Study On The Mechanisms Of Hepatotoxicity Induced By Isoniazid And Rifampicin And The Hepatoprotective Effect Of Selective Inhibitors Of CYP450 In Mouse Hepatocytes

Objective: The present study was carried out to research the hepatocyte toxicity and the effect of isoniazid and rifampicin on the activities of CYP2E1 and CYP3A in mouse primary hepatocytes. Furthermore, the role of CYP2E1, CYP3A and their inhibitors in the occurrence and preservation of isoniazid-rifampicin induced hepatoxicity was observed.Methods:1 Isolation and culture of mouse hepatocytes Hepatocytes were isolated from mice using a two-step liver collagenase perfusion method. Mice were anesthetized and a midline laparotomy was performed. The inferior vena cava was cannulated and perfused with Hepes solutionⅠ(NaCl 160.8 mmol/L, KCl 3.15 mmol/L, Na2HPO4·12H2O 0.7 mmol/L, Hepes 33 mmol/L, pH 7.4)followed by collagenase solutionⅡ(Hepes solutionⅠwith 0.566g/L CaCl2, 0.1g/L collagenaseⅣ, pH 7.4). All solutions were kept at 37°C. After removing the liver from the mouse, the liver capsule was disrupted with a pair of forceps. The cells were suspended in Williams′E culture medium (with 10% fetal bovine serum and 100 U/mL penicillin–streptomycin) and filtered through sterile sieves (160 meshes). Hepatocytes were washed twice by centrifugation at 600r/min for 5 min, and resuspended in Williams′E culture medium. Cell count and viability were assessed by 0.4% trypan blue exclusion. Preparations showing greater than 85% were used for all experiments. The cells were seeded in collagen-coated 24-well plates at a density of 2×105 cells/mL. The plates were cultured at 37°C under an atmosphere consisting of 5% CO2 : 95% air. The medium was renewed for 24 hours by fresh Williams′E culture medium.2 Measurement of lactate dehydrogenase activity in culture media Cells was cultured in Williams′E culture medium with drugs for 48h, culture media was collected for dehydrogenase assay. The lactate dehydrogenase activity in culture media were determined with spectrophotometric method using a lactate dehydrogenase activity kit. The lactate dehydrogenase activity was calculated.3 Determination of 4-nitrophenol and midazolam concentration in culture media with HPLC-MSA Gemini C18 analyse column (2.0×50 mm, 5μm)was used with C18 guard column (3.0×4.0 mm, 5μm). Mobile phase was consist with acetile: water (60:40, v/v, containing 0.2% glacial acetic acid ) and was pumped in 0.2 mL/min. Ten milliliters sample was analysed on column. ESI ionization was used in mass detection in selected ion monitoring. The ions selected for 4-nitrophenol and midazolam were m/z [138.1]- and m/z [326.0]+ respectively.4 Grouping and drug treatment4.1 Effect of isoniazid and rifampicin on lactate dehydrogenase activity Cells were planted in 24-well plates with density of 2×105 cells per well. Cells were designated to ten groups with six in each. Blank veichle, isoniazid (150μg/mL,500μg/mL,1000μg/mL ), rifampicin(100μg/mL, 200μg/mL, 300μg/mL), rifampicin + isoniazid (100μg/mL+50μg/mL, 200μg/mL + 100μg/mL, 300μg/mL+150μg/mL) were added in culture media. After 48 hours culture, Culture media was collected for lactate dehydrogenase assay.4.2 Effect of isoniazid and rifampicin on the activity of CYP2E1 and CYP3A Cells were designated to three groups with six in each. Blank veichle, isoniazid (100μg/mL), rifampicin (200μg/mL), rifampicin + isoniazid (200μg/mL+100μg/mL) were added in culture media. After 48 hours culture, Culture media was desposed. 4-nitrophenol (200 ng/mL) and midazolam (200 ng/mL), substrates of CYP2E1 and CYP3A respectively, were added and cultured for additional 2 hours. Concentration of 4-nitrophenol and midazolam were determined with HPLC-MS. The activity of CYP2E1 and CYP3A was calculated by the decreasing of 4-nitrophenol and midazolam.4.3 Effect of sanchinoside and narigenin on isoniazid and rifampicin induced lactate dehydrogenase releasingCells were designated to eight groups with six in each. Blank veichle, rifampicin + isoniazid (200μg/mL+100μg/mL), rifampicin + isoniazid + sanchinoside (20μg/mL), rifampicin + isoniazid + sanchinoside (100μg/mL), rifampicin + isoniazid + sanchinoside (500μg/mL), rifampicin + isoniazid + narigenin (5μg/mL), rifampicin + isoniazid + narigenin (50μg/mL), rifampicin + isoniazid + narigenin (500μg/mL), were added in culture media. After 48 hours culture, Culture media was collected for lactate dehydrogenase assay.4.4 Effect of sanchinoside on the activity of CYP2E1Cells were designated to five groups with six in each. Blank veichle, rifampicin + isoniazid (200μg/mL+100μg/mL), rifampicin + isoniazid +sanchinoside (20μg/mL), rifampicin + isoniazid + sanchinoside (100μg/mL), rifampicin + isoniazid + sanchinoside(500μg/mL), were added in culture media. After 48 hours culture, Culture media was collected for the activity of CYP2E14.5 Effect of narigenin on the activity of CYP3ACells were designated to five groups with six in each. Blank veichle, rifampicin + isoniazid (200μg/mL+100μg/mL), rifampicin + isoniazid + narigenin (5μg/mL), rifampicin + isoniazid +narigenin (50μg/mL), rifampicin + isoniazid + narigenin (500μg/mL), were added in culture media. After 48 hours culture, Culture media was collected for the activity of CYP3A.5 Data and statisticsStatistics was performed with SPSS 13.0 software, and data were expressed in mean±sd. Significance between groups was analyzed by t test or ANOVA analysis with Dunnett t test; and binary variable correlation analysis. Significance was considered when P<0.05.Results:1 Effect of isoniazid and rifampicin on lactate dehydrogenase activity After 48 hours treatment with isoniazid (500μg/mL, 1000μg/mL) and rifampicin-isoniazid (200μg/mL+100μg/mL, 300μg/mL+150μg/mL), the lactate dehydrogenase activity increased significantly compared with control group (P<0.05, P<0.01). The lactate dehydrogenase activity increased significantly in rifampicin (300μg/mL) group (P<0.05).Conadministration of isoniazid and rifampicin dose dependently increased lactate dehydrogenase activity and Pearson correlation coefficient was 0.708(P<0.05).2 Effect of isoniazid and rifampicin on the activity of CYP2E1 and CYP3A Compared with control group, the activity of CYP2E1 increased significantly in isoniazid group and rifampicin-isoniazid group (P<0.05); the activity of CYP3A increased significantly in rifampicin group and isoniazid-rifampicin group (P<0.05).3 Effect of sanchinoside and narigenin on isoniazid and rifampicin induced lactate dehydrogenase releasingCompared with rifampicin-isoniazid group, the lactate dehydrogenase releasing decreased significantly in sanchinoside (20μg/mL, 100μg/mL, 500μg/mL) treated groups (P<0.05, P<0.01, P<0.01). Treated with narigenin (50μg/mL, 500μg/mL), the lactate dehydrogenase releasing decreased significantly ( P<0.01, P<0.01).4 Effect of sanchinoside on the activity of CYP2E1 Compared with control group, the activity of CYP2E1 of rifampicin -isoniazid group increased significantly (P<0.01). Compared with rifampicin -isoniazid group, the activity of CYP2E1 of sanchinoside (100μg/mL, 500μg/mL) groups decreased significantly (P<0.01, P<0.01).5 Effect of narigenin on the activity of CYP3A Compared with control group, the activity of CYP3A of isoniazid- rifampicin group increased significantly (P<0.05). Compared with rifampicin -isoniazid group, the activity of CYP3A of narigenin (500μg/mL) group decreased significantly (P<0.05).Conclusions:1 The mouse hepatocyte toxicity could be induced by isoniazid and rifampicin alone and their cotreatment. The effect of coadministration is more intensive than isoniazid or rifampicin.2 The activity of CYP2E1 and CYP3A could be enhanced by isoniazid and rifampicin alone and their cotreatment in mouse primary hepatocytes,which may be responsible for isoniazid and rifampicin inducing hepatocyte toxicity.3 The activity of CYP2E1 could be depressed by sanchinoside and CYP3A could be depressed by naringenin. Moreover, sanchinoside and naringenin attenuate isoniazid and rifampicin cotreatment induced lactate dehydrogenase releasing through their effect on CYPs.