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Effect of ethanol on some brain and hepatic parameters in male and female spontaneously hypertensive rats (SHRs)
Abstracts / Toxicology Letters 196S (2010) S37–S351
in few patients. We have investigated acute and chronic hepatic effects of amiodarone using the well differentiated human hepatoma HepaRG cell line. This cell line is composed of both mature hepatocyte-like and biliary-like cells (50/50) and remains functionally stable for several weeks. HepaRG cells were exposed to amiodarone for 24 h (single addition) or 14 days (every 2day addition). After a 24 h exposure this drug was found to induce, in the two cell types, formation of intracytoplasmic vesicles that, at the electron microscopic level, appeared as lamellar bodies, the hallmark of phospholipidosis. No Oil-Red O staining was observed. By contrast, after 14 days of repeated treatment, additional vesicles labelled with Oil-Red O, indicative of lipid accumulation, were evidenced in hepatocyte-like cells. Theses results were confirmed by quantification of neutral lipids and phospholipids. Indeed, a dose-dependent induction of triglycerides, phosphatidylethanolamine and phosphatidylcholine was observed in amiodarone-treated HepaRG cells. Moreover, a transcriptomic study showed that expression of several genes involved in lipid metabolism was modulated by amiodarone. In addition, a reduction of fatty acid oxidation was evidenced by measuring the [14C] labelled acid-soluble -oxidation products in amiodarone-treated cells. These results show that amiodarone induces phospholipidosis after a short-term treatment and both phospholipidosis and steatosis after a long-term exposure in HepaRG cells. They support the conclusion that these cells represent a suitable in vitro model for investigating long-term drug-induced liver injury in humans. This study was supported by the European Community contract LIINTOP-STREP-037499. doi:10.1016/j.toxlet.2010.03.704
P206-016 Effect of ethanol on some brain and hepatic parameters in male and female spontaneously hypertensive rats (SHRs) R. Simeonova, V. Vitcheva, M. Mitcheva Faculty of Pharmacy, Medical University – Sofia, Bulgaria Ethanol addiction, developed after multiple ethanol administration, is a complex phenomenon that apart of tolerance and dependence development is characterized with neurotoxicity and hepatotoxicity. Hypertension is a pathophysiological status that additionally contributes to damages in central nervous system and to liver disfunction. The aim of our study was to investigate the effects of multiple ethanol administration on some brain and hepatic biochemical parameters in male and female SHRs, compared to their agematched normotensive controls Wistar Kyoto rats (WKY). The animals from both strains were treated with ethanol (3 g/kg p.o. 14 days). Twenty-four hours after the last administration, the samples were taken for measuring the following parameters: nNOS activity (brain), MDA quantity and GSH level (both in brain and liver). On the brain level, ethanol affected both male and female WKY rats in a similar way: increased nNOS activity by average 80%, decreased GSH level by 40% and increased MDA quantity. In the liver GSH level was depleated by 35% and by 28% in male and female WKY respectively. MDA quantity was increased by 53% in male and by 36% in female rats. In SHRs multiple ethanol treatment resulted in less pronounced effect on the assessed parameters, both in brain and liver, especially in female SHRs – nNOS activity was not changed and GSH level was less affected, compared to male SHR and to female WKY.
S209
These results, on one hand, might be due to a protective effect on the oxidative stress of the estrogens and to a stimulation of the antioxidant defence systems, both in brain and liver. On the other hand, the less affected nNOS activity could be explained by the less active reward system in SHR than in WKY. doi:10.1016/j.toxlet.2010.03.705
P206-017 Comparative changes in gene expression profiles induced by PPAR␥ AND PPAR␣/␥ agonists in primary human hepatocytes and HepaRG cells A. Rogue 1 , C. Spire 2 , N. Claude 2 , A. Guillouzo 1 1
INSERM U991, France, 2 SERVIER Group, France
Peroxisome proliferator-activated receptor gamma (PPAR␥) agonists (glitazones) are used to treat type 2 diabetes mellitus. However, the first compound of this class troglitazone was withdrawn from the market shortly after approval because of severe hepatotoxicity cases. The second generation of PPAR␥ agonists, such as rosiglitazone, is less toxic but rare and less severe incidences of hepatotoxicity have been reported. A variety of dual PPAR alpha and gamma (PPAR␣/␥) agonists (glitazars), has also been developed but has been stopped during clinical trials due to some cardiac and renal side-effects. The mechanisms underlying these toxicities still remain largely unknown. We have analyzed changes in gene expression profiles induced by two PPAR␥ agonists (troglitazone and rosiglitazone) and two PPAR␣/␥ dual agonists (muraglitazar and tesaglitazar) in both primary human hepatocytes and the well differentiated human hepatoma HepaRG cells. The cells were exposed to different concentrations of each chemical for 24 h and their transcriptomes were analyzed using one or two-color human pangenomic Agilent microarrays. Principal Component Analysis, hierarchical clustering and Ingenuity Pathway Analysis were performed. The gene expression profiles identified with HepaRG cells represented the average profiles of those obtained with the five hepatocyte populations which showed a large inter-individual variability. A number of genes involved in lipid and carbohydrate metabolism were commonly modulated by all the compounds. However, we also identified several genes specifically modulated by either glitazones or glitazars. In addition, expression of few genes was altered by only one of the four compounds. In conclusion, our study demonstrates that PPAR agonists can induce differential changes in gene profiles in human hepatocytes depending on the compound and/or the class. doi:10.1016/j.toxlet.2010.03.706
P206-018 Evaluation of potential biomarkers for thioacetamide-induced hepatotoxicity using siRNA J.S. Kang 1 , Y.N. Yum 2 , S.N. Park 2 Namseoul University, South Korea, 2 National Institute of Food and Drug Safety Evaluation, Korea Food and Drug Administration, South Korea
1
In our previous publication we compared the gene expression profiles on hepatotoxicants exposure to assess the comparability between in vivo and in vitro test systems. We investigated global gene expression from both mouse liver and mouse hepatic cell line treated with thioacetamide (TAA) and identified several common
in few patients. We have investigated acute and chronic hepatic effects of amiodarone using the well differentiated human hepatoma HepaRG cell line. This cell line is composed of both mature hepatocyte-like and biliary-like cells (50/50) and remains functionally stable for several weeks. HepaRG cells were exposed to amiodarone for 24 h (single addition) or 14 days (every 2day addition). After a 24 h exposure this drug was found to induce, in the two cell types, formation of intracytoplasmic vesicles that, at the electron microscopic level, appeared as lamellar bodies, the hallmark of phospholipidosis. No Oil-Red O staining was observed. By contrast, after 14 days of repeated treatment, additional vesicles labelled with Oil-Red O, indicative of lipid accumulation, were evidenced in hepatocyte-like cells. Theses results were confirmed by quantification of neutral lipids and phospholipids. Indeed, a dose-dependent induction of triglycerides, phosphatidylethanolamine and phosphatidylcholine was observed in amiodarone-treated HepaRG cells. Moreover, a transcriptomic study showed that expression of several genes involved in lipid metabolism was modulated by amiodarone. In addition, a reduction of fatty acid oxidation was evidenced by measuring the [14C] labelled acid-soluble -oxidation products in amiodarone-treated cells. These results show that amiodarone induces phospholipidosis after a short-term treatment and both phospholipidosis and steatosis after a long-term exposure in HepaRG cells. They support the conclusion that these cells represent a suitable in vitro model for investigating long-term drug-induced liver injury in humans. This study was supported by the European Community contract LIINTOP-STREP-037499. doi:10.1016/j.toxlet.2010.03.704
P206-016 Effect of ethanol on some brain and hepatic parameters in male and female spontaneously hypertensive rats (SHRs) R. Simeonova, V. Vitcheva, M. Mitcheva Faculty of Pharmacy, Medical University – Sofia, Bulgaria Ethanol addiction, developed after multiple ethanol administration, is a complex phenomenon that apart of tolerance and dependence development is characterized with neurotoxicity and hepatotoxicity. Hypertension is a pathophysiological status that additionally contributes to damages in central nervous system and to liver disfunction. The aim of our study was to investigate the effects of multiple ethanol administration on some brain and hepatic biochemical parameters in male and female SHRs, compared to their agematched normotensive controls Wistar Kyoto rats (WKY). The animals from both strains were treated with ethanol (3 g/kg p.o. 14 days). Twenty-four hours after the last administration, the samples were taken for measuring the following parameters: nNOS activity (brain), MDA quantity and GSH level (both in brain and liver). On the brain level, ethanol affected both male and female WKY rats in a similar way: increased nNOS activity by average 80%, decreased GSH level by 40% and increased MDA quantity. In the liver GSH level was depleated by 35% and by 28% in male and female WKY respectively. MDA quantity was increased by 53% in male and by 36% in female rats. In SHRs multiple ethanol treatment resulted in less pronounced effect on the assessed parameters, both in brain and liver, especially in female SHRs – nNOS activity was not changed and GSH level was less affected, compared to male SHR and to female WKY.
S209
These results, on one hand, might be due to a protective effect on the oxidative stress of the estrogens and to a stimulation of the antioxidant defence systems, both in brain and liver. On the other hand, the less affected nNOS activity could be explained by the less active reward system in SHR than in WKY. doi:10.1016/j.toxlet.2010.03.705
P206-017 Comparative changes in gene expression profiles induced by PPAR␥ AND PPAR␣/␥ agonists in primary human hepatocytes and HepaRG cells A. Rogue 1 , C. Spire 2 , N. Claude 2 , A. Guillouzo 1 1
INSERM U991, France, 2 SERVIER Group, France
Peroxisome proliferator-activated receptor gamma (PPAR␥) agonists (glitazones) are used to treat type 2 diabetes mellitus. However, the first compound of this class troglitazone was withdrawn from the market shortly after approval because of severe hepatotoxicity cases. The second generation of PPAR␥ agonists, such as rosiglitazone, is less toxic but rare and less severe incidences of hepatotoxicity have been reported. A variety of dual PPAR alpha and gamma (PPAR␣/␥) agonists (glitazars), has also been developed but has been stopped during clinical trials due to some cardiac and renal side-effects. The mechanisms underlying these toxicities still remain largely unknown. We have analyzed changes in gene expression profiles induced by two PPAR␥ agonists (troglitazone and rosiglitazone) and two PPAR␣/␥ dual agonists (muraglitazar and tesaglitazar) in both primary human hepatocytes and the well differentiated human hepatoma HepaRG cells. The cells were exposed to different concentrations of each chemical for 24 h and their transcriptomes were analyzed using one or two-color human pangenomic Agilent microarrays. Principal Component Analysis, hierarchical clustering and Ingenuity Pathway Analysis were performed. The gene expression profiles identified with HepaRG cells represented the average profiles of those obtained with the five hepatocyte populations which showed a large inter-individual variability. A number of genes involved in lipid and carbohydrate metabolism were commonly modulated by all the compounds. However, we also identified several genes specifically modulated by either glitazones or glitazars. In addition, expression of few genes was altered by only one of the four compounds. In conclusion, our study demonstrates that PPAR agonists can induce differential changes in gene profiles in human hepatocytes depending on the compound and/or the class. doi:10.1016/j.toxlet.2010.03.706
P206-018 Evaluation of potential biomarkers for thioacetamide-induced hepatotoxicity using siRNA J.S. Kang 1 , Y.N. Yum 2 , S.N. Park 2 Namseoul University, South Korea, 2 National Institute of Food and Drug Safety Evaluation, Korea Food and Drug Administration, South Korea
1
In our previous publication we compared the gene expression profiles on hepatotoxicants exposure to assess the comparability between in vivo and in vitro test systems. We investigated global gene expression from both mouse liver and mouse hepatic cell line treated with thioacetamide (TAA) and identified several common