Study On Bioactivity Of CYP3A65 Enzyme In Zebrafish

Objective:Zebrafish has been widely used for pharmaceutical research as a model animal,but it has rarely reported in domestic and foreign literature about its enzyme bioactivity.In this study,we investigated the expression of the drug-metabolizing enzyme CYP3A65 in zebrafish at different growth stages,and select the most suitable zebrafish growing season for drug metabolism research.Further evaluation were carried out for the application of zebrafish as a drug metabolism model.Methods:First,the fluorescent quantitative PCR were used to detect zebrafish drug-metabolizing enzyme gene CYP3A65 mRNA expression in juvenile zebrafish(72hpf,96 hpf,120 dpf,7dpf,21dpf),young zebrafish(30 dpf),45 dpf,60 dpf,75 dpf)and adult zebrafish(90 dpf)in the whole growth stage of fish,liver and intestine tissue.The activity of zebrafish liver microsome CYP3A65 was measured using the contents of NADPH-cytochrome C reductase,erythromycin-N-demethylase(ERND)and testosterone hydroxylase.UHPLC quantitative analysis was performed to detect testosterone and its metabolites 6?-hydroxytestosterone.Results:The CYP3A65,PXR and AHR2 mRNA expression was highest in the zebrafish at 75dpf in adolescence.During the growth and development period of zebrafish,the gene expression trends of metabolic enzymes and their nuclear receptors were consistent,and they were affected by liver tissue and intestinal tissues.Therefore,the 75dpf zebrafish in youth may be most suitable for CYP3A65 metabolism studies.Analysis of CYP3A65 and upstream nuclear receptors PXR and AHR2 for correlation analysis,there was a correlation between CYP3A65 and PXR mRNA expression,R~2=0.806,there was a correlation between CYP3A65 and AHR2 mRNA expression,R~2=0.750.To evaluate the activity of the drug-metabolizing enzyme CYP3A65 in the zebrafish liver microsomes during the optimal growth period.The cytochrome b5content of zebrafish liver microsomes was determined to be 0.134 nmol/mg,erythromycin-N-demethylase activity was 0.862 nmol/(min·mg),and testosterone hydroxylase activity was 0.066 nmol/(min·mg).Conclusion:The expression levels of the main drug metabolism enzymes CYP3A65,PXR and AHR2 in the whole fish,liver and intestine of 75dpf zebrafish are the highest.The 75dpf zebrafish may be best suited for CYP3A65-related drug metabolism experiments.There is a correlation between CYP3A65 and upstream nuclear receptor PXR and AHR2 mRNA expression.Zebrafish liver microsomes can metabolize testosterone,which are substrates for human CYP3A4.This study preliminarily verified the applicability of zebrafish as a drug metabolism model.