| AimsThe aim of this paper is to study the transporters and bile acids(BAs) based mechanisms of isoniazid(INH)- and acetaminophen(APAP)- induced Hepatotoxicity, the hepatic epression of bile acids(BAs) transporter and the plasma, bile and liver concentration of BAs of rats were investigated.Methods1. A simple and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS) method was developed and applied for simultaneous determination of 15 BAs in liver, bile, urine and plasma of liver.2. A total of 14 male Wistar rats were randomly divided into INH group and control group. Rats in INH group were orally administered with INH(100 mg/kg/d) for successive 60 days, and rats in control group were orally treated with saline. After drug administration, all the rats were fasted overnight, anesthetized and the bile duct was cannulated. After 60 days, all the rats were selected and fasted overnight. Bile was collected for 10 min from the cannula for LC-MS/MS analysis. After that, blood samples(5 mL) were collected though abdominal aortic with heparinized syringes and centrifuged, the plasma was collected for biochemical and LC-MS/MS analysis. Liver tissue was collected, one part was fixed in 10% formaldehyde for histological evaluation, and the other part was kept in-80 oC for BAs detection and Western blot analysis on protein expression of hepatic transporters.3. A total of 14 male Wistar rats were randomly divided into APAP group and control group. Rats in APAP group were orally treated with APAP(500 mg/kg), and rats in control group were administered with saline. Rats were all given a single treatment. At 1 day post treatment, rats were fasted overnight, anesthetized and the bile duct was cannulated. Bile was collected for 10 min from the cannula for LC-MS/MS analysis. Blood samples(5 mL) were collected though the abdominal aortic with heparinized syringes and centrifuged, the plasma was collected for biochemical and LC-MS/MS analysis. Liver tissue was collected, one part was fixed in 10% formaldehyde for histological evaluation, and the other part was kept in-80 oC for BAs detection and Western blot analysis on protein expression of hepatic transporters.Results1. The results showed that higher peak responses and shorter analysis time can be achieved when using Agilent HC-C18 column. The total run time for BAs determination was 21 min.This method was valid and sensitive with a large dynamic range(from 4.30 ng/mL to 200 μg/mL for various analytes). Intra-day(inter-day) accuracy were in the range of 80%~104%, and precision were all less than 15% obtained in rat liver, bile, urine and plasma. Extraction recoveries for all analytes in the 5 matrices were all exceed 80%. The analytes in rat plasma, liver, bile and urine showed good stability for all the analytes over four storage condition.2. Compared to the normal rats, plasma total bilirubin(TBIL) and alkaline phosphatase(ALP) activity in INH-treated rats were all significant elevation(P<0.05); plasma level of chenodeoxycholic acid(CDCA), tauro-chenodeoxycholic acid(TCDCA), tauro-ursodeoxycholic acid(TUDCA), ursodeoxycholic acid(UDCA), tauro-deoxycholic acid(TDCA), tauro-cholic acid(TCA) and lithocholic acid(LCA) were all significantly increased(P<0.05); while bile level of TUDCA, UDCA, TDCA and deoxycholic acid(DCA) were markedly decreased(P<0.05); and hepatic level of cholic acid(CA), TCDCA, glyco-chenodeoxycholic acid(GCDCA), glyco-cholic acid(GCA) and glyco-lithocholic acid(GLCA) were significantly increased. Moreover, the hepatic expression of and multidrug resistance-associated protein 2(Mrp2), multidrug resistance-associated protein 4(Mrp4), and organic anion transporter(Oatp2) were all significantly decreased in INH-treated rats when compared to that of the normal rats(P<0.05), no significant difference was found in the protein expression of bile salt export pump(Bsep) and polypedtides Na+/taurocholate Cotransporting Polypeptide(Ntcp)(P>0.05).3. Compared to the normal rats, plasma alanine aminotransterase(ALT) and aspartate aminotransferase(AST) activity in APAP-treated rats were all significant elevated(P<0.05); plasma level of DCA, LCA, TDCA, glyco-deoxycholic acid( GDCA) and GLCA were all marked increased(P<0.05); and bile level of CDCA, GDCA, DCA and GLCA were all significantly increased, but bile TUDCA were significantly decreased(P<0.05); and hepatic level of GDCA, DCA, GLCA and LCA were all significantly increased(P<0.05). Moreover, compared to the normal rats, the hepatic expression of Mrp4 and Oatp2 were all significantly decreased in APAP-treated rats, while Cyp7a1 expression was significantly increased(P<0.05), no significant difference was found in the protein expression of Mrp2, Bsep and Ntcp(P>0.05).Conclusions1. The established method provided high sensitivity and specificity as well as relatively short run-time for quantitative determination of 15 individual BA species in rat plasma, liver, bile and urine samples. In addition, the validated method showed acceptable precision and accuracy for simultaneous quantification of BAs.2. INH-induced liver injury may be resulted from the accumulation of BAs in the plasma and liver, the underlying mechanisms may have correlation with down–regulated protein expression of Mrp2, Mrp4 and Oatp2 in the liver treatment with INH for rats.3. APAP-induced liver injury may be resulted from the accumulation of BAs in the plasma, bile, and liver, and the underlying mechanisms may have correlation with down–regulated protein expression of Mrp4 and Oatp2 as well as up–regulated protein expression of Cyp7a1 in the liver. |
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