Elsevier

Journal of Hepatology

Volume 40, Issue 3, March 2004, Pages 454-463
Journal of Hepatology

Toxicity of low dose azathioprine and 6-mercaptopurine in rat hepatocytes. Roles of xanthine oxidase and mitochondrial injury

https://doi.org/10.1016/j.jhep.2003.11.024Get rights and content

Abstract

Background/Aims

To study effects of pharmacologic concentrations of azathioprine and 6-mercaptopurine (6-MP) on rat hepatocytes.

Methods

Hepatocytes cultured on matrigel were incubated with azathioprine or 6-MP; effects of putative protective agents were studied. Viability (LDH leakage), reduced (GSH) and oxidized glutathione (GSSG), mitochondrial (mt) GSH, ATP and ultrastructural changes were determined.

Results

Azathioprine and 6-MP (0.5–5 μmol/l) reduced viability 5–34% at day 1 and 42–92% by day 4. Allopurinol (20 μM) (xanthine oxidase inhibitor) and 2 mM Trolox (vitamin E analog) together provided near complete protection. During culture with azathioprine, GSSG increased before cell death and there was a disproportionate reduction of mtGSH and ATP, together with ultrastructural abnormalities in mitochondria. All changes were prevented by allopurinol and trolox. Discontinuation of 1 μmol/l azathioprine restored ATP levels and arrested cell injury, while culture in glucose-enriched media augmented ATP levels and ameliorated cell death.

Conclusions

Clinically relevant concentrations of azathioprine and 6-MP are toxic to rat hepatocyte cultures by a mechanism that involves oxidative stress, mitochondrial injury and ATP depletion. This can lead to irreversible de-energization and cell death by oncosis (necrosis).

Introduction

Liver injury is an important adverse effect of azathioprine or its active moiety 6-mercaptopurine (6-MP) [1], [2]. The estimated frequency of liver test abnormalities ranges from <1 to 9% [1], [2]. While disorders such as venoocclusive disease, nodular regenerative hyperplasia and peliosis hepatis implicate the microvasculature as a possible site of liver injury [1], [2], [3], [4], [5], [6], [7], hepatocellular injury and cholestasis are more common and indicate toxic effects focussed on hepatocytes.

In earlier work, we and others showed that hundred micromolar concentrations of azathioprine exert acute (less than 24 h) dose-dependent hepatotoxicity to liver cells in primary culture [8], [9], [10]. Cell death was preceded by profound depletion of reduced glutathione (GSH), while addition of GSH or N-acetylcysteine protected against azathioprine-induced cell death [8], [10]. We attributed such acute azathioprine toxicity to consumption of GSH by glutathione S-transferase-mediated conjugation of the nitroimidazole moiety, the first step of azathioprine metabolism (Fig. 1) [10]. However, although some evidence implicates higher doses of azathioprine in hepatotoxicity in man [11], tissue levels after therapeutic doses (2–3 mg/kg body weight) are unlikely to exceed 10 μmol/l [12], [13], [14]. This calls into question the relevance of the earlier mechanistic studies.

Azathioprine-induced bone marrow toxicity and skin reactions are related to thiopurine methyltransferase (TPMT) polymorphisms, and possibly other factors which modulate the activity of the metabolic pathways that determine clearance of azathioprine and 6-MP [11], [12], [13], [14]. However, TPMT polymorphisms do not appear to predispose to azathioprine-induced liver injury [1], [2]. Xanthine oxidase is involved with at least two steps in azathioprine and 6-MP metabolism (Fig. 1). The possibility that this ubiquitous enzyme may play a role in azathioprine-induced tissue injury has been raised by the observation that patients taking allopurinol, a xanthine oxidase inhibitor, experienced less nephrotoxicity during rejection episodes after renal transplantation [15].

The present studies were designed to elucidate whether pharmacological concentrations of azathioprine or 6-MP injure well-differentiated rat hepatocytes in stable culture on matrigel. Under these conditions, viability of control hepatocytes consistently exceeds 95% after 5 days. During exposure to 0.1–5 μmol/l azathioprine or 6-MP, we noted a progressive decline of hepatocyte viability over 4 days, associated with a rise in cellular oxidized glutathione (GSSG), a disproportionate fall in mtGSH compared to total cellular GSH, ultrastructural evidence of mitochondrial injury and decline in cellular ATP content. We then designed experiments to test the hypothesis that azathioprine and 6-MP cause reversible or irreversible de-energization of hepatocytes by a process related to xanthine oxidase-mediated generation of oxidant stress.

Section snippets

Reagents

Fine chemicals were obtained from Sigma-Aldrich Chemical Co. (St Louis, MO), except for insulin (Actrapid) from Novo Nordisk Pharmaceutical (Bagsvaerd, Denmark), and collagenase (Worthington Biochemical Corporation, Lakewood, NJ, USA). Matrigel was prepared in-house from murine Engelbreth–Holm–Swarm tumors [16].

Hepatocyte preparation, culture and determination of viability

Male Wistar rats weighing 300–350 g were obtained from the Westmead Hospital Animal House. Study protocols were approved by the Animal Care and Ethics Committee of the Western Sydney

Low-dose azathioprine and 6-MP produce time-dependent necrosis to well-differentiated primary cultures of rat hepatocytes

The viability of control hepatocytes was >95% over 5 days. As shown in Fig. 2, low concentrations of azathioprine caused time- and dose-dependent toxicity. At 24 h, 5 μmol/l azathioprine produced substantial (34%) loss of viability compared with controls, and by 48 h 0.5–2μmol/l azathioprine significantly reduced viability (Table 1). When cultured hepatocytes were exposed to equivalent molar concentrations of 6-MP, similar loss of hepatocyte viability was observed (Fig. 3A).

With all doses and

Discussion

The present study shows that the mechanism by which clinically relevant concentrations of azathioprine and 6-MP (0.5–5 μmol/l) injure cultured hepatocytes differs from that caused by brief exposure to supra-pharmacological (100–500 μmol/l) concentrations. In earlier high-dose studies [8], [10], 1 mM 6-MP was not toxic, and azothioprine profoundly depleted total cellular GSH before the onset of cell death; further, cytotoxicity was accentuated by prior depletion of GSH and prevented by augmenting

Acknowledgements

This work was supported by project grant 211173 of the Australian National Health and Medical Research Council. The authors are grateful to Dr John Duley, Purine Research Laboratory, Guy's Hospital for helpful discussions, and thank Mrs Krishanthi Gunaratnam for preparing the sections for EM study and Mr Ross Boadle of the Electron Microscope Unit, Westmead Hospital for analysing the EM specimens and photograph preparation. Ms Debbie Docherty provided valued administrative assistance.

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