Age-Related Changes In Hepatic Drug Metabolizing Enzymes In Wild-type And Breast Cancer Resistance Protein 1 Knockout(Bcrp1^-/-) FVB Mice

Background and ObjectivesMost drugs and endogenous undergo metabolic biotransformation by drug metabolizing enzymes(DMEs).Morever,the feature of DMEs are not only regulated with xenobiotic but also endogenous factors.Many studies revealed that DMEs showed different activities and expression levels in different periods of age.Because of the ethical reciew of human sample,it is contributed to the rise of animal model,which provide a good platform to research the mechanism of drug metabolism.Wild type FVB(WT FVB)mice are commonly used to study the role of DMEs on pharmacokinetic,which the optimal period of age of FVB mice for experiment hasn’t study clearly.In addition,many studies have revealed the interplay between breast cancer resistance protein(BCRP/Bcrp)and DMEs for specific compounds.Moreover,the FVB mice are used extensively in transgenic research especially for efflux transporters knockout FVB mice.And there were also few reports that Bcrpl knockout wether it would affect the properties of DMEs.Therefore,this study revealed that the alteration of DMEs affected by age and knockout Bcrpl.According to the above studies,we comprehensively investigated the development regulations of activities and expression of seven isoforms(Cyp3a11,Cyp2d22,Cyp2e1,Cypla2,Ugtla1,Ugtla6a,Ugtla9)in FVB and Bcrpl-/-mice,which are considered to be the ortholog of DMEs of humans.Through our research could find a period of maximally expression for the study.And we also demonstrated the role of BCRP at each period to do further research.MethodsWe investigated the activities and expression of seven isoforms in male FVB and Bcrp1-/-mice aged newborn(1 day),1 and 3 weeks(weaning period),6 and 9 weeks(puberty),17 and 27weeks(adulthood),42 weeks(reproductive senescence),52 weeks(old age).We used probe substrates method in activity assay,isotope label-free UHPLC-MS/MS in protein expression and Rea-time PCR in mRNA expression.We also showed the turnover number(TON)to indicate the catalytic capacity of enzyme and the correlations between activities,TON,protein and mRNA levels.Results1 Changes in activities of seven DMEs with increasing age in FVB mice and Bcrp1-/-mice1.1 In FVB mice,the activities of Cyp3a11,Cyp2d22 and Cye2el depicted maximum metabolic rate in weaning period and Ugts matured in the period of puberty.1.2 In Bcrp1-/-mice,the activities of Cyp3a11,Cyp2d22 and Cye2el also depicted maximum metabolic rate in weaning period and Ugts matured in the period of puberty.1.3 The activity of Cyp3a11 decreased 61%in newborn Bcrp1-/-mice.The activity of Cyp2e1 in newborn,and in 9 and 17 weeks increased by 1.61-,2.11-and 2.36-fold respectively,compared with that of WT FVB mice.On the contrary,in Bcrp1-/-mice,the activity of Cyp2d22 from newborn to 9 weeks decreased by 82.5%,53.5%,37.1%,62.9%and 54.3%,respectively.The activities of Ugtlal and Ugtla6a in 9 weeks decreased by 50-52%,and Ugt1a6a in 6 weeks increased by 2.18-fold.And Ugt1a9 in newborn and lweek increased by 1.19 and 1.56-fold,respectively.2 Changes in protein expression of seven DMEs with increasing age in FVB and Bcrp1-/-mice2.1 In FVB mice,the protein expression mat ured in puberty in Cyp3a11,Cyp2d22,,Cyp2e1,Cypla2,Ugtlal,and Ugtla6a,but the protein levels of Cyp2el and Ugt1a9 increased with age.2.2 In Bcrp1-/-mice,Cyp3a11 showed maximum expression levels in 3 weeks.The preoti n levels of Cyp2d22 and Cypla2 increased with ages.However,Utla1 Showed maximum expression levels in 1 week.2.3 The protein expression of Cyp3a11 in 6 weeks decreased by 51%compared with that of FVB mice.The protein expression of Cypla2 in 9 weeks decreased by 73.5%,and increased by 1.20-foldin 52 weeks.We found that most ratios were in the range of 0.5-2.The protein levels of Ugtlal in 6,9,and 26 weeks decreased by 59.2%,84.7,and 56.3%,respectively.3 Changes in mRNA expression of four Cyps and three Ugts enzymes with increasing age in FVB and Bcrp1-/-mice3.1 In FVB mice,the mRNA expression of Cyp3a11,Cyp2e1,Cypla2 and Ugtla9 gradually increased expression to the maximum in 17 weeks.And the mRNA expression of Ugtlal showed maximum in 3weeks and decreased with ages.However,the mRNA expression of Cyp2d22 and Ugtla6a showed different development with increasing ages.3.2 In Bcrp1-/-mice,the mRNA expression level of Cyp3a11,Cyp2e1,Cypla2,Ugtlal,Ugtla6a and Ugtla9 gradually increased to 17 weeks to be maximized.And the Cyp2d22 showed maximum in 3 weeks.ConclusionIn conclusion,we demonstrated the regulation of seven DMEs with increasing ages.And we also showed the role of Bcrp at each period.We found the activities of Ugts matured in puberty both in FVB and Bcrp1-/-mice.And it is surprised that the activities of Cyps(except Cypla2)showed maximum metabolic rate in weaning period.The protein expression also matured in puberty in all seven isoforms in FVB mice.The mRNA expression matured in 17weeks in Cyp3a11,Cyp2el,Cyp1a2,Ugtla6a and Ugtla9.These results revealed the age-related changes in hepatic DMEs and would lay the foundation for the research on interplay between BCRP and enzymes.