehyde, eugenol, hydroquinone) and 4 non-sensitizing (methyl salicylate, sodium lauryl sulfate, benzalkonium chloride, dimethyl sulfoxide) by the standard in vivo assays were added just prior to anti-dinitrophenyl antibody sensitization. Concentrations of the chemicals in the culture were 2X, 1X, 0.5X, or 0.1X of IC50 determined by MTT assay. No significant modulation of interleukin-4 production from MC / 9 mast cells was observed in the presence of the test chemicals. Whereas, elevation in the level of tumor necrosis factor-alpha (TNFα) productioan was apparent in the sensitizing chemicals, but not in the non-sensitizers compared with vehicle treatment. If threshold value to classify chemicals into sensitizer were set at 125%, the highest TNFα production rate among the non-sensitizing chemicals tested, 1-chloro-2,4-dinitrobenzene, hexyl cinnamic aldehyde, and hydroquinone was positive for enhancement of TNFα production, resulting in classification as sensitizers. In conclusion, being considered a valuable alternative tool for screening chemicals for their sensitizing potential, production of TNFα from MC / 9 mast cell line should be investigated further using more sensitizing or irritant chemicals.
MC/9 마우스 비만세포주의 피부감작능 대체시험을 위한 활용도평가에 대한 연구
허용 ( Yong Heo ),임연미 ( Yeon Mi Lim ),박귀례 ( Kui Lea Park ),이종권 ( Jong Kwon Lee ) 한국동물실험대체법학회 2007 동물실험대체법학회지 Vol.1 No.1
We attempted to develop an in vitro assay to classify chemicals into contact sensitizers or irritants. For this purpose, a protocol for optimal production of histamine from MC / 9 murine mast cell line was established as follows: in vitro sensitization of MC / 9 (0.5x106 cells / well) with anti-dinitrophenyl (DNP) antibody (1㎍ /0.1㎖) overnight followed by DNP-albumin (0.1㎍ /0.1㎖) challenge for 1 hour. Using the protocol, chemicals known as skin sensitizer (1-chloro-2,4-dinitrobenzene, hexyl cinnamic aldehyde, eugenol, hydroquinone) or irritant (methyl salicylate, sodium lauryl sulfate, benzalkonium chloride, dimethyl sulfoxide) based on animal results were tested for their sensitizing capacity. Concentrations of the chemicals in the culture were 2X, 1X, 0.5X, 0.1X, 0.01X, or 0.001X of IC50 determined by MTT assay. No significant modulation of histamine release from MC / 9 mast cells sensitized overnight in the presence of the test chemicals was observed. Furthermore, when the chemicals were added 24 hour before initiation of anti-DNP antibody sensitization and have been present during the period of sensitization, histamine release was not significantly enhanced from MC / 9 cells activated in the presence of the sensitizing chemicals. In conclusion, being considered a valuable alternative tool for screening chemicals for their sensitizing potential, other immunologic markers of MC / 9 mast cell including newly synthesized mediators or cell surface molecules should be further evaluated.
연구논문 : MC/9 마우스 비만세포주의 피부감작능 대체시험을 위한 활용도평가에 대한 연구
허용 ( Yong Heo ),임연미 ( Yeon Mi Lim ),박귀례 ( Kui Lea Park ),이종권 ( Jong Kwon Lee ) 한국동물실험대체법학회 2007 동물실험대체법학회지 Vol.1 No.1
We attempted to develop an in vitro assay to classify chemicals into contact sensitizers or irritants. For this purpose, a protocol for optimal production of histamine from MC / 9 murine mast cell line was established as follows: in vitro sensitization of MC / 9 (0.5x106 cells / well) with anti-dinitrophenyl (DNP) antibody (1㎍ /0.1㎖) overnight followed by DNP-albumin (0.1㎍ /0.1㎖) challenge for 1 hour. Using the protocol, chemicals known as skin sensitizer (1-chloro-2,4-dinitrobenzene, hexyl cinnamic aldehyde, eugenol, hydroquinone) or irritant (methyl salicylate, sodium lauryl sulfate, benzalkonium chloride, dimethyl sulfoxide) based on animal results were tested for their sensitizing capacity. Concentrations of the chemicals in the culture were 2X, 1X, 0.5X, 0.1X, 0.01X, or 0.001X of IC50 determined by MTT assay. No significant modulation of histamine release from MC / 9 mast cells sensitized overnight in the presence of the test chemicals was observed. Furthermore, when the chemicals were added 24 hour before initiation of anti-DNP antibody sensitization and have been present during the period of sensitization, histamine release was not significantly enhanced from MC / 9 cells activated in the presence of the sensitizing chemicals. In conclusion, being considered a valuable alternative tool for screening chemicals for their sensitizing potential, other immunologic markers of MC / 9 mast cell including newly synthesized mediators or cell surface molecules should be further evaluated.
Macrophage inflammatory protein-1β 생성 수준 분석을 통한 피부감작물질 분류에 대한 시험기관간 신뢰성 검증
안수선 ( Su Sun An ),임연미 ( Yeon Mi Lim ),문성준 ( Seong Joon Moon ),김서영 ( Seo Young Kim ),이수진 ( Soo Jin Lee ),이승혜 ( Seung Hye Lee ),김소남 ( So Nam Kim ),허용 ( Yong Heo ) 한국동물실험대체법학회 2008 동물실험대체법학회지 Vol.2 No.1
The study was performed to test the value of the macrophage inflammatory protein-1β(MIP-1β) assay for testing chemicals` skin-sensitizing potentials, and to test its reproducibility and validity. The assay was performed using 5 sensitizing(dinitrofluorobenzene, nickel sulfate hexahydrate, benzocaine, 5-chloro-2-methyl-isothiazolion-3-one, photassium dichromate) and 3 non-sensitizing chemicals(lactic acid, isopropanol, salicylic acid) judged by the standard in vivo assays. THP-1 cells were cultured in the presence or absence of four doses, 0.01X, 0.1X, 0.5X, or 1X IC50(50% inhibitory concentration for THP-1 cell proliferation) of these chemicals for 24 hr, and MIP-1β level in the supernatants was determined. Two research laboratories in Korea were participated in the inter-laboratory validation process. Skin sensitization by test chemicals was decided by MIP-1β production rates, which was expressed as the relative increase in MIP-1β production in response to chemical treatment compared with vehicle treatment. When the threshold MIP-1β production rate used was 100%, all the sensitizing chemicals were positive for both laboratories, but all the non-sensitizing chemicals were negative for the one laboratory and positive for the other laboratory. In addition, inter-laboratory consistency was 63%. When the threshold MIP-1β production rate used was 97% or 144%, which was the highest level among the non-sensitizing chemicals tested by each laboratory, all the non-sensitizing chemicals were negative for both laboratories. Whereas, all the sensitizing chemicals were positive for the one laboratory, but benzocaine and 5-chloro-2-methyl-4-isothiazolion-3-one were negative for the other laboratory, which resulted in 75% inter-laboratory consistency. Our study suggests that MIP-1β could be a biomarker for classification of chemicals as sensitizers or non-sensitizers, but more standardized assay procedure may be needed to increase the reproducibility or validity.
연구논문 : 비육돈 및 산란계 밀집사육과 스트레스 대응 호르몬의 상관성에 대한 연구
구중회 ( Jung Hoi Koo ),이승혜 ( Seung Hye Lee ),임연미 ( Yeon Mi Lim ),김소남 ( So Nam Kim ),허용 ( Yong Heo ) 한국동물실험대체법학회 2007 동물실험대체법학회지 Vol.1 No.2
Prior to systemic investigation on intense husbandry environment-mediated stress outcome in pig or hen, we compared serum levels of epinephrine, norepinephrine, and cortisol in pigs or hens housed in intense husbandry confinement building or environmentally friendly husbandry facility. Pigs were accommodated under density of 3 per 3.3 m2 and were kept on wet floors in the intense husbandry confinement building with fan-ventilation system. Meanwhile, the environmentally friendly husbandry facility with open ventilation system accommodated 2 pigs per 3.3 m2 and pigs were kept on dry floors. Hens were housed under density of 3 per 0.1 m2 in wire-partitioned battery cage at the intense husbandry confinement building with fan-ventilation system. Meanwhile, the environmentally friendly husbandry facility with open ventilation system accommodated 11 hens per 3.3 m2 on flat ground. Five animals from each facility were randomly chosen and their peripheral blood was collected from jugular vein in pig or wing vein in hen. Levels of epinephrine, norepinephrine, and cortisol were determined using commercially available ELISA kit. Pigs housed in the intense husbandry confinement building demonstrated 10-fold and 6-fold lower level of epinephrine and norepinephrine, respectively, than those in the environmentally friendly husbandry facility. Furthermore, level of cortisol was significantly lowered in both pigs and hens housed in the intense husbandry confinement building, compared with that in the environmentally friendly husbandry facility. Overall our results suggest that pigs and hens housed in the intense husbandry confinement building are not adequately responding to extrinsic stress such as manual bleeding, eventually leading to vulnerability to various diseases.