Impact Of Neonatal Drug Exposure And High-Fat Diet On Hepatic Expression Of Drug-Metabolizing Enzymes And Underlying Mechanism

BackgroundDrug-metabolizing enzymes(DMEs)play key roles in the biotransformation process of endogenous and exogenous substances.The expression level and enzyme activities of DMEs are highly associated with drug efficacy and toxicity.During postnatal liver maturation,the majority of DMEs appear to follow a distinct pattern of ontogenic expression.Dose extrapolation from adults to neonates or children is not straight-forward,which presents a major clinical challenge to pediatric medication.The expression of DMEs can be influenced by physiological and pathological conditions.Mounting evidence indicates that aberrant environmental factors during early life can trigger alterations of epigenetic modifications and form "epigenetic memory",which leads to long-term changes of gene expression and have a persistent effect on health during later life.Whereas,limited information is available about the influence of early-life environmental factors on the expression of DMEs in adulthood and the ontogeny of DMEs.It is well studied that the expression of DMEs can be induced or suppressed by xenobiotics.Pregnane X receptor(PXR),an important transcription factor in the liver,is considered as "xenobiotic sensor".PXR can be activated by a large number of clinical-used drugs(including pediatric drugs),herbs or herb extracts,and environmental pollutants,which is the molecular basis of clinical drug-drug interaction.However,far less is known about the impact of PXR activation during early life on the expression of DMEs in adulthood.The previous work of our group has confirmed that epigenetic modifications,histone H3 lysine 4 and 27 trimethylations(H3K4me3 and H3K27me3,respectively),contribute to PXR-mediated induction of CYP3A4 by rifampicin.Based on the evidence,we hypothesize that exposure of PXR activator during early life can trigger the alteration of histone modifications and format epigenetic memory subsequently,which leads to altered expression of DMEs and drug sensitivity in later life.Owing to the change in diet,the prevalence of overweight and obesity,especially in child populations,has become a serious epidemic health issue in the last decade.Moreover,childhood obesity is highly associated with the family-based lifestyle and parental obesity.Evidence from a few clinical studies has shown that the in vivo activities of DMEs in the obese population are altered,which leads to decreased efficacy or increased toxicity of drugs metabolized by these enzymes.However,contradictory results on the alteration tendency were found in these studies.There is a clue indicating that the degree of obesity affects the alteration extent of DMEs,which may provide an explanation for the inconsistent results of previous studies.Whereas,far less information is available on the impact of obese levels(overweight,obesity,and severe obesity)on the expression of DMEs.Childhood obesity is highly associated with parental obesity and the family-based lifestyle.However,there is limited information on the impact of parental obese levels or family-based lifestyle on the ontogeny of DMEs.Given the crucial roles of transcription factors in the regulation of DMEs as well as in many physiological and pathological conditions,we hypothesize that obese levels influence the hepatic expression and ontogeny of DMEs by altering the expression of key transcription factors in adult and child populations.To test the hypotheses,the current study was divided into two parts:(1)Impact of Pxr agonist exposure during early life on the drug metabolism in adult mice and underling epigenetic mechanism;(2)Consequence of high-fat diet feeding durations on the hepatic expression of drug-metabolizing enzymes and transcription factors in adult and offspring mice.This study not only provides a new explanation for the inter-individual expression of drug-metabolizing enzymes but also provides more theoretical information for optimizing drug dosage according to the medication history and obese levels of individuals.Part 1 Epigenetic memory is involved in the persistent alterations of drug metabolism in adult mice due to PCN-activated Pxr during early lifeObjectiveThis study is aimed to investigate the impact of Pxr agonists exposure during early life on the hepatic expression of drug-processing genes(DPGs),including DMEs and transporters,and drug efficacy in adult mice as well as the underlying mechanism of histone modifications.This knowledge may prompt understanding of the long-term effect of drug administration during early life on drug metabolism and provide more theoretical information for precision medication.Methods1.Impacts of Pxr agonist exposure during early life on the hepatic expression of DPGs in adulthoodC57BL/6 mice were used as an animal model and pregnenolone-16α-carbonitrile(PCN),a prototype agonist of mouse Pxr,was served as a typical xenobiotic.To access the role of PCN treatment dose,mice at age days 5-8 were administered with different doses of PCN(50,100,150,or 200 mg/kg/day)or corn oil(vehicle control),once daily for 4 constitutive days intraperitoneally(i.p.);To study the effect of PCN exposure age,mice were injected with 200 mg/kg/day PCN or corn oil from different ages(postnatal day 5,10,15,or 25),once daily for 4 constitutive days.All the mice were sacrificed atpostnatal day 60 and liver tissues were collected.The mRNA expression of Pxr and its targeted DPGs,including drug-metabolizing enzymes Cyp3a11,Cyp2b10,Cyp1a2,Cyp2a4,Cyp2b13,Sult2al,and Ugtlal,as well as drug transporters Abcc4,Oatp1a4,and Papss2 was determined by a quantitative real-time PCR(qRT-PCR)method.The protein expression of Cyp3a11 was detected by the Western blot assay.2.Effects of PCN exposure during early life on the drug sensitivity in adulthoodSixty-day mice which were administrated with 200 mg/kg/day PCN or corn oil at days 5-8 were referred to as PCN(5-8d)or NC(5-8d)mice,respectively.Primary mouse hepatocytes(PMHs)from female PCN(5-8d)mice and NC(5-8d)mice were isolated by a collagenase perfusion method.PMHs were treated with different doses of PCN(0.1,0.5,1,or 10 μM)or 0.1%DMSO(vehicle control)for 48 h and harvested afterward.The mRNA expression of Cyp3a11 in PMHs was determined using the qRT-PCR method.3.Chromatin Immunoprecipitation(ChIP)-qPCR analysis of histone modifications in the promoter of Cyp3a11The ChIP-qPCR analysis was conducted in liver tissues from female PCN(5-8d)mice and NC(5-8d)mice.The enrichment levels of H3K4me3 and H3K27me3 in the promoter of Cyp3a11 were determined.Results1.The dose of PCN exposure during early life contributes to the persistently changed expression of DPGs in adult mouse liverCompared with the control group,lower dose(≤100 mg/kg/day)of PCN exposure at days 5-8 led to higher expression of Cyp2b10,Cyp2a4,Cyp2b13,Sult2a1 and Oatpla4 in mRNA level,and high dose(≥150 mg/kg/day)resulted in increased mRNA expression of Pxr,Cyp3a11,Cyp1a2,Cyp2a4,Ugt1a1,Abcc4,and Oatpla4 as well as the protein expression of Cyp3a11 in adult mouse liver.In addition,gender-different effects were observed.2.Age of PCN exposure during early life contributes to the persistently changed expression of DPGs in adult mouse liverCompared with the control groups,PCN treatment before 15-day-old mice resulted in persistently altered mRNA expression of Pxr and its target genes,including Cyp3a11,Cyp2b10,Cyp2a4,Cyp2b13,Ugt1a1,Abcc4,Oatpla4,and Papss2.Moreover,elevated expression of Pxr,Cyp3a11,Sult2a1,Ugt1a1,and Papss2 was found in the liver of day 60 mice which were exposed to PCN after postnatal 15-day age.Gender different effects were observed as well.3.PCN exposure during early life attenuated in vitro sensitivity to PCN in adultDose-dependent induction of Cyp3a11 mRNA expression by PCN was observed in PMHs isolated from both PCN(5-8d)mice and NC(5-8d)mice.PMHs from NC(5-8d)mice were sensitive to as low as 0.5 μM PCN with a 3.6-fold induction of Cyp3a11 compared with the control group.Whereas,only the highest concentration(10 μM))of PCN treatment induced the mRNA expression of Cyp3al 1 in the PMHs isolated from PCN(5-8d)mice.4.Histone modifications contribute to the persistently altered expression of Cyp3a11 following Pxr activation during early lifeIn the liver of adult mice with PCN exposure at days 5-8,statistically increased enrichment levels of H3K4me3(P<0.05),an active epigenetic mark,and a trend of decreased enrichment levels of H3K27me3(P>0.05),a gene silencing mark,in the promoter of Cyp3a11 were observed,compared with the control group.ConclusionsActivation of Pxr via transient PCN exposure during early life can persistently alter the expression of DPGs and attuned drug sensitivity in adult mice,in which treatment dose and exposure age are two key factors and histone modifications can be an underlying mechanism.Part 2 Impact of high-fat diet on the hepatic expression of drug-metabolizing enzymes and transcription factors in adult and offspring miceObjectiveThe goal of this work is to investigate the consequence of high-fat diet feeding durations and family-based HFD lifestyle on hepatic expression of DMEs and transcription factors in adult and offspring mice.This work will provide reference to the rational use of drugs in overweight and obesity populations.Methods1.Mouse modelMale and female C57BL/6 mice(4-5 weeks old)were maintained on either a low-fat diet(LFD)containing 10 kcal%from fat or a high-fat diet(HFD)containing 60 kcal%from fat.After fed for different durations(4,8,or 18 weeks),some mice were sacrificed by carbon dioxide asphyxiation and liver tissues were collected to assess the consequence of obese levels on hepatic expression of drug-metabolizing enzymes and transcription factors.The remaining mice were set up as breeding pairs to obtain offspring mice;offspring mice were sacrificed at different ages(postnatal days 5-60)and liver tissues were collected to study the impact of family-based HFD lifestyle and obese levels of parental mice on the ontogeny of drug-metabolizing enzymes in offspring mice.2.Determination of gene expression levelsThe hepatic mRNA expression of nine DMEs,including Cyp1a1,Cyp1a2,Cyp2b10,Cyp2c29,Cyp2e1,Cyp3a11,Cyp3a16,Ugtlal,and Sult1a1,and five transcription receptors,including Car,Pxr,Hnf4a,Ppara,and Ahr was determined by a qRT-PCR method.3.Enzyme activitiesEnzyme activities of Cypla2,Cyp2e1,Cyp2b10,and Cyp3a11 were determined by an UPLC-MS method.ResultsIn this study,mice maintained with a LFD or HFD for 4,8,and 18 weeks were referred to as 4-LA or 4-HA,8-LA or 8-HA,and 18-LA or 18-HA mice,respectively.Offspring of these mice were referred to as O-4-LA or O-4-HA,O-8-LA or O-8-HA,and O-18-LA or O-18-HA mice,respectively.1.Consequences of high-fat diet feeding durations on hepatic expression of drug-metabolizing enzymes and transcription factors in adult miceWith the prolongation of feeding duration,the differences in body weight gain between HFD and the related LFD groups were greater indicating the obese levels of mice increased.More severe lipid accumulation was observed in the 18-HA group than the 4-HA group.In addition,male mice were more sensitive to the HFD than female mice.HFD consumption had an inductive effect on the mRNA expression of DMEs in the mouse liver,which was influenced by feeding duration and was gene-and gender-specific.Compared with the corresponding LFD groups,in male mice,the mRNA expression of Cyp1a2 and Cyp2e1 was only increased in 4-HA group,while Cyp1a1 in 18-HA groups and Cyp2b10 in all HFD groups.In female mice,the mRNA expression of Cyp1a1 in the 4-HA group as well as Cyp1a1,Cyp1a2,and Cyp2b10 in the 8-HA group was higher than the corresponding LFD groups.However,no statistical difference in the expression of DMEs was observed between the 18-HA group and the 18-LA group in females.Feeding durations were found to affect the inductive effect of HFD on the hepatic expression of transcription factors,which effect was gender-and gene-specific as well.The mRNA expression of Car and Ppara was only higher in male HFD groups,while Ahr in female HFD groups compared with the corresponding LFD groups.In males,consumption with a HFD for 4 weeks resulted in elevated expression of Hnf4α,whereas 18-week HFD feeding decreased its expression compared with the corresponding LFD groups.In females,the mRNA expression of Hnf4α and Ppara was higher in the 8-HA group than in the 8-LA group.2.Consequences of family-based HFD consumption on the ontogeny of drug-metabolizing enzymes and transcription factors in the liver of offspring miceFamily-based HFD consumption resulted in heavier body weight and liver weight of 5-day-old O-8-HA mice as well as larger body weight of 60-day-old male and 20-day-old female O-8-HA mice.However,the liver weight of 30-day O-8-HA mice was lighter than 30-day O-8-LA mice.Family-based HFD lifestyle led to abnormal ontogeny of Cypla1,Cyp1a2,Cyp2e1,Ugt1a1,and Sultlal in the liver of offspring mice.Compared with the corresponding LFD groups,the mRNA expression of Cypla1,Cyp1a2,Cyp2e1,and Ugtla1 was increased in the liver of O-8-HA mice at age day 30 and/or day 60.However,the mRNA expression of Cyp1a1 in the 5-day O-8-HA group was lower than in the corresponding LFD groups.Decreased mRNA expression of Sultlal in 15-day female and 30-day male O-8-HA groups was also observed in comparison with the corresponding LFD groups.Moreover,family-based HFD lifestyle changed the ontogenic expression pattern of Cypla2 and Cyp2e1 changed from an adolescent-enriched pattern to an adult-enriched pattern in both male and female mice.There was no effect of family-based HFD lifestyle on the ontogeny of Cyp2b10,Cyp2c29,and Cyp3a11 in the liver of offspring mice.Family-based HFD lifestyle resulted in higher mRNA expression of Car,Pxr,Pparα,Hnf4a,and Ahr in the liver of 30-day and/or 60-day O-8-HA mice,which was consistent with elevated expression of Cypla1,Cyp1a2,Cyp2e1,and Ugtla1 in these groups.3.Consequences of high-fat diet feeding durations of parental mice on the hepatic expression of drug-metabolizing enzymes and transcription factors in offspring miceIn the three HFD feeding duration groups,the body weight of all 60-day offspring mice was higher than the corresponding LFD groups,while the liver weight was changed.Compared to the related LFD groups,the mRNA expression of Cypla1,Cyp1a2,Cyp2e1,and Ugt1a1 was increased in the 60-day O-8-HA groups,whereas no changed in the 60-day O-4-HA and O-18-HA groups.Family-based HFD lifestyle had no effects on the ontogenic expression of Cyp2b10,Cyp2c29,and Cyp3a11 in the O-8-HA mice.However,decreased mRNA expression of Cyp2b10,Cyp2c29,Cyp3a11,and Sultlal was observed in the O-4-HA mice compared with the related LFD groups.The expression of Car in 60-day O-8-HA groups was 2.5-fold higher than the related LFD groups,whereas no changes in the 60-day O-4-HA and O-18-HA group compared with the corresponding LFD groups.In the three HFD feeding duration groups,the mRNA expression of Pxr and Ppara in the liver of 60-day offspring mice was higher than the related LFD groups.For the 60-day offspring mice,family-based HFD consumption did not affect the expression of Hnf4α in the O-8-HA and O-4-HA groups but,induced its expression on the O-18-HA in comparison with the related LFD groups.Family-based HFD lifestyle induced the expression of Ahr in the liver of O-8-HA mice,whereas suppressed its expression in the liver of 20-day O-4-HA male mice.ConclusionsHigh-fat diet feeding durations influence the impacts of HFD on the hepatic expression of DMEs(Cypla1,Cyp1a2,Cyp2b10,Cyp2e1,Cyp3a11,Ugt1al,and Sult1a1)in mice.Family-based HFD lifestyle and obese levels of parental mice affect the ontogeny of DMEs in the liver of offspring mice.Altered expression of DMEs by HFD lifestyle is associated with changed expression of transcription factors.