| Isoniazid (INH) in the treatment of all types of tuberculosis (TB) is associated with mild to moderate elevation of liver enzyme activity in plasma, and severe hepatotoxicity in approximate 1 % - 2 % of patients. Although near 30 years after the toxicity was detected in INH-treated patient, the mechanism is still unknown. And there is no special treatment for INH-induced hepatotoxicity in clinic. Cytochrome P450 2E1 (CYP2E1) is constitutively expressed in human liver and is responsible for the metabolic bioactivation of a wide variety of xenobiotics (including hepatotoxin CCl4 and acetaminophen hepatotoxicity). It is well known that CYP2E1 can be induced by INH, but its role in INH-induced hepatotoxicity is unclarified.Rifampin (RFP) is usually co-used with INH to treat TB. The combination of INH and RFP was reported to result in an increase in liver injury, and cytochrome P450 was thought to be involved in the synergistic effect of RFP on INH.Thus, in present study, we investigated the role of CYP2E1 in INH-induced hepatotoxicity and the influence of RFP on the hepatotoxicity of INH in rats. Meanwhile, the study on mechanism-based intervention in INH-induced hepatotoxicity was carried out in rats.This study includes three parts.PART OneAIM: A reversed phase HPLC method was established to determine hydrazine in plasma that is the toxic metabolite of isoniazid. METHODS: The samples were analyzed on C18 column, with the mobile phase of 5 mmol L-1 sodium acetate (adjusted pH to 5.0 with 1 mol L-1 glacial acetic acid) -CH3CN (35:65) at the detection wavelength of 300 nm. RESULTS: The linear range for hydrazine in plasma was from 8.5 to 85.2 umol L-1 (r = 0.999). The average recovery rate was 100.2%, and the relative standard deviation of intra-day and inter-day were all less than 2 %. CONCLUSIONS: The RP-HPLC method is asimple way to determine hydrazine in plasma for evaluating the toxicity of isoniazid.PART TwoAIM: To investigate the role of CYP2E1 in isoniazid (INH)-induced hepatotoxicity and the influence of rifampin (RFP) on INH-induced liver injury. METHODS: Rats were treated with INH alone (100 mg kg-1, ip) or co-administered with RFP (100 mg-kg-1, ig) for 10 d and 21 d. Hepatotoxicity was assayed by plasma enzymes (sALT, sAST), liver index, and histopathological examinations. The activity of hepatic CYP1A1, CYP2E1, and CYP3A were measured by 7-ethoxyresorufin O-deethylase (EROD), aniline hydroxylase (ANH) and erythromycin N-demethylase (ERD), respectively. Meanwhile, 1-chlor-2, 4-dinitrobenzene, ethacrynic acid, cumene peroxide and bromsulfophthalein were respectively used as special substrates to determine cytosolic sGST, aGST, uGST and GST. And 7-hydroxy-4-methylcoumarine and 4-phenylphenol were also used as special substrates to determine the activities of isoforms of UDP-glucuronosyltransferase. CYP2E1 and CYP3A1 mRNA expression were determined by RT-PCR. Plasma hydrazine concentration was determined by RP-HPLC. RESULTS: For 10 d INH-treatment, sALT, sAST and liver index were not significantly changed, but hepatic CYP2E1 level was increased to 3.7-fold over the control. However, in INH-RFP group for 10 d, CYP2E1 and plasma hydrazine were respectively decreased by 13 % and 19 % over INH group. In INH group for 21 d. liver impairment appeared, while CYP2E1 and plasma hydrazine were, respectively, increased to 4.6-fold and 1.7-fold. And sGST, aGST, uGST and GST in INH group were respectively decreased by 21 %, 39 %, 40 % and 39 % over the control. Similarly, hepatic injury in INH-RFP group for 21 d is equal to INH group appeared, and CYP2E1 was further decreased by 24 % over INH-RFP group for 21 d. While sGST, aGST, uGST and GST in INH-RFP group for 21 d were increased of 14 %, 9 %, 64 % and 32 % over the INH group, respectively. Nevertheless, the activity of CYP3A and isoforms of UDP-glucuronosyltransferase were at the same level in all the rats pretreated for 10 days and 21 days. Correlation analysis showed that sALT had a positive correlation with plasmahydrazine and with CYP2E1 activit...
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