Study On The Mechanisms Of Hepatotoxicity Induced By Isoniazid And Rifampicin And The Role Of Hepatic Protective Drugs

Tuberculosis (TB) is one of the major causes of death from infectious disease. Recommended standard treatment by the World Health Organization (WHO) for adult respiratory TB is a regimen of isoniazid, rifampicin, and pyrazinamide for 2 months, followed by 4 months of isoniazid and rifampicin. The most frequent and most serious adverse effect of anti-tuberculosis treatment is hepatotoxicity. Evidence accumulated during the last years documented that cytochrome enzymes that mediate the metabolism of isoniazid play an important role in the development of hepatotoxicity. Moreover, the cytochrome enzymes CYP 3A4 and 2E1 are thought to mediate the hepatotoxicity. However, mechanisms that mediate anti-tuberculosis treatment induced hepatotoxicity are still not clear.Studies by others and our previous studies in mice suggested that hydrazine, the metabolite of isoniazed, was the main hepatotoxic compound. And drugs administered, such as sodium ferulate, baicalin and silibinin are protective in isoniazid and rifampicin exacerbated heptotoxicity.The present study was carried out to research the levels of isoniazid and its metabolites in the plasma of lung tuberculosis patients administered with isoniazid with or without rifampicin for two weeks. And the hepatocyte toxicity of isoniazid, its metabolites and rifampicin was investigated in cultured hepatocytes by using cell viability assays and lactate dehydrogenase release, a marker of cell death. Furthermore, the role of CYP 3A4, 2E1 and their inhibitors in the occurrence and preservation of isoniazid-rifampicin induced hepatoxicity was demonstrated.Methods1. Patients and drugs administrationTwenty-four lung tuberculosis patients were designated to three groups randomly. In the isoniazid group, patients were administered with isoniazid, levofloxacin and glucurolactone. In the rifampicin group, patients were administered with rifampicin, levofloxacin and glucurolactone. In the isoniazid and rifampicin group, patients were administered with isoniazid, rifampicin and glucurolactone.2. Plasma sample collection and disposalPlasma samples were collected before and 2 hours, 4 hours and 12 hours after the last administration of isoniazid and/or rifampicin following a two week duration of therapy and the samples were then stored at -70℃until HPLC-MS analysis. Alanine aminotransferase, aspartate aminotransferase and total bilirubin levels in serum were determined before and after therapy.3. Culture of QSG-7701 hepatocellularsThe QSG-7701 cell strain was cultured in DMEM culture media that contained high glucose at a cell density of 1×105 cells/mL. Pancreatin (0.25%) was used for trypsinization and sub-culturing.4. Cell viability assayThe MTT assay was used to evaluate the viability of the hepatocytes cultured in 96-well plates. Briefly, MTT was added to the culture media with a final concentration of 0.05%. After 4 hours of culture, the culture media was removed and 150μL DMSO was added to dissolve the crystals. The absorbance of the solution was read at 570nm on a spectrophotometer and the cell viability was calculated.5. Measurement of lactate dehydrogenase activity in cells and culture mediaThe cells in the culture plate were flushed with sodium solution and scraped off. After centrifugation, the cells were lyzed with lysis buffer that was incubated with the cells for 30 minutes. The resultant supernatant was used for the assay. Lactate dehydrogenase activity in cell lysates and culture media were determined using a lactate dehydrogenase activity kit and absorbance spectrophotometery. The ratio of extracellular and intracellular activity of lactate dehydrogenase was calculated.6. Determination of plasma isoniazid, acetylhydrazine and hydrazine concentrations using HPLC-MS Internal standard and methanol were added to plasma samples that were then vortexed for 3 minutes. After centrifugation of the samples, the supernatants were incubated with derivative agent for 24 hours and then analyzed with HPLC-MS using a 10μL on each HPLC run. An Agilent C18 analyze column (5μm, 2.1mm×150 mm ID) was used with an Agilent SB C18 guard column (5μm). The mobile phase consisted of methanol: water (containing 0.1% glacial acetic acid) and was pumped into the system at a rate of 0.15 mL·min-1. ESI ionization was used in the mass detection in selected ion monitoring. The ions selected for isoniazid, acetylhydrazine and hydrazine- were m/z [256.1]+, m/z [193.2]+ and m/z [151.1]+ , respectively.7. Determination of midazolam and 4-nitrocatichol concentrations in culture media using HPLC-MSA Gemini C18 analyze column (2.0×50 mm, 5μm) was used with a C18 guard column (5μm,3.0×4.0 mm). The mobile phase consists of acetonitrile: water (60:40, v/v, containing 0.2% glacial acetic acid) and was pumped in 0.2 mL·min-1. Ten milliliters of sample was analyzed on the column. ESI ionization was used in mass detection in selected ion monitoring. The ions selected for 4-nitrocatechol and midazolam- were m/z [154.0]- and m/z [326.0]+, respectively.8. Grouping and drug treatment8.1 Effect of isoniazid, its metabolites and rifampicin on cellular viabilityThe cells were plated in 96-well plates at a cell density of 1×104 cells per well. The cells were designated to sixteen groups with six wells in each group. The six treatment groups consisted of the blank vehicle, isoniazid (250μg/mL, 500μg/mL, 2500μg/mL), rifampicin (100μg/mL, 200μg/mL, 300μg/mL), acetylhydrazine (125μg/mL, 250μg/mL, 1250μg/mL), hydrazine (0.25μg/mL, 2.5μg/mL, 25μg/mL) and isoniazid+rifampicin (100+50μg/mL, 200+100μg/mL, 300+150μg/mL). All treatments were added to the culture media and after 48 hours of culture, cell viability was determined using the MTT method.8.2 Effect of isoniazid, its metabolites and rifampicin on lactate dehydrogenase activityCells were plated in to 6-well plates. The groups and treatments were the same as item"8.1". Cells and culture media were collected for assay of lactate dehydrogenase.8.3 Effect of isoniazid and rifampicin on the activity of CYP 2E1 and 3A4Cells were designated to eight treatment groups with six wells in each group. The eight treatment groups consisted of the blank vehicle, isoniazid (100μg/mL), rifampicin (200μg/mL), isoniazid (100μg/mL) and rifampicin (200μg/mL) that were added to the culture media. After a 48 hour incubation period the culture media was removed. 4-nitrophenol (15μg/mL) and midazolam (1μg/mL), substrates of CYP 2E1 and 3A4 respectively, were added to the wells and cultured for an additional 2 hours. The concentration of 4-nitrocatechol, a metabolite of 4-nitrophenol, and midazolam were determined using HPLC-MS. The activity of CYP 2E1 and 3A4 were calculated by the increasing level of 4-nitrocatechol and the decreasing level of midazolam.8.4 Effect of CYP 2E1 and 3A4 inhibitors on isoniazid and rifampicin on lactate dehydrogenase releaseCells were plated onto 24-well plates, and were designated to fourteen groups with six-wells in each group. Three of the fourteen groups consisted of the blank vehicle, isoniazid (100μg/mL) and rifampicin (200μg/mL) treatments that were added to the culture media. The other groups consisted of the naringenin and erythromycin groups where naringenin (1μg/mL, 5μg/mL, 25μg/mL) or erythromycin (1μg/mL, 5μg/mL, 25μg/mL) were added in addition. In the sodium ferulate and sanchinoside groups, sodium ferulate (10μg/mL, 50μg/mL, 250μg/mL) and sanchinoside (4μg/mL, 20μg/mL, 100μg/mL) were added in addition. After 48 hours of culture, lactate dehydrogenase activity in the cells and culture media were determined.8.5 Effect of CYP 2E1 and 3A4 inhibitors on isoniazid and rifampicin decreased cellular viabilityCells were plated in 24-well plates, and were designated to eight treatment groups with six-wells in each group. The eight treatment groups consisted of the blank vehicle, isoniazid (100μg/mL) and rifampicin (200μg/mL) that were add to the culture media. In naringenin and erythromycin groups, naringenin (5μg/mL) or erythromycin (5μg/mL) were added in addition. In sodium ferulate and sanchinoside groups, sodium ferulate (50μg/mL) and sanchinoside (20μg/mL) were added in addition. After 48 hours of culture, the cell viability was determined using the MTT method.8.6 Effect of enzyme inhibitors on the activity of CYP 2E1 and 3A4Cells were plated in 24-well plates, and were designated to eight treatment groups with six-wells in each group. The eight treatment groups consisted of the blank vehicle, isoniazid (100μg/mL) and rifampicin (200μg/mL) that were added to the culture media. In the naringenin and erythromycin groups, naringenin (5μg/mL) or erythromycin (5μg/mL) were added in addition. After 48 hours of culture, the culture media was removed and the activity of CYP 3A4 determined. In the sodium ferulate and sanchinoside groups, sodium ferulate (50μg/mL) and sanchinoside (20μg/mL) were added in addition. After 48 hours of culture, the culture media was removed and the activity of CYP 2E1 determined.9 Data and statisticsStatistic was performed with SPSS 11.5 for windows software, and data were expressed in mean±SD. Significance between the groups was analyzed by independent samples t test or ANOVA with the Dunnett t test and binary variable correlation analysis. Significance was considered when P<0.05.Results1. Concentrations of isoniazid, acetylhydrazine and hydrazine in the plasma of lung tuberculosis patientsThe concentration of hydrazine increased significantly 2 h, 4 h and 12 h (P<0.05 or P<0.01) in plasma after isoniazid and rifampicin were co-administered to patients with lung tuberculosis compared with treatment with isoniazid alone. There were no significant differences between the concentrations of isoniazid and acetylhydrazine between the groups (P>0.05). There was no significant abnormality in the levels of GPT, GOT and total bilirubin before and after two weeks of drug therapy (P>0.05).2. Viability of isoniazid, rifampicin, acetylhydrazine and hydrazine treated hepatocytesAfter 48 hours of treatment with isoniazid (500μg/mL or 2500μg/mL), the viability of cultured QSG-7701 cells decreased significantly compared with the control group with the value of 64.3% (P<0.01) and 9.9% (P<0.01). The Pearson correlation coefficient was -0.959 (P<0.01) between the concentration of isoniazid and cell viability. The viability of cells decreased to 56.2% (P<0.01), 53.5% (P<0.05) and 46.0% (P<0.01) in the rifampicin (300μg/mL) and hydrazine (2.5μg/mL and 25μg/mL) treated groups. Co-administration of isoniazid and rifampicin dose dependently decreased cell viability and the Pearson correlation coefficient was -0.892 (P<0.01).3. Activity of lactate dehydrogenase released from isoniazid, rifampicin, acetylhydrazine and hydrazine treated hepatocytesIsoniazid, rifampicin, acetylhydrazine, hydrazine, isoniazid and rifampicin co-treatment increased the release of lactate dehydrogenase from cells after 48 hours of culture (P<0.01 or P<0.01). The effect of acetylhydrazine and isoniazid-rifampicin co-treatment was dose dependent and the Pearson correlation coefficient was 0.674 (P<0.01) and 0.907 (P<0.01), respectively.4. Effect of isoniazid and rifampicin on activity of CYP 3A4 and 2E1 in hepatocytesThe activity of CYP 2E1 increased significantly in QSG-7701 cells cultured for 48 hours with isoniazid or isoniazid and rifampicin (P<0.05) compared with the control group. There was no significant change in CYP 2E1 activity in the rifampicin alone treated group (P>0.05). Treatment with isoniazid, rifampicin alone or isoniazid and rifampicin all increased the activity of CYP 3A4 in QSG-7701 cells compared with the control group(P<0.05 or P<0.01).5.Effect of CYP 3A4 inhibitors on the hepatotoxicity of isoniazid and rifampicin Naringenin and erythromycin inhibited the activity of CYP 3A4 enhanced by isoniazid and rifampicin, and attenuated the decrease in cell viability and depressed lactate dehydrogenase release (P<0.01) from cultured QSG-7701 cells. The effect of naringenin on lactate dehydrogenase release was dose dependent and the Pearson correlation coefficient was -0.712 (P<0.01).6. Effect of CYP 2E1 inhibitors on the hepatotoxicity of isoniazid and rifampicinSodium ferulate and sanchinoside inhibited the activity of CYP 2E1 enhanced by isoniazid and rifampicin, attenuated the decrease in cell viability and depressed the increase in lactate dehydrogenase release (P<0.05 or P<0.01) from cultured QSG-7701 cells. The effect of sanchinoside on lactate dehydrogenase release was dose dependent and the Pearson correlation coefficient was -0.659 (P<0.01).Conclusions1 The concentration of hydrazine increased significantly in the plasma of lung tuberculosis patients that were co-administered isoniazid and rifampicin compared with patients that were treated with isoniazid alone, and the co-treatment may be the mechanism by which isoniazid and rifampicin induce hepatotoxicity.2 Different co-administered doses of isoniazid, hydraine, rifampicin, isoniazid and rifampicin result in increasing lactate dehydrogenase release and decrease the viability of hepatocytes. The effect of co-administration is more intensive than isoniazid or rifampicin treatment alone. And hydrazine, the metabolite of isoniazid, induced more severe hepatocytes toxicity than its precursor, which suggests a pivotal and considerable role of hydrazine in anti-tuberculosis induced hepatotoxicity.3 The activity of CYP 3A4 and 2E1 could be enhanced by isoniazid and rifampicin alone and their co-treatment, which may be responsible for the excessive production of hydrazine and the hepatocyte toxicity following isoniazid and rifampicin co-therapy.4 The activity of CYP 3A4 could be depressed by naringenin and erythromycin and the activity of CYP 2E1 could be depressed by sodium ferulate and sanchinoside. Moreover, naringenin, erythromycin, sodium ferulate and sanchinoside attenuate isoniazid and rifampicin co-treatment induced hepatocyte viability and decrease lactate dehydrogenase release through their effect on CYPs.