ABSTRACT
Cholestasis develops during inflammation and is characterized as occurring under oxidative stress. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid or lipopolysaccharide (LPS)-induced acute oxidative stress in rat liver. However, it remains unclear whether canalicular Mrp2 internalization is observed in human liver under conditions of acute oxidative stress. In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and found in traditional Chinese medicine, on endotoxin-induced Mrp2 internalization in rat and human liver slices. At 1.5h following LPS treatment (100microg/mL), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of filamentous actin in the rat and human liver slices. Pretreatment with DMA (10microM) counteracted LPS-induced subcellular distribution of Mrp2. Our data clearly indicated that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress in rat and human liver slices.
Subject(s)
Antioxidants/pharmacology , Cholestasis/metabolism , Diketopiperazines/pharmacology , Hydroxamic Acids/pharmacology , Liver/drug effects , Multidrug Resistance-Associated Proteins/metabolism , Oxidative Stress , Animals , Antioxidants/chemistry , Cryopreservation , Diketopiperazines/chemistry , Glutathione/metabolism , Humans , Hydroxamic Acids/chemistry , Lipopolysaccharides , Liver/metabolism , Multidrug Resistance-Associated Protein 2 , RatsABSTRACT
Oxidative stress is known to be a common feature of cholestatic syndrome. Lipopolysaccharide (LPS) induces cholestasis, causing multidrug resistance-associated protein 2 (Mrp2) downregulation in two different ways: early retrieval from the canalicular membrane and the latter event of reduced mRNA expression. However, the triggering factor for LPS-induced cholestasis is not fully understood. In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and traditional Chinese medicine, on endotoxin-induced Mrp2 downregulation in rat liver. At 3h following LPS injection (4mg/kg body weight), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of tight junctions in the liver. Pretreatment with DMA (12mg/kg body weight) counteracted LPS-induced subcellular distribution, and decreased the bile flow rate and biliary glutathione (GSH) excretion. At 12h following LPS injection, Mrp2 protein and mRNA expression were significantly decreased by 58% and 7%, respectively. In contrast, pretreatment with DMA did not have any effect on the decreased Mrp2 expression and biliary excretion of GSH induced by LPS exposure. Taken together, our data clearly indicate that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress, while the long-term transcriptional regulation of Mrp2 expression did not depend on the intracellular redox status.
Subject(s)
Down-Regulation/drug effects , Lipopolysaccharides/pharmacology , Multidrug Resistance-Associated Proteins/metabolism , Piperazines/pharmacology , Animals , Gene Expression Regulation/drug effects , Liver/drug effects , Male , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/genetics , Rats , Rats, WistarABSTRACT
PURPOSE: Methotrexate (MTX) causes intestinal damage, resulting in diarrhea. The side effects often disturb the cancer chemotherapy. We previously reported that AGE protected the small intestine of rats from the MTX-induced damage. In the present paper, the mechanism of the protection of AGE against the MTX-induced damage of small intestine was investigated, using IEC-6 cells originating from rat jejunum crypt. METHODS: The viability and apoptosis of IEC-6 cells were examined in the presence of MTX and/or AGE. RESULTS: The viability of IEC-6 cells exposed to MTX was decreased by the increase of MTX concentration. The MTX-induced loss of viable IEC-6 cells was almost completely prevented by the presence of more than 0.1% AGE. In IEC-6 cells exposed to MTX, the cromatin condensation, DNA fragmentation, caspase-3 activation and cytochrome c release were observed. These were preserved to the control levels by the presence of AGE. MTX markedly decreased intracellular GSH in IEC-6 cells, but the presence of AGE in IEC-6 cells with MTX preserved intracellular GSH to the control level. IEC-6 cells in G2/M stage markedly decreased 72 h after the MTX treatment, which was preserved to the control level by the presence of AGE. These results indicated that AGE protected IEC-6 cells from the MTX-induced damage. CONCLUSIONS: The MTX-induced apoptosis of IEC-6 cells was shown to be depressed by AGE. AGE may be useful for the cancer chemotherapy with MTX, since AGE reduces the MTX-induced intestinal damage.
Subject(s)
Antimetabolites, Antineoplastic/therapeutic use , Apoptosis/drug effects , Garlic , Intestinal Mucosa/drug effects , Intestine, Small/drug effects , Methotrexate/therapeutic use , Plant Extracts/pharmacology , Animals , Blotting, Western , Caspase 3/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Cytochromes c/metabolism , Cytosol/drug effects , Cytosol/metabolism , Glutathione/metabolism , Intestinal Mucosa/pathology , Intestine, Small/pathology , Mitochondria/drug effects , Mitochondria/metabolism , RatsABSTRACT
Dimerumic acid (DMA) is contained in Monascus anka and Monascus pilosus fermented products. The purpose of this study was to evaluate the effect of DMA against salicylic acid (SA)- and tert-butylhydroperoxide (t-BHP)-induced oxidative stress and cytotoxicity in the liver, using rat liver microsomes and isolated rat hepatocytes. DMA was extracted from monascus-garlic-fermented extract using M. pilosus. In rat liver microsomes, 1 microM DMA decreased SA-induced lipid peroxidation but did not affect the production of the oxidative metabolite of SA via CYP. In isolated rat hepatocytes, 1 microM DMA decreased SA-induced lipid peroxidation and chemiluminescence (CL) generation and the intracellular glutathione-reduced form/oxidized form (GSH/GSSG) ratio in the presence of 1 microM DMA was higher than that without DMA; however, 100 microM DMA suppressed the leakage of lactate dehydrogenase (LDH). On the other hand, t-BHP-induced lipid peroxidation, CL generation, and LDH leakage were prevented by 100 microM DMA. Thus, DMA showed an antioxidative effect in hepatocytes and protected against hepatotoxicity by suppressing oxidative stress without affecting CYP enzymes.
Subject(s)
Antioxidants/pharmacology , Hepatocytes/drug effects , Oxidative Stress/drug effects , Piperazines/pharmacology , Animals , Cell Survival , Cytochrome P-450 Enzyme System/metabolism , Cytoprotection , Dose-Response Relationship, Drug , Glutathione/metabolism , Hepatocytes/enzymology , Hepatocytes/metabolism , Hepatocytes/pathology , L-Lactate Dehydrogenase/metabolism , Lipid Peroxidation/drug effects , Male , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Oxidants/toxicity , Rats , Rats, Sprague-Dawley , Salicylates/toxicity , tert-Butylhydroperoxide/toxicityABSTRACT
Gastrointestinal toxicity is one of the most serious side effects of methotrexate (MTX) treatment. The side effects often disrupt the cancer chemotherapy. We previously reported that aged garlic extract (AGE) protects the small intestine of rats from MTX-induced damage. In this study, the protection of AGE against MTX-induced damage of IEC-6 cells originating from the rat jejunum crypt was investigated. MTX decreased the viability of IEC-6 cells, but this effect was prevented by AGE (0.5%). The MTX-induced apoptosis of IEC-6 cells was depressed by AGE. These results indicated that AGE protects IEC-6 cells from the MTX-induced damage. AGE may be useful in cancer chemotherapy with MTX because it reduces MTX-induced intestinal damage.