Effects Of Intermittent Hypoxia On The Activity Of Hepatic Cytochrome P450 1A2 (CYP1A2) And The Metabolism Of Relevant Drugs

Background: Hepatic cytochrome P450(CYP)is a significant enzyme for human drug metabolism.It can be influenced by several factors.Hypoxia is one of the most significant ones.Previous experimental studies showed that chronic or acute sustained hypoxia can influence the activity of various CYP isoforms and the bio-metabolism of relevant drugs.Clinical studies also found that the bio-metabolism of drugs in patients with some diseases was also affected by chronic hypoxia.The drug dose should be regulated in such patients.Obstructive sleep apnea(OSA)syndrome is a common medical condition which presenting as snoring,apnea,or extreme daytime sleepiness.The incidences of hypertension and cardiovascular diseases in OSA population are obviously high.Two of the pathophysiological hallmarks of OSA are intermittent hypoxia(IH)and sleep fragmentation.Evidence showed that IH contributes to oxidative stress,system inflammation,cell apoptosis,and endothelial injury.Our previous animal study demonstrated that IH can lead to changes in hepatic ultrastructure.However,it is unclear whether IH can influence the activity of hepatic CYPs isoforms and the metabolism of relevant drugs.Purpose: This study created an IH mouse model mimicking OSA and figured out whether IH influences the activity of various isoforms of hepatic CYPs.We confirmed those findings in an IH cell model;Drugs concentration which metabolizing by the different expression CYPs were also detected in the supernatants.The underlying molecular mechanisms of IH accelerating changes of hepatic CYPs were also analyzed in vitro.Furthermore,an IH New Zealand rabbit model was established;serum drug concentration which metabolized through different expression CYPs was detected.Weaim to confirm the influence of IH on hepatic CYPs activity and relevant drugs metabolism via the studies evidence from mouse,cell and rabbit,and to provide experimental evidence for the clinician to figure out whether the drug doses should be adjusted or not in OSA patients.Materials and Methods: 1.A total of 24 male C57BL/6J mice were assigned to normoxia(CTL)group and IH group of 12 mice per group.The mice in the IH group were exposed to IH condition for 12 weeks.After sacrificed,the blood of mice was obtained for alanine transaminase(ALT)and aspartate transaminase(AST)detection.The liver was isolated and observed by a microscope.Hepatic CYPs m RNA(Abcb1a,CYP1A2,CYP2A5,CYP2C29,CYP2E1,CYP3A11,and CYP3A25)were detected by real time-polymerase chain reaction(RT-PCR).The CYP1A2 protein expression levels from hepatic homogenates and microsome were analyzed by western blot.NF-?B was also assessed by western blot.The CYP1A2 expression levels from liver tissue were detected by immunochemistry.2.(1)Human LO-2 hepatic cells were used and divided into the normoxia(CTL)group and IH group.The LO-2 hepatic cell models of IH were established.The cells or culture supernatants were collected from the CTL and IH groups at baseline,24 h,and 48 h.The cellular activity was detected.The differences in cell apoptosis between two groups were analyzed by flow cytology.CYP1A2 m RNA was detected by the RT-PCR.The different expression of CYP1A2 protein between groups was detected by western blot and flow cytology.(2)Aminophylline and warfarin were added into the cell culture supernatants.The supernatants in the CTL and IH groups were collected for the analysis and comparison of aminophylline and warfarin concentrations via the high-performance liquid chromatography(HPLC).3.(1)Human LO-2 hepatic cells were exposed to IH or normoxia condition.The following reagents were added to the supernatants: HIF-1? inhibitor(YC-1),NF-?B inhibitor(SC75741),Ah R inhibitor(SR1),and Ah R nature ligand(NOR).Cells were divided into 13 groups according to the abovementioned experimental condition,and the experiment period lasts for 48 h.Cells from each group were collected for analysis ofCYP1A2,HIF-1?,NF-?B,Ah R,and ARNT in both m RNA and protein levels via the RT-PCR and western blot,respectively.(2)The cells were clarified to normoxia,IH,IH+YC-1+NOR,and IH+SC75741+NOR groups according to the results of part(1);Aminophylline was also added to the supernatants.After 48 h of the experiment,the supernatants of all cells were collected for detection of aminophylline concentration via the HPLC.(3)In order to verify whether IH regulates CYP1A2 gene expression through the transcription factor Ah R and to figure out the Ah R enrichment to CYP1A2 gene promoter under different conditions,the following experiment were conducted: Cells were exposed to IH or normoxia condition and divided into 7 groups after being added with the following reagents: YC-1,SC75741,NOR,and SR1.The Ah R relative enrichment to CYP1A2 gene promoter sequence was detected and compared between groups via chromatin immunoprecipitation(Ch IP)assay.4.A total of 20 New Zealand rabbits were randomly assigned to normoxia and IH groups of 10 rabbits per group.Rabbits in each group were administrated aminophylline(n=5)or warfarin(n=5).Rabbits in the IH group were exposed to the IH environment.Excepted of the IH exposure,the living and feeding conditions of rabbits in both groups were similar.The period of the IH was 5 weeks.Aminophylline or warfarin was given via intravenous injection or oral administration at the end of the experiment.The blood from each rabbit was drawn at a series of time points for the analysis of aminophylline and warfarin concentrations via the HPLC.After sacrificed the rabbits at the end of the IH exposure,the livers were isolated for the CYP1A2 protein detection via western blot and immunochemistry.Results: 1.After 12 weeks of the IH exposure,there were no differences in serum ALT and AST values between the CTL and IH groups(p>0.05).No significant changes in liver structures were observed under the examination of the microscope.The m RNAs levels of hepatic CYP isoforms,namely Abcb1 a,CYP2A5,CYP2C29,CYP2E1,CYP3A11,and CYP3A25,were no differences between groups,while the CYP1A2 m RNA levels were significantly lower in the IH group than those of the CTL group(p<0.05).Western blot results showed that the CYP1A2 protein levels of hepatic homogenates andmicrosomes were lower in the IH group than those of the CTL group(all p<0.05).The NF-?B was also increased in the IH group(p<0.01).There was a similar finding of the CYP1A2 expression in liver tissue between the two groups via immunochemical examination(p<0.05).2.(1)there was no difference of hepatic cellular activity between the CTL and IH groups at 24 h.After 48 h of the IH exposure,however,the cellular activity of the IH group was significantly lower than that of the CTL group.The flow cytology results demonstrated that in comparison with the CTL group,the cellular apoptotic levels were increased in the IH group after the IH exposure for 48 h.When compared with the CTL group at 48 h,both the CYP1A2 m RNA(RT-PCR)and protein(western blot and flow cytology)levels were decreased in the IH group(all p<0.05);the similar findings were failed to be found between group after the IH exposure for 24 h.(2)At 24 h,the differences of aminophylline and warfarin concentrations at the cell culture supernatants did not reach statistical significance(p>0.05).However,at 48 h,there was significantly lower aminophylline concentration in the IH group than that of the CTL group(p<0.05);meanwhile,the warfarin concentration between the two groups was no significant difference(p>0.05).3.(1)The IH inhibits the m RNA and protein expressions of CYP1A2.After adding YC-1,SC75741,NOR,YC-1+NOR,and SC75741+NOR,the CYP1A2 m RNA expression increased under the IH condition.The CYP1A2 protein levels,however,only increased at the YC-1+NOR and SC75741+NOR groups under the IH condition(p<0.001 when compared with the solitary IH group).The CYP1A2 m RNA and protein expressions were similar between groups under normoxia condition.(2)The mean aminophylline concentration was higher in the solitary IH group than that of the normoxia group(p<0.05).Compared with the solitary IH group,decreased aminophylline concentration were found in both the YC-1+NOR and SC75741+NOR groups under the IH condition(all p<0.05).(3)The results of Ch IP with Ah R antibody from RT-PCR and agarose electrophoresis showed that compared with normoxia group,the amount of CYP1A2 promoter was decreased under the IH condition,and the difference reached statistical significance(p<0.01).Under the IH condition,theCYP1A2 promoter amount was increased after adding YC-1,SC75741,SR1,NOR,or the combination(all p<0.05).4.Western blot and immunochemical results showed that there was significant low CYP1A2 expression in the IH group than that of the CTL group after 5 weeks of the IH exposure.After one dose of aminophylline administration,rabbits in the IH group had higher serum aminophylline concentrations than those of the CTL group at several time points(all p<0.05).The pharmacokinetic values of aminophylline,T1/2(prolong 1.26 hours in the IH group),AUC0-t,and AUC0-?,were higher in the IH group than in the CTL group(all p<0.001).Regarding warfarin,there were no statistic differences in serum drug concentrations at different time points,T1/2,AUC0-t,and AUC0-? between the CTL and IH groups(all p>0.05).Conclusions: 1.The IH selectively induces the decreased expression of hepatic CYP1A2 m RNA and protein levels in a mouse model.2.Decreased CYP1A2 expression and increased aminophylline concentrations were detections in LO-2 cells after the IH exposure.3.Molecular mechanism study confirmed that HIF-1? and NF-?B influence the expression of CYP1A2 gene by interrupting the transcription factor-Ah R.4.IH exposure contributes to increased aminophylline concentrations and a longer half-life in a rabbit model.