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Validation of Alternative Method for Evaluating Effects of Drug Metabolism on Cytotoxicity
- ZHOU Jing-jing1，YANG Ming2，MAO Yu-chang3，LU Jie-qi2，YANG You-ming3，HU Zhuo-han1,3
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2010, 30(2):
87-94.
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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.
