Isoniazid- And Rifampicin- Induced Hepatotoxicity And Its Mechanism

Background and objective:Both isoniazid (INH) and rifampicin (RIF) are widely used drugs in antituberculosis therapy recommended by World Health Organization (WHO) and the main reason of acute drug-induced liver injury in China. INH and RIF has been known to be hepatotoxic but the interaction between INH and RIF is still not entirely clear. INH-induced hepatotoxicity was associated with INH-induced hepatic oxidative stress via hepatic CYP 2E1 metabolism. Starvation upregulates the expression of CYP 2E1 in liver. However, whether starvation aggravate INH-induced hepatotoxicity is still not entirely clear. The treatment of RIF combined INH significantly increased hepatic TG and TCH. However, whether RIF or INH alone affects lipid metabolism needs to be further studied. The purpose of the present study was to (1) the effects of starvation on INH-induced hepatotoxicity, (2) the effects of RIF alone on hepatic lipid metabolism, and (3) the efffects of RIF on INH-induced oxidative stress in liver. Methods: The present study includes three separate experiments. (1) To study INH -induced liver injury, ICR female mice were divided into two groups: the control group and the INH group. Mice were orally administered with saline or INH (100 mg/kg, dissolved in saline) daily for seven consecutive days and sacrificed at 6 h after the last INH administration. To investigate the effects of starvation on INH-induced liver oxidative stress in mouse liver, mice were divided into four groups: control group, starvation group, INH alone group and INH plus starvation group. In INH alone group, mice were administered with INH (100 mg/kg, dissolved in saline) daily by gavage for four consecutive days. In INH plus starvation group, mice were starved for 12 h before INH and 6 h after INH. Control mice received saline only. At sacrifice, the mice were fasted for 6 h after the last INH administration. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP) were measured using standard clinical methods. Liver sections were stained with hematoxylin and eosin to evaluate the hepatic histology. The glutathione (GSH) was determined by the method of Griffith. Lipid peroxidation was quantified by measuring thiobarbituric acid-reactive substance (TBARS). RT-PCR and Western blotting were used to detect the expression of cytochrome P450 2E1. (2) To study the effects of a seven-day treatment with RIF on lipid metabolism in mouse liver, ICR female mice were divided into two groups: the control group and the RIF group. Mice were respectively administered with saline or RIF (200 mg/kg) daily by gavage for seven consecutive days. To study the effects of a single dose of RIF on lipid metabolism in mouse liver, mice were administered with saline or RIF (200 mg/kg) and sacrificed at 2h, 6h, 24h after RIF administration. Serum ALT, AST, AKP, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (TCH), triglyceride (TCH), high density lipoprotein (HDL), very low density lipoprotein (VLDL) and hepatic TCH, TG, HDL, VLDL were measured using standard clinical methods. Liver sections were stained with hematoxylin and eosin to evaluate hepatic histology. Liver lipid accumulation was observed by Oil Red O staining. RT-PCR was used to detect mRNA levels of fatty acid synthetase (Fasn), acetyl-CoA catboxyla-se(Acc), stearoyl-Co A desaturase 1 (Scd-1)and scavenger receptor (CD36). (3) To investigate the efffects of RIF on INH-induced oxidative stress, ICR female mice were divided into four groups. Mice received RIF (200mg/kg) and/or INH(100mg/kg) daily by gavage for seven consecutive days. Control mice received saline only. The mice were sacrificed at 6 h after the last administration. The GSH content in liver was determined by the method of Griffith and lipid peroxidation was quantified by measuring TBARS. RT-PCR and Western blotting were used to detect the expression of CYP 2E1. Results: (1) INH administration induce necrosis and inflammation in mouse liver. In addition, a slight increase in serum ALT and AST was also observed among mice treated with INH. Either INH or a short term of starvation resulted in oxidative stress in mouse liver, as determined by the decreased hepatic GSH level and increased TBARS content. And compared with the mice received INH alone, a short term of starvation significantly aggravated INH-induced decrease in hepatic GSH and increase in TBARS. Furthermore, either INH or starvation significantly upregulated protein expression of hepatic CYP 2E1. A short term of starvation aggravated the upregulation of INH-induced hepatic CYP 2E1. (2) In the rifampicin group, serum ALT and AST were slightly increased. Serum TBA and TBIL were significantly increased. Hepatic histology showed a steatosis associated with mild necrosis and inflammation. In addition, hepatic TG and VLDL were significantly increased after a single dose of rifampicin. And hepatic TG, TCH and VLDL were significantly increased after a seven-day treatment with rifampicin. Moreover, the level of hepatic Fasn and CD36 mRNA significantly upregulated in liver of rifampicin-treated mice. (3) RIF significantly attenuated the INH-induced hepatic oxidative stress and downregulated the expression of CYP 2E1 in mouse liver. Compared with the mice received INH alone, RIF significantly reduced the expression of CYP 2E1 in mouse liver, suggesting that RIF could significantly attenuate the INH-induced oxidative stress via downregulating the expression of CYP 2E1. Conclusion: (1) A short term of starvation aggravates INH-induced hepatic oxidative stress in starved mice via upregulating the level of hepatic CYP 2E1; (2) RIF alone increases lipid accumulation in livers, and the key enzymes of fatty acid synthesis and transport, Fasn and CD36, play an important role in the process of RIF-induced hepatic lipogenesis. (3) RIF attenuates INH-induced oxidative stress through downregulating the expression of CYP 2E1 in liver.