摘要
目的探索邻氟硝基苯和2,4-二氟硝基苯对大鼠肝脏中药物代谢酶的影响。方法建立大鼠肝微粒体中睾酮、非那西丁、氯唑沙宗和香豆素的RP-HPLC定量分析方法基础上,制备大鼠肝微粒体,检测底物浓度变化,根据Lineweaver-Burk方程计算酶反应动力学参数Km和Vm值。结果结果表明睾酮、非那西丁、氯唑沙宗和香豆素分别在0.5~100μg·mL^(-1)浓度范围内,线性关系良好(r>0.999),回收率90%以上,日内和日间RSD小于10%。酶反应动力学结果表明两种氟化物对CYP3A1、CYP2E1和CYP2A6起显著促进作用,Km值显著降低;同时对CYP2A1起显著抑制作用,Km值显著升高。结论本研究建立的RP-HPLC检测方法灵敏、准确、可靠,能够满足不同肝药酶底物定量检测和肝药酶活性评价,结果表明两种氟化物能够促进CYP3A1、CYP2E1和CYP2A6活性,抑制CYP2A1酶活性。
OBJECTIVE In this study,the effects of fluoronitrobenzene and difluoronitrobenzene on drug metabolizing enzymes in liver were investigated by the usage of rat as experimental subjects.METHODS On the basis of establishing the RP-HPLC quantitative analysis method for testosterone,phenacetin,chlorzoxazone and coumarin in rat hepatomicrosome,the concentrations of all these enzyme substrates were determined after the treatment 20 min by in vitro hepatomicrosome acquired from normal control rat and oral fluoronitrobenzene/difluoronitrobenzene rat.And testosterone,phenacetin,chlorzoxazone and coumarin were used as the substrates of CYP3A1,CYP1A2,CYP2E1 and CYP2A6,respectively.Then the reaction kinetics parameters(Vm and Km)were calculated by the Lineweaver-Burk equation.RESULTS The results showed that the linear relationship was well(r>0.999)in all the substrate concentration ranges of 0.5-100μg·mL^(-1)and all the recoveries were more than 90%.Moreover,all the intra-day and inter-day variable coefficients(RSD)were less than 10%.The results of enzyme reaction kinetics showed that the Km values from CYP3A1,CYP2E1 and CYP2A6 were significantly decreased in oral fluoronitrobenzene/difluoronitrobenzene rats by the comparison with the normal control rats,while the Km values were markedly increased.That is to say,the enzyme activities of CYP3A1,CYP2E1 and CYP2A6 were induced by fluoronitrobenzene/difluoronitrobenzene,while the activity of CYP1A2 was inhibited.CONCLUSION The RP-HPLC method established in this study was sensitive,accurate and reliable,which has satisfied the quantitative detection of different P450 enzyme substrates and the evaluation of liver drug enzyme activity.The evaluation results of liver drug enzyme indicated that the two kinds of fluorides promoted the activities of CYP3A1,CYP2E1 and CYP2A6,and inhibit the activity of CYP2A1 enzyme.
作者
万旺军
何坚刚
葛建
邓同乐
WAN Wang-jun;HE Jian-gang;GE Jian;DENG Tong-le(Technology centre of Hangzhou Customs District P.R.China,Hangzhou 310007,China;Institute of Drug Quality and Safety Evaluation,China Jiliang University,Hangzhou 310018,China)
出处
《海峡药学》
2021年第6期8-11,共4页
Strait Pharmaceutical Journal
基金
国家质量监督检验检疫总局科技计划项目资助(T2017-ZJCIO-0001)
海关总署科技计划项目(2019HK010)。