Mitochondrial oxidative stress and permeability transition in Isoniazid and Rifampicin induced liver injury in mice
Introduction
Anti tubercular (AT) drugs are the commonest agents causing serious, clinically significant drug induced liver disease in the developing countries [1], [2], [3]. Most commonly used AT drugs like Isoniazid (INH), Rifampicin (RMP) and Pyrazinamide are hepatotoxic. Various factors predisposing to ATD hepatotoxicity, both genetic and acquired, are well delineated [4], [5], [6], but little is known about the cellular and biochemical mechanisms of AT drugs induced hepatotoxicity.
The hepatotoxicity of INH is thought to be initiated by cytochrome P450 (CYP) mediated metabolism of INH to acetylhydrazine and hydrazine [7], [8], [9]. RMP, which is generally co-administered with INH in the treatment of tuberculosis, enhances hydrazine production [10] by enzyme induction. The high reactivity of hydrazine with sulfhydryl groups results in glutathione (GSH) depletion within the hepatocytes [7] leading to cell death [11], [12]. We have demonstrated oxidative stress in patients having AT drugs induced hepatotoxicity [13]. A few experimental studies have also demonstrated the critical role of GSH in AT drugs induced hepatotoxicity [14], [15]. But only depletion of hepatic GSH by INH or its metabolites, could not explain the AT drug induced hepatotoxicity.
Mitochondrion is an important organelle for cell survival and functions. Mitochondrial dysfunctions have been observed in diclofenac and acetaminophen hepatotoxicity [16], [17]. Mitochondrial permeability transition (MPT) is recently focused as a mechanism for drug induced liver cell injury [16], [17], [18], causing both necrotic and apoptotic cell death. INH could produce apoptosis in Hep G2 and AHH1 cell line [19] as well as necrosis in rabbit liver [8]. However, there is lack of experimental data that supports the role of mitochondria in AT drugs induced liver injury. In the present study, we have investigated the possible role of mitochondria, particularly oxidative stress and involvement of MPT in AT drugs induced liver cell injury in BALB/c mice.
Section snippets
Materials
Reagents and kits were purchased from the following sources: ALT kit from Boehringher Mannheim, Germany, TUNEL staining and AMV RT-PCR kits from Roche Diagnostics GmbH, Mannheim, Germany, anti cytochrome c antibody (clone 7H8.2C12) from R&D Systems, USA, anti mouse IgG, AP conjugates from Promega, Madison, USA, nitrocellulose membrane from Pall Gelman Laboratory, MI, USA, TRIzol reagent from Invitrogen, Carlsbad, USA and other chemicals from Sigma Chemical Co. (St. Louis, MO, USA).
Mice
Male BALB/c
INH and RMP induced liver injury
In BALB/c mice, co-administration of INH-RMP for 3 days induced liver injury, as indicated by a marked increase of serum ALT (Fig. 1A). Hepatic histology had revealed predominantly macrovesicular steatosis (steatosis score 2.46 ± 0.74), associated with mild necrosis and inflammation (Fig. 1B). Biochemically, a 3-fold increase of hepatic triglyceride content was observed (Table 1). A less marked increase in serum ALT (Fig. 1A) and hepatic triglyceride (Table 1) was observed when INH50 was
Discussion
Although, the role of mitochondria in programming, regulating and executioning of cell injury and death in drug induced hepatotoxicity is established, its precise role in INH-RMP hepatotoxicity has not been documented previously. The major goal of our study has been to elucidate the mechanism of INH-RMP hepatotoxicity. Here, we provide experimental evidences that are informative in improving our understanding of pathogenesis of AT drug-induced hepatotoxicity. Apart from increased oxidative
Acknowledgements
The authors would like to acknowledge Prof. Neil Kaplowitz, Los Angeles, CA for critical review of the manuscript. They also acknowledge the sincere help of Ayan Biswas, Sumanta Sarkar, and Pratap Pandit for conducting this experimental study. This work was supported by a grant in aid from Indian Council of Medical Research, New Delhi, India (5/8/5/2/2003-ECD-I) to AS and AC.
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