The Effects Of Cytochrome P450 Isozymes On Aristolochic Acid Renal Toxicity

Since the report from Belgium's Vanherweghem that taking the weight loss drug containing aristolochic acid composition led to kidney damage and cancer in 1993,aristolochic acid renal toxicity problems caused by chinese medicine containing aristolochic acid has become hotspot in the domestic and foreign medical research fields.To avoid such adverse drug reactions from happening again,the FDA has already banned products containing aristolochic acid to enter the american market and also has prohibited the use of medicine containing aristolochic acid confused species since 2001.Many countries in the international also has banned on imports of chinese herbal medicines containing aristolochic acid.The commissioner of china has issued a document to abolish the medicinal standards of aristolochia manshuriensis,Green costas,widely Menispermaceae and to restrict the use of chinese medicine and related agents containing aristolochic acid component in 2003,2004.However,in our country, there are still a variety of chinese medicines containing aristolochic acid using in clinical,such as aristolochic,Tianxian rattan,cinnabar-lian and so on.Although the content of aristolochic acid in these varieties is low,the security dose of aristolochic acid is still unclear at present,therefore,it is very important to study the toxicity mechanism of aristolochic acid in-depth and to understand the factors which impacts the toxicity of aristolochic acid.In the so-called "herbal nephropathy" incident,the incidence rate of aristolochic acid nephropathy was 5%.This showed that different groups of people had different sensitivity to aristolochic acid.Because of the genetic polymorphism for the important drug metabolizing enzymes in human(the liver cytochrome P450 enzyme),some CYP450 isozymes of different individuals has a lot of difference,thus the ability of drug metabolism for different individual is different.Whether the differences in aristolochic acid kidney toxicity among different individuals relate to the differences in CYP450 isozymes or not,which will be worth to study in-depth.Furthermore,there are many drugs that can affect the activity of some CYP450 isozymes.Moreover,the combinations of multi-drugs are widespread in clinical,in particular,the combination of chemical medicine with chinese medicine are used more and more.Whether the kidney toxicity of aristolochic acid will change when chinese medicine containing aristolochic acid combined with those drugs which can inhibit or induce CYP450 isozymes.It is worthy of study in depth.This study funded by the Beijing Municipal Science(issue No.:7052058).ObjectiveBy using cell culture technology in vitro and and animals as experimental methods,to investigate the effects of cytochrome P450 isozymes on aristolochic acid renal toxicity and to screen the major CYP450 isozymes that impact AA renal toxicity,which will provide reference for the safety of clinical drug-using and to find ways to prevent aristolochic renal toxicity.MethodsChapter 1:CYP450 isoforms that participate in the drug metabolism of aristolochic acid were screened by CYP450 enzymes inhibitory effect in vitroWe chose several of classic inhibitors of CYP450 isoenzymes,including sodium diethyldithiocarbamate(CYP4502A6 and 2E1 inhibitors),quinidine(CYP4502D6 inhibitor),α-lipoic acid(NADPH:P450 reductase inhibitor),α-naphthoflavone(CYP4501A1 and 1A2 inhibitors),α-lipoic acid(CYP4502C inhibitor),ketoconazole(CYP3A4 inhibitor).Aristolochic acid renal toxicity was tested by using human renal epithelial cells (HK-2) as model in vitro.The metabolism environment in vivo simulated by adding the liver microsomal mixed enzymes(S9) into HK-2 cell culture system.The inhibitors were added into HK-2 cell culture system respectively to form different activity inhibition situation for CYP450 isoenzymes.The cytotoxicity of AA alone and the combination of AA with inhibitors were determined by inhibition of cell proliferation(MTT) test and LDH release test.In this experiment,we used 5×4×2 factorial experimental design to initially screen CYP450 subtypes that impacted AA renal toxicity.Chapter 2:The effects of combination of aristolochic acid with recombinant protein CYP4501A2/CYP4503A4 on AA renal cytotoxicityAccording to the preliminary screening results in chapter 1,we will further verify the effects of the purification CYP450 isozymes(CYP1A2 and CYP3A4 enzymes) on aristolochic acid renal cytotoxicity.In this experiment,we used 4×4 factorial experimental design with AA three concentrations,CYP1A2/CYP3A4 three concentrations and detected that through LDH release test.Chapter 3:The combination of aristolochic acid with different cytochrome P450(CYP450) inducers(phenobarbital,rifampicin,omeprazole) effects AA acute toxicity.Based on the experimental results in vitro,mice experiment in vivo were carried out to verify and identify the major CYP450 isozymes that effects aristolochic acid renal toxicity.we selected three CYP450 isozymes inducers—phenobarbital(PB)(CYP450 inducer),rifampicin(RFP)(CYP3A inducer),omeprazole(OM)(CYP1A inducer).206 mice were randomly divided into 20 groups,namely the control group,that groups of four AA concentration,including AA1(60 mg·kg^-1),AA2(48 mg·kg^-1), AA3(38.4 mg·kg^-1),AA4(30.72 mg·kg^-1),the PB group,the PB+AA four concentration's groups,the RFP group,the RFP+AA four concentration's groups,the OM group,the OM + AA four concentration's groups.The experiment began after the mice were fasted for 16 hours,free of drinking.For the first three days,the normal control group and the aristolochic acid group were given 0.5%sodium carboxymethyl cellulose 10 mL·kg^-1,the induction groups were given inducers as PB 40 mg·kg^-1,RFP 40 mg·kg^-1,OM 200 mg·kg^-1 respectively.The mice were fasted on the third day.On the fourth day,three hours after giving the inducers,the aristolochic acid were given and the fresh water and food were supplied.During the experiment,general conditions of mice had been observed for 14days.Weighing body weight in mice once every two days to observe drug-treated animals' weight changes.The changes in the various organs of the dead animals were observed and death records were maken.After a period of observation,the mice were weighed body weight after fasting for 16 hours.The blood was gathered for determination of CRE,UREA and the liver,stomach,kidney were removed for the calculation of organ index.ResultsChapter 1:CYP450 isoforms that participate in the drug metabolism of aristolochic acid were screened by CYP450 enzyme inhibitory effect in vitroThe experimental results showed that:adding liver microsomal enzymes(S9) into the HK-2 culture system,AA cytotoxicity on renal tubular cell had reduced to a certain extent.Compared with AA group,cell proliferation rate and the percentage changes in LDH release rate of AA + S9 group decreased,there is a significant difference between them(P＜0.05).MTT assay and LDH release found that the inhibiting effect formed by sodium diethyldithiocarbamate,quinidine,α-lipoic acid and sulfaphenazole may be not obvious on the AA cytotoxicity.However,under the circumstances of adding ketoconazole without obvious cytotoxicity into the HK-2 culture system,there was a significant difference between AA+ketoconazole+S9 group and AA+S9 group.Compared with AA+S9 group,AA+ketoconazole+S9 can significantly enhanced the proliferation inhibition of aristolochic acid on HK-2 cell(P＜0.05),the relative multiples of inhibition increased.The results showed that ketoconazole maybe influence AA cytotoxicity through inhibiting the activity of CYP3A4.At the same time,the result of this study also showed that under the circumstances of addingα-naphthoflavone with low cytotoxic effects into the HK-2 culture system,there was a significant difference between AA+α-naphthoflavone + S9 group and AA+S9 group.Compared with AA+S9 group,AA+α-naphthoflavone +S9 can significantly increased LDH release rate of aristolochic acid on HK-2 cell(P＜0.05),the relative multiple of LDH release changes increased.Experimental results indicated that:α-naphthoflavone maybe influence AA cytotoxicity through inhibiting the activity of CYP1A2.So we got preliminary screening results:CYP4503A, CYP4501A were the major CYP450 isozymes that affected the metabolic activation of AA.Chapter 2:The effects of the combination of aristolochic acid with recombinant protein CYP4501A2/CYP4503A4 on AA renal cytotoxicityThe experimental results showed that:compared with the same concentration of AA groups,the LDH release rate percentage changes of the groups that AA combined with recombinant protein CYP3A4 reduced.Compared with the same concentration of AA groups,the LDH release rate percentage changes of the groups that AA combined with recombinant protein CYP1A2(97.5 pmol·mL^-1) increased,with release relative multiples＞1,but the LDH release rate percentage changes of the groups that AA combined with recombinant protein CYP1A2(25 pmol·mL^-1)reduced,with release relative multiples＜1.The experimental results showed that:CYP3A4 and CYP1A2 were the major CYP450 isoforms that affected HK-2 cell toxicity caused by AA.CYP3A4 could reduce HK-2 cell toxicity;CYP1A2 with high activity maybe increase HK-2 cells toxicity and CYP1A2 with lower activity maybe reduce HK-2 cells toxicity.Chapter 3:The effects of the combination of aristolochic acid with cytochrome P450(CYP450) inducers(Phenobarbital,rifampicin,omeprazole) on AA acute toxicity.The determination of LD₅₀ was calculated by bliss method,the LD₅₀ of the AA alone group was 55.01 mg·kg^-1.The toxicity of the PB-induced mice significantly decreased after its taking AA.the induction of PB reduced the mortality rate in mice and delayed the death of the mice.The results suggested that the induction for CYP450 enzymes in the liver caused by PB can significantly reduce the toxicity.Compared with the LD₅₀ of AA alone group,the LD₅₀ of RFP + AA group was 33.82 mg·kg^-1,which decreased by about 38.5 percents.The results showed that the induction for CYP3A4 enzyme caused by RFP significantly enhanced the AA kidney toxicity.The LD₅₀ of AA + OM group was 56.22 mg·kg^-1.compared with the LD₅₀ of AA alone group,there was not obvious difference.The results showed that the induction for CYP1A enzyme caused by OM had no obvious effect on AA kidney toxicity.Conclusion1.the increasing activity of the total cytochrome P450 can reduce the toxicity of aristolochic acid to a certain extent.2.different cytochrome P450 enzymes have different effects on aristolochic acid renal toxicity,and the increasing activity of cytochrome P4503A4 may increase aristolochic acid toxicity.The results of this study suggested that the combination of chinese medicine containing aristolochic acid with drugs that can induce or inhibit some CYP450 enzymes maybe impact aristolochic acid toxicity.Therefore,the safety of drugs combined should be noted.Furthermore,CYP450 enzymes activity from different individuals are different,which may cause the AA sensitivity different and show differences in toxicity after taking drugs containing aristolochic acid.