[304] OPIOID AND NITRIC OXIDE MODULATION OF PENTYLENETETRAZOLE-INDUCED SEIZURE SUSCEPTIBILITY IN A REVERSIBLE MODEL OF CHEMICAL CHOLESTASIS

s120 zyxwvutsrqpo zyxwvutsrqponml zyxwvu zyxwvutsrqpon zyxwvutsr zyxwvutsrqpo POSTERS 13031 CHRONIC OPlOlD ADMINISTRATION INDUCES OXIDANT AND APOPTOTIC DAMAGE IN THE MICE LIVER S. Payabvash’ , A. Beheshtian’ , A.H. Salmasi’ , S. Kiumehr’, M.H. Ghahremani’, S.M. Tavangar3, A.R. Dehpour’ . ’Department of Pharmucologj~,School of Medicine; 2Depurtment of Phurmacology and Toxicology, Faculty of Pharnzacy; 3Department of‘ Pathology, Shariati Hospital, RJhrun Uniuersity of Medical Sciences, RJhrun, lrun E-mail: dehpour@jyahoo.com Aim and Background: The increment of opioidergic neuromodulation is a systemic phenomenon in cholestatic liver disease. Previous studies have revealed the increment of plasma and liver concentrations of opioid peptides in animal models of cholestasis and human cholestatic cirrhosis. The present study was conducted to clarify the effects of chronic opioid administration on liver antioxidant defence and hepatocytes vitality Methods: Mice were divided into four groups: Control group, received vehicle (normal saline); Mor group, received morphine injections at dose of 10 mg/kg; Mor+Nal group, received 10 mgikg morphine and 10 mgikg naltrexone injections. In order to investigate whether morphine-induced oxidative stress contributes to induction of apoptosis in hepatocytes we treated the mice in Mor+NAC group with 500mgikg intragastric dose of N-acetylcysteine, 30 min prior to subcutaneous injection of 10 mgikg morphine. All agents were injected intraperitoneally for 9 consecutive days. The changes in liver glutathione concentration and its synthesis pathway were determined. The activities of hepatic antioxidant enzymes were also measured. Induction of apoptosis was examined by in situ determination of the fragmented DNA. The enzyme activities of aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transpeptidase were also determined in plasma of mice. Results: Following 9 days of morphine injection hepatocytes apoptosis index and biochemical markers of liver damage increased significantly. Liver glutathione concentration as well as catalase, glutathione peroxidase, and superoxide dismutase activities were significantly decreased in Mor group. Hepatic glutathione biosynthesis pathway was changed in the way to compensate for reduced hepatocytes glutathione concentrations. In Mor+Nal group, co-treatment of mice with naltrexone normalized the aforementioned changes. Interestingly, the pro-apoptotic effects of morphine were antagonized by co-administration of thiol antioxidant in Mor+NAC group. Co-treatment of mice with N-acetylcysteine also normalized other oxidant and hepatotoxic effects of morphine. Conclusion: Since the effects of chronic morphine treatment were opioid receptor-mediated and were antagonized by co-administration of an exogenous thiol antioxidant, our findings propose that chronic opioidergic neuromodulation induces oxidative stress in the liver, which may contribute to induction of apoptosis in hepatocytes. These data are valuable in interpretation of the opioid-dependent liver damage during cholestatic liver disease. 13041 OPlOlD AND NITRIC OXIDE MODULATION OF PENTYLENETETRAZOLE-INDUCED SEIZURE SUSCEPTIBILITY IN A REVERSIBLE MODEL OF CHEMICAL CHOLESTASIS A.R. Dehpour, A. Nourian, A. Jannati, H. Honar, K. Riazi, T. Gholipour. Depurtment of Pharmucology, School of Medicine, RJhrun Uniuersity, RJhrun,lrun E-mail: dehpour@yahoo.com Background and Aims: Elevated plasma level of endogenous opioids is thought to be the cause for some pathological processes observed in cholestasis. Studies on models of well-known surgical bile duct-ligated animal support this idea. Endogenous opioids have been shown to have modulatory epileptiform activities both in evoking and inhibiting convulsions. Opioid system and nitric oxide (NO) have been coupled in many physiological and pathological processes including some opioidergic central nervous system manifestations of cholestatic animals. In one study, significant increase in clonic seizure threshold (CST) in mice after surgical cholestasis suggested that elevated plasma levels of endogenous opioids and overproduction of NO may have been involved in this phenomenon. In this study, we used a reversible model of chemically-induced cholestasis to investigate the effect of chemical cholestasis on CST and also whether this change in seizure threshold would return to normal level after the cholestasis resumes or not. Methods: Male Swiss mice underwent fasting before a-naphthylisothiocyanate (ANIT) administration. ANIT was dissolved in corn oil and delivered by gavage directly into the animal stomach with a concentration of l00mgikg. In the sham group, corn oil was gavaged. The threshold of PTZ-induced seizures was measured by an infusion of I % PTZ into the tail vein of freely moving mice at a constant rate of I mlimin. Results: The ANIT-treated animals expressed cholestatic signs which were maximally evident 72 hours after the administration and then slowly decreased until the 7th day. Time-dependent changes in cholestasis-related biochemical markers and CST were shown. Increase in CST could be reversed by chronic naltrexone pretreatment or acute administration of L-NAME. Coadministration of subeffective doses of L-NAME and naltrexone could also additively reverseCST increase. Administration of an anticonvulsant dose of morphine to cholestatic mice on day 7 after ANIT treatment did not increase CST, which suggest downregulation of receptors even after cholestasis resolution. Conclusions: The present study demonstrates that chemical cholestasis induced by ANIT increases CST in mice in a reversible manner through an opioid/NO mediated pathway, and is probably accompanied by a downregulation of opioid receptors, few days after restoring of CST to baseline level. 13051 PERIPORTAL DOWNREGULATION OF Bsep AND Mrp2 IN EXPERIMENTAL OBSTRUCTIVE CHOLESTASIS IS MEDIATED BY TNF-ALPHA AND ILI-BETA AND IS ASSOCIATED WITH PERIPORTAL INFLAMMATION AND CYTOKINE SECRETION M.G. Donner, S. Schumacher, U. Warskulat, J. Heinemann, D. Haeussinger. Department of Gastroenterology, Hepatology and Infictious Diseases, Heinrich Heine Uniuersity DuexwAkvf,’ DnexwAkvf,’ Germuny E-mail: Markus-Donner@yahoo.de Background and Aims: Previous studies have shown that down-regulation of Bsep (Abcbl 1) and multidrug resistance protein 2 (Mrp2, Abcc2) in obstructive cholestasis mainly occurs in periportal hepatocytes. We studied the mechanisms of this diverse zonal transporter regulation in rat liver. Methods: Hepatocellular transporter zonation was analyzed by densitometry of the peak immunofluorescent membrane signals in periportal and pericentral hepatocytes following 7 d of bile duct ligation (BDL). The contribution of TNF-a and Il-l[3 to transporter zonation in obstructive cholestasis was studied by cytokine inactivation in bile duct-ligated rats with etanercept and anakinra. Protein and mRNA expression was quantitied by western blotting and real time PCR, respectively. Results: In cholestatic rat liver Bsep peak immunofluorescence in periportal hepatocytes significantly decreased to 6 6 f 4 % (p < 0.01), whereas peak immunofluorescence in pericentral hepatocytes was not significantly altered. Mrp2 peak immunofluorescence in periportal and pericentral hepatocytes decreased to 28+1% (p <0.001) and 55+2% (p i0.001), respectively. Cytolcine inhibition in bile duct-ligated rats largely restored Bsep expression in periportal hepatocytes. Peak immunofluorescence in periportal hepatocytes following BDL and etanercept or BDL and anakinra treatment was 96+5% and 9 9 f 3 % (p 0.001 compared to BDL animals), respectively. Mrp2 zonal down-regulation was partially reversed by cytolcine inhibition. In etanercept-treated cholestatic rat liver periportal and pericentral peak immunofluorescence was 7 5 f 2 % and 90&1‘%,(p < 0.001 compared to BDL animals), respectively. In anakinra-treated cholestatic