240 Inhibition of cjun and FADD protects hepatocytes against azathioprine-induced necrosis

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AASLD ABSTRACTS

239

INTERINDIVIDUAL VARIABILITY OF CANALICULAR ATPBINDING-CASSETTE (ABC)-TRANSPORTER EXPRESSION IN H U M A N LIVER. Yvonne Meier, Bruno Stieger, Karin Fa~'nger,

Ulrich Zanger, University Hospital Ziirich, Ziirich, Switzerland; Ulrich Zanger, Dr. Margarete Fischer-Bosch Institut fiir Klinische Pharmakologie, Stuttgart, Germany; Peter J Meier, Christiane PauliMagnus, University Hospital Ziirich, Ziirich, Switzerland Background: Acquired liver disease such as drug induced liver injury is an important clinical problem. Many of these patients suffer from cholestasis because of impaired bile formation. The primary driving force for hepatocellular bile formation are canalicular ATP-Binding Cassette (ABC) transporters such as the bile salt export p u m p (BSEP, ABCBll), the multidrug resistance protein 3 (MDR3, ABCB4) and the multidrug resistance associated protein 2 (MRP2, ABCC2). Impaired expression and function of these transporters have b e e n shown to be the cause for different hereditary cholestatic syndroms. Additionally, various BSEP-inhibiting drugs such as cyclosporine can induce cholestasis in suceptible patients. Hence interindividual differences in expression levels and function of these ABC-transporters could lead to differences in susceptibility to drug induced liver injury. The aim of this project was to determine the extent of interindividual variability in expression of BSEP, MDR3 and MRP2 in normal h u m a n liver tissue. Methods: Samples of normal liver tissue (0.8 - 1,2 gr) were obtained from 100 patients (median age 59 (51.5; 66.5), 44 female) undergoing liver resection because of liver metastasis of extrahepatic tumors (56), hepatocellular carcinoma (21), Klatskin t u m o r (8) and other causes (15). Canalicular liver plasma m e m b r a n e (cLPM) were isolated by a modification of the method described by H. Wolters et al. (Biochim Biophys Acta. 1991; 1069:61-9). The degree of purification of cLPM was estimated by the enrichment of the specific activity of alkaline phosphatase (AP) and aminopeptidase N (APN) relative to the homogenate. Western Blotting of BSEP, MDR3 and MRP2 was performed using an affinity purified antibody against the C-terminal e n d of h u m a n BSEP and two commercial antibodies against MDR3 and MRP2. Blots were analysed semiquantitatively by densitometry (area u n d e r the curve, AUC). To account for interblotting differences, transporter expression in individual patients (sample) was normalised by the m e a n AUC of each blot (20 lanes). Analysis was done on Intransformed data. Results: Isolated cLPMs (n-100) were enriched 4.0 ± 1.0 fold for AP and 3.0 ± 1.1 fold for APN. The reproducibility of Western blot analysis between individual liver samples was 1.09 ± 0.56 for BSEP-expression, 1.15 ± 0.46 for MDR3 and 1.15 ± 0.37 for MRP2. In all samples normalised BSEP-expression ranged from 0.13 to 2.40, 0.07 to 2.74 for MDR3 and 0.01 to 3.65 for MRP2. After logarithmic transformation m e a n expression levels were -0.15 ± 0.56 for BSEP, -0.13 ± 0.55 for MDR3 and -0.24 ± 0.81 for MRP2. These data follow normal distribution and indicate that interindividual variability was similar for all transporters. Neither gender nor underlying disease correlated with transporter expression. Interestingly 3 patients were identified with especially low BSEP expression in the presence of normal (within one SD) MDR3 and MRP2 expression. Similar "low expressor'patients were found for MDR3 (1) and MRP2 (2). Conclusions: The results indicate a normal rather t h a n a bimodial distribution of BSEP-, MDR3- and MRP2-expression in the population. Thus, there appears to exist no relevant polymorphism > 5% for "low expressors'. Only I to 3 out of 100 patients had low expression for either BSEP, MDR3 or MRP2. Wether such "low expressors" of canalicular ABC-transporters have an increased risk for drug induced cholestasis remains to be investigated. Disclosures: Karin Fattinger - No relationships to disclose Peter J Meier - No relationships to disclose Yvonne Meier - No relationships to disclose Christiane Pauli-Magnus - No relationships to disclose Bruno Stieger - No relationships to disclose Ulrich Zanger - No relationships to disclose Ulrich Zanger - No relationships to disclose

HEPATOLOGY, October 2003

24O INHIBITION OF CJUN AND FADD PROTECTS HEPATOCYTES AGAINST AZATHIOPRINE-INDUCED NECROSIS. Brett~ Jones, Wan M Wu, Michael J Tapner, Geoffrey

C Farrell, Westmead Millennium Institute, Westmead, Australia Objectives: We studied hepatocyte injury with pharmacologic concentrations of azathioprine and investigated the role of NFKB, cJun and Fas associated protein with death domain (FADD). Methods: Rat hepatocytes were isolated by 2-stage collagenase perfusion, cultured on matrigel and treated for up to 4 days with azathioprine. Viability, ATP and glutathione (GSH) [total & mitochondrial] were quantified. Western immunoblotting was performed to assess caspase activation. Electron microscopy evaluated hepatocyte ultrastructure. Adenoviral-mediated expression of super-repressor m u t a n t IKB, dominant negative forms of cJun and FADD were used to inhibit NFKB, cJun and FADD function respectively. Results: I and 5 /~mol/L azathioprine induced 10 and 33% cell death at 24h and 25 and 83% cell death by 48h of culture. Microscopy confirmed hepatocyte death occurred by necrosis. Western immunoblots demonstrated preserved pro-caspase-3 b a n d s and absence of cleaved caspase products confirming the absence of caspase-3 activation. Azathioprine reduced total cellular glutathione and ATP levels in proportion to loss of hepatocyte viability, but there was accentuated depletion of mitochondrial (mt) GSH that coincided with cell death. Addition of either 20/~M allopurinol or 2 m M Trolox reduced azathioprine-induced cell death by 50%; whereas combined allopurinol/Trolox pre-treatment of hepatocytes provided complete protection. Pre-treatment of hepatocyte cultures with either GSH ethyl ester or N-acetyl cysteine improved cell viability by less t h a n 10% following exposure to 5/~M azathioprine. Caspase inhibition failed to protect hepatocytes against azathioprine-induced toxicity. Azathioprine induced dose- and t i m e - d e p e n d e n t changes in mitochondrial ultrastructure that were completely prevented by combined allopurinol and Trolox pre-treatment. C o m b i n e d allopurinol/Trolox pre-treatm e n t not only preserved mt GSH following exposure to azathioprine but e n h a n c e d mt GSH levels to greater t h a n 125% of control values. Adenoviral-mediated inhibition of cJun or FADD reduced cell death by 60% at 24 h and 45% at 48h following exposure to azathioprine (5/~M). Inhibition of NFKB had similar efficacy at 24h but minimal effect by 48h. There was no benefit from dual adenoviral infections. Inhibition of cJun combined with Trolox increased ceil viability from 17% to 92% providing almost complete protection against azathioprine-induced cell death. Conclusion: Clinically relevant concentrations of azathioprine induced necrosis in rat hepatocyte cultures by mechanisms that involved oxidative stress, GSH and ATP depletion and mitochondrial injury. Allopurinol and Trolox protected against azathioprine toxicity by i n d e p e n d e n t but additive pathways. FADD and cJun are important mediators of azathioprine-induced hepatocyte necrosis. Disclosures: Geoffrey C Farrell - No relationships to disclose Brett E Jones - No relationships to disclose Michael J Tapner - No relationships to disclose W a n M W u - No relationships to disclose