Effects of ethanol on antioxidant capacity in isolated rat hepatocytes

PO Box 2345, Beijing 100023, China魔ol20051146):72727276orld Jourmal ofenterology IssN 1007-9327wjgwwjgnet.comELSEVIER62005 The wiG Press aner Inc. All rights reservedBASIC RESEARCHEffects of ethanol on antioxidant capacity in isolated rathepatocytesSien-Sing Yang, Chi-Chang Huang, Jiun-Rong Chen, Che-Lin Chiu, Ming-Jer Shieh, Su-Jiun Lin, Suh-Ching YangSien-Sing Yang, Liver Unit, Cathay General Hospital, Taipei CONCLUSION: These results suggest that long106, Taiwan. Chinatime incubation with higher concentration of ethanolChi-Chang Huang, Graduate Institute of Pharmacy, School of(100 mmol/L)decreased the cell viability by meansPharmacy, Taipei Medical University, Taipei 110, Taiwan, Chinalun-Rong Chen, Che-Lin Chiu, Ming-Jer Shieh, Suh-Chingreducing GRD and CAT activities and increasing lipidYang, School of Nutrition and Health Sciences, Taipei MedicalUniversity, Taipei 110, Taiwan, ChinaSu-Jiun Lin, Graduate Institute of Biology and Environment 2005 The WJG Press and Elsevier Inc. All rights reservedScience, School of Cellular and Molecular Biology, University ofNew Haven, CT 06516. United StatesKey words: Hepatocyte; Ethanol; Lipid peroxidationSupported by the Research Fund from Cathay General Hospital Antioxidant capacityin Taiwan. 93CGH-TMU-15Co-first-authors: Sien-Sing Yang and Chi-Chang HuangYang SS, Huang CCr Chen JR, Chiu CL, Shieh M, Lin SJCorrespondence to: Dr Suh-Ching Yang, School of Nutrition Yang SC. Effects of ethanol on antioxidant capacity inand Health Sciences, Taipei Medical University, Taipei 110, 250 isolated rat hepatocytes. World J Gastroenterol 2005;Wu-Hsing Street, Taipei 110, Taiwan, China. sokei@tmu. edu. twTelephone:+886-2-27361661Rax:+886-2-2737311211(46):7272-7276Received: 2005-03-18 Accepted: 2005-09-12http://www.wjgnet.com/1007-9327/11/7272.aspAbstractINTRODUCTIONAIM: To investigate dose-response and time-course of Ethanol is metabolized to acetalde by some enzymethe effects of ethanol on the cell viability and antioxidant in the body, including alcohol dehydrogenase(ADH),capacity in isolated rat hepatocytescytochrome P450 2E1(CYP 2E1), catalase(CAT), xanthineoxidase (XO), etc. Then acetaldehyde is decomposed toMETHODS: Hepatocytes were isolated from male adult acetic acid by acetaldehyde dehydrogenase(ALDH)inWistar rats and seeded into 100-mm dishes. hepatocytes mitochondria. Several studies have provided evidences forwere treated with ethanol at concentrations between 0 reactive oxygen species (ROS) generation during ethanol(C),10(E10), 50(E50), and 100(E100)mmol/L(dose metabolism, including superoxide radical", hydrogerresponse)for 12, 24, and 36 h(time course). Then, peroxide", hydroxyl radical ", and 1-hydroxyethyl radicallactate dehydrogenase(LDH)leakage, malondialdehyde Numerous studies have indicated that excessive ethanol(MDA) concentration, glutathione(GSH) level, and intake induces the mass production of free radicals in theactivities of glutathione peroxidase(GPX), glutathione body, which are considered to be associated with alcoholicreductase(GRD), superoxide dismutase(SoD), and liver disease 5l. Furthermore, a number of experimentalcatalase(CAT) were measuredstudies demonstrated that either acute or chronicalcohol administration to experimental animals increasesRESULTS: Our data revealed that LDH leakage was the formation of lipid peroxidation products, such assignificantly increased by about 30% in group E100 over malondialdehyde (MDA), and decreases tissue levels ofthose in groups C and E10 at 24 and 36 h, The MDa antioxidants, such as glutathione(GSH), in the liverlower than that in group E100 at 36 h. Furthermore, Bailey and Cunningham also indicatsignificantly higher by 4.5-and 1.7-fold, respectively, ROS, which correlated with decreased cell viability).Thethan that at 12 and 24 h. on the other hand. the gsh impairment of cellular antioxidant defenses along with thelevel in group E100 at 24 and 36 h was significantly formation of oxygen-derived radicals has been proposeddecreased, by 32% and 28%, respectively compared to pl中国煤化工 damage associated withto that at 12 h. the activities of GRD and CAT in group alcohe individual differencesE100 at 36 h were significantly less than those in groups in aCN Inconsistent: thereforeC and E10. However, The GPX and SoD activities showed the purpose of this study was to investigate and clarify theno significant change in each groupinfluences of different concentrations of ethanol on theYang Ss et al. Ethanol, hepatocyte and antioxidant capacity7273cell viability, antioxidant capacity, and antioxidant enzymes sample was added to 400 HL of 2.4 mmol/L GSSG bufferactivities in the isolated liver parenchymal cells during(dissolved in 125 mmol/L potassium phosphate buffer, pHdifferent incubation times.7.5, 2.5 mmol/L EDTA). Four hundred microliters of 0.55mmol/L NADPH (dissolved in deionized water)was addedMATERIALS AND METHODSto the mixture and grd activity was measured at 340 nmfor 5 min on a Hitachi U-2000 Spectrophotometer. ThePreparation of isolated rat hepatocytesactivity was expressed as mU/mg protein in hepatocytes.Hepatocytes were isolated from rats according to the two-step collagenase perfusion technique described by Berry Superoxide dismutase(SOD )activity of hepatocytesand Friend. Isolated cells were cultured as monolayers SOD activity of hepatocytes was measured with ain Williams medium E with 5% fetal bovine serum and commercial kit(SD 125; Randox Laboratories). FiftyAfter 24 h of incubation at 37 C in 50 mL/L CO2, of mixed substrate(50 umol/L xanthine and 25 Hmol/Lhepatocytes were treated with ethanol at concentrations 2(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazoliumbetween 0(C), 10(E10), 50(E50), and 100(E100)mmol/L chloride, INT). Two hundred and fifty microliters of Xo(dose response)for 12, 24, and 36 h(time course). Then, was added to the mixture and SOD activity was measuredthe cells were collected using a scraper and resuspended in at 37C on a Hitachi U-2000 Spectrophotometer at 505Tris buffer(50 mmol/L Tris-HCL, 5 mmol/L EDTA, and nm for 3 min The activity was expressed as U/ mg protein1 mmol/L DTT, pH 7. 5)for analysis as followsin hepatocytes.Lactate dehydrogenase(LDH) leakageCatalase(CAT) activity of hepatocytesThe viability of hepatocytes was expressed as the CAT activity of hepatocytes was determined at 25Cpercentage of LDH leakage, which was the LDH activity with Hitachi U-2000 Spectrophotometer UV-VISin the culture medium relative to the total LDH activity Spectrophotometer according to the previous study o.including the culture medium and cytosolic fraction. The Diluted sample was added to 59 mmol/L H2O2(dissolvedLDH level was determined using the method described by in 50 mmol/L potassium phosphate buffer, PH 7.0)anda previous studCAT activity was measured at 240 nm for 3 min. Oneunit of CAt activity was defined as the mmol of H2O2peroxidation of hedegraded/min/mg protein. The activity was expressed asThe MDA concentration of hepatocytes was assessed U/mg protein in hepatocytescolorimetrically at 586 nm using a commercial kitCalbiochem 437634; Calbiochem-Novabiochem, La Jolla, Total protein concentrationCA, USA). The concentration was expressed as nmol/mg In order to express the antioxidant enzymes activitiesprotcin in hepatocytesper gram of protein, total protein concentration ofhepatocytes was determined colorimetrically by using aGlutathione(GSH)concentration of hepatocytesBio-Rad dC protein assay kit(Cat. No. 500-0116; Bio-RadThe concentration of reduced GSH in hepatocytes Laboratories, Hercules, CA, USA)was measured spectrophotometrically at 400 nm usinga commercial kit( Calbiochem 354102; Calbiochem- Statistical analysisNovabiochem). The concentration was expressed as nmol/ Values were expressed as mean+SD. To evaluate themg protein in hepatocytesdifferences between the groups studied, two-way analysisof variance(ANOVA) with Fisher's post boc test was used.Glutathione peroxidase(GPX) activity of hepatocytesThe SAS software (Vers. 8.2, SAS Institute Inc., Cary, NC,GPX activity of hepatocytes was determined with a USA)was used to analyze all the data Differences werecommercial kit(RS 504; Randox Laboratories, Antrim, considered statistically significant when P<0.05UK. Twenty microliters of the diluted sample was addedto 1 mL of mixed substrate(4 mmol/L GSH, 0.5 U/L GRDand 0.34 mmol/L NADPH dissolved in 50 mmol/L RESULTShosphate buffer, PH 7. 2 4.3 mmol/L EDTA). Forty Effect of ethanol on LDH leakagemicroliters of cumene hydroperoxide(diluted in deionized LDH leakage into the culture media of hepatocyte waswater)was added to the mixture and GPX activity wused to assess the hepatotoxicity of ethanol(Figure 1)measured at 37 C on a Hitachi U-2000 Spectrophotometer LDH leakage in group E10 was nearly the same as thatat 340 nm for 3 min. The activity was expressed as mU/mg in groun G at 12 24. and 36 h. In contrast, LDH leakageprotein in hepatocytes中国煤化工 creased by about30%more24and36h(P<005)Glutathione reductase(GRD ) activity of hepatocytesFurtCNMHGowed dose-dependentGRD activity of hepatocytes was measured with a correlation with the ethanol concentrations(P= 0.0026)commercial kit(Calbiochem 359962; Calbiochem- However, there was no significant correlation betweeNovabiochem). Two hundred microliters of the diluted LDH leakage and incubation time of ethanol(P>0.05)7274 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol December 14, 2005 Volume 11 Number 464.5150305s3.0505E100Figure 1 Lactate dehydrogenase( LDH) leakage of primary rat hepatocytes in each Figure 3 Glutathione(GSH) concentrations of primary rat hepatocytes in eachgroup. Data are mean*sD for 3 hepatocyte preparations. Cultures were incubated group Data are mean*SD for 3 hepatocyte preparations. Cultures were incubatedwithout ethanol(C), with 10 mmol/L ethanol(E 10), with 50 mmolL ethanol (E 50), without ethanol ( C), with 10 mmolL ethanol (E 10), with 50 mmoWL ethanol(E 50).and with 100 mmo/L ethanol (E 100)for 12, 24, and 36 h, respectively. '"p<0.05 vs and with 100 mmo//L ethanol(E 100)for 12, 24, and 36 h, respectively. "P