CAJ | 학술논문

目的随着近20年实验动物伦理原则的逐步普及,学术界、工业界和政府管理部门开始研究和推广动物试验的替代方法,用于药品、食品和化妆品的安全评价.美国15个政府管理机构和研究机构成立了"替代评价方法协调委员会(ICCVAM)"并指定和验证了评价方法.作者按照ICCVAM推荐的方法,应用体外细胞毒性评价方法和代谢性相互作用评价方法,研究出入境天然产物制品的毒性作用及其代谢性活化或代谢性解毒机制.方法用人肝微粒体,研究了天然产物制品对肝细胞色素P450亚酶的作用.用超低温冻存的人肝原代细胞,评价了5个进口天然产物制品的细胞毒性(MTT方法).应用化学抑制法,研究了细胞色素P4502A6,2C9,2C19和3A4对天然产物制品细胞毒性的影响.结果天然产物制品TA-07-004和TA-07-005对CYP1A2有明显的抑制作用,它们的IC50分别为初始浓度的0.22%和0.03%,且它们对CYP3A4也有明显的抑制作用,它们的IC50分别为初始浓度的0.49%和0.20%.天然产物制品TA-07-003没有肝细胞毒性,其余4个天然产物制品观察到有肝细胞毒性,它们的IC50分别为初始浓度的0.37%,0.26%,0.62%和0.19%.在研究与天然产物制品TA-07-005细胞毒性有关的细胞色素P450亚型酶中,发现CYP2C9、CYP2A6、CYP2C19和CYP3A4活性被抑制后能减轻TA-07-005对肝细胞的毒性,说明TA-07-005被CYP450生物活化后毒性增加.结论用人原代肝细胞模型为基础的替代方法研究细胞毒性及代谢性相互作用对细胞毒性的影响是可行的.
목적 수착근20년실험동물윤리원칙적축보보급,학술계、공업계화정부관리부문개시연구화추엄동물시험적체대방법,용우약품、식품화화장품적안전평개.미국15개정부관리궤구화연구궤구성립료"체대평개방법협조위원회(ICCVAM)"병지정화험증료평개방법.작자안조ICCVAM추천적방법,응용체외세포독성평개방법화대사성상호작용평개방법,연구출입경천연산물제품적독성작용급기대사성활화혹대사성해독궤제.방법 용인간미립체,연구료천연산물제품대간세포색소P450아매적작용.용초저온동존적인간원대세포,평개료5개진구천연산물제품적세포독성(MTT방법).응용화학억제법,연구료세포색소P4502A6,2C9,2C19화3A4대천연산물제품세포독성적영향.결과 천연산물제품TA-07-004화TA-07-005대CYP1A2유명현적억제작용,타문적IC50분별위초시농도적0.22%화0.03%,차타문대CYP3A4야유명현적억제작용,타문적IC50분별위초시농도적0.49%화0.20%.천연산물제품TA-07-003몰유간세포독성,기여4개천연산물제품관찰도유간세포독성,타문적IC50분별위초시농도적0.37%,0.26%,0.62%화0.19%.재연구여천연산물제품TA-07-005세포독성유관적세포색소P450아형매중,발현CYP2C9、CYP2A6、CYP2C19화CYP3A4활성피억제후능감경TA-07-005대간세포적독성,설명TA-07-005피CYP450생물활화후독성증가.결론 용인원대간세포모형위기출적체대방법연구세포독성급대사성상호작용대세포독성적영향시가행적.
Objective With increased concerns and gradual progress on the ethical use of experi-mental animals in the past decades, the development, and validation of new and revised non-animal and reliable alternative methods have been approached by academia, industry and government regulators in order to reduce the number of animals, and refine the procedures to lessen or eliminate pain and distress to animals, and replace animals with non-animal systems. According to the methods recommended by "The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM)" in USA,priority for alternative methods has been placed on basal cytotoxicity methods and additional efforts are on incorporating in vitro cytotoxic data with in vitro parameters of absorption, distribution, metabolism,excretion, and toxicity(ADMET). This study was to validate an alternative ADMET method by using human primary hepatocytes. Method Human liver microsomes were used to evaluate the effects of natural products on CYP450 isoforms. Basal cytotoxicity of 5 imported natural products was estimated by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and effects of metabolism by CYP450 isoforms on cytotoxicity of natural products was evaluated by using chemical inhibitors of CYP2A6, 2C9, 2C19, and 3A4 preincubation with human primary hepatocytes. Results The inhibitory potential(IC50) of natural products (TA-07-004, TA-07-005) on CYP1A2 were 0.22%and 0.03%, CYP 3A4 were 0.49% and 0.20% of the initial concentration, respectively.The hepatotoxicity,IC50 values were 0.37%, 0.26%, 0.62%, 0.19% of original concentrations for TA-07-001, TA-07-002,TA-07-004, and TA-07-005, respectively. No cytotoxicity was observed for TA-07-003. Preincubation of selective inhibitors for CYP450 isoforms with primary hepatocytes showed that cytotoxicity of TA-07-005 was reduced by inhibition of CYP2A6, 2C9, 2C19, 3A4, suggesting that TA-07-005 might be bioactivated by CYP450. Conclusions The In vitro hepatotoxic model with in vitro metabolism assay provided a valuable and feasible alternative procedure for estimating metabolism dependent toxic potentials of test materials as natural products.