| ObjectiveThe study aims to investigate the effects of traditional Chinese medicine containing arsenic and mercury on organic anion transporters (Oatl, Oat3), the cytochrome 450 members (Cyp3a, Cyp2e1) and P-glycoprotein (P-gp) associated with first pass effect in mice.MethodsNIH mice were randomly divided into pure water group, CMC group, probenecid (50 mg·kg-1) group, high and low dosages of realgar group (60 mg·kg-1 and 15 mg·kg-1), grinding realgar group (60 mg·kg-1 and 15 mg·kg-1), cinnabar group (120 mg·kg-1 and 30 mg·kg-1) and HgCl2 group (0.2 mg·kg-1 and 0.05 mg·kg-1). They were orally treated with investigational samples two times a day for continuous five days. On the experiment day, gavaged the last administration of investigational samples, all mice were i.v. /rara-aminohippuric acid (PAH) at a dose of 30 mg·kg-1 after 60 minutes. The blood samples were collected at 1.0,2.5,5.0,7.5,10,20,30 min with euthanization and then the kidneys were quickly removed.NIH mice were randomly divided into pure water group, CMC group, rifampicin group (40 mg·kg-1), ketoconazole group (24 mg·kg-1), high and low dosages of realgar group (60,15 mg·kg-1), grinding realgar group (60,15 mg·kg-1), cinnabar group (120,30 mg·kg-1) and HgCl2 group (0.2,0.05 mg·kg-1). They were orally treated with investigational samples two times a day for continuous five days. On the experiment day, gavaged the last administration of investigational samples, all mice were i.g. acetaminophen (APAP) at a dose of 20 mg·kg-1 after 60 minutes. The blood samples were collected at 5,15,30,45,60, 75,90,120 min with euthanization and then the liver and intestine were quickly removed.The concentrations of PAH in sera and renal homogenates were quantified by probe drug testing method. Pharmacokinetic analysis of PAH in sera was done by pharmacokinetics software (DAS 2.0). Homogenized kidneys were used for the PAH accumulation and PAH uptake tests in renal slices. The activities of Cyp3a in liver and intestine were detected by erythromycin and aminopyrine. The activities of Cyp2e1 in liver were detected by aniline hydrochloride. APAP concentration in sera and hepatic homogenate was detected by probe drug. Pharmacokinetic analysis of APAP in sera was done by DAS 2.0. The activities of P-gp coupled ATPase in intestinal homogenates were detected by minimum ATP enzyme test kit. Protein concentrations were quantified by Folin-phenol. The expression of Oat1, Oat3, Cyp3al1, Cyp2e1 and Abcb1b mRNA was estimated by real-time PCR.ResultsThe results of mOats uptake activity assay:compared with control group, pharmacokinetic parameters were significantly changed by both high and low dosage of investigational samples:t1/2β prolonged, VdT decreased, CLT reduced, AUC0-30min increased (P<0.05); The PAH uptake in renal slices was remarkable reduced by investigational samples (P<0.05); The PAH ratios of renal AUC1-20min and serous AUC1-20min were significantly reduced by investigational samples (P<0.05).The results of liver and intestinal microsomal assay:compared with control group, the liver and intestinal microsomal Cyp3a were significantly induced by the investigational samples (P<0.05), when both erythromycin and aminopyrine were as the probe drugs. Liver microsomal Cyp2el activities were significantly induced by them (P<0.05), when aniline was used as the probe drugs.The results of P-gp efflux activity assay:compared with control group, major pharmacokinetic parameters of APAP in sera were significantly changed by both high and low dosage of investigational samples:Vdx decreased, CLT reduced, AUC5-120min increased (P<0.05). However, the t1/2(absoiption) of the high dosage cinnabar and HgCl2 were prolonged; Liver APAP accumulations in investigational groups were significantly increased (P<0.05); The P-gp coupled ATPase activities were reduced by investigational samples significantly (P<0.05).The results of RT-PCR assay:compared with control group, Cyp3a11 and Cyp2el mRNA expression in the liver could be induced by investigational samples significantly (P<0.05). However, the Oatl, Oat3 mRNA expression in kidney and the Abcblb mRNA expression in intestine could be inhibited by investigational samples significantly (P<0.05).The aboved experiment showed that there were no significant difference between the CMC group and pure water group.ConclusionTraditional Chinese medicine containing arsenic and mercury (realgar and cinnabar) probably induced kidney damage through inhibiting OAT1 and OAT3. The CYP3A and CYP2E1 activities can be induced obviously and the P-gp in mice liver and intestine was inhibited strongly, which regulated the first-pass effect. |
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