Effects of Ethanol on Isolated Hepatocytes : Alteration in Cell Surface and Intracellular ATP

Orrc o.f the urost serious lrcput()toci.t og,ents to the liver is ethanol. Alrhough irs ro.riciry' hos been investigated, the to.tic tnechanisnr itself retnains conrroy'ersial. The ains of the present w,ork are to investigare the effect of etlnnol on the su.rface of freshly isolated hepaîrtc1.tes qfter incubatiotr v,ith ethanol, and its itdluence on cytosolic ATP-concentration. Ittcuburion with ethanol led to the specific fornation of ret'ersible blebs otr rhe surface of hepatoc)'les and a signif cant decrease (p < 0,05) o.f c1'tosolic ATP-concentratiott. Ke y v, o rd s : he pal o I o B)', e I han o I t o r i c i t1', ble b fo r n nt i o tt Liver is the main organ which metabolizes ethanol. Although the liver is at the beginning resistant against the influence of ethanol, the continous uptake of ethanol in high doses can damage liver cell producing a pathological state such as "cirrhosis". Many studies on ethanol-toxicity has been carried out, however its mechanism is still not clear, The present paper will describe two studies about the influence of ethanol on hepatocytes. Firstly, its influence to the cell surface as pictured by scarnning electron n.ricroscopy and secondly its influence on the intracellular ATP concentration. MATERIALS AND METHODS Sprague-Dawley rats (ca. 22O g) were obtained from Savo, nredizinische Versuchstierzuchten GmbH, Kissl egg/Al I gaeu, Germany. Isolated hepatocytes were prepared by collagenase perfr,rsion.l C"ll uiobility, as judged by trypan blue exclusion,' was between 85 To 95% for all preparations. Following isolation, a suspension of hepatocytes containing I 1.5 million cells/ml was transfered to plastic vials (10 ml). Ham's Fl2 medium and ethanol of different concentrations (0.3 2.6 mol/l) were added, so that the final volume was 1.0 ml in all experiments. The contents were n.rixed gently, and the vials were kept in a waterbath at temperature of 25" C for 30 min. Scanning electron microscopy I to 1.5 million hepatocytes for electron microscopic scanning were fixed in 1% glutaraldehyde in 0. 1 mol cocodylate-buffer, washed washing (2 times) with cocodylate-buffer for 15 min each, followed by the fixation with 0.5% Os (VIII)-oxide for lh. Dehydration was carried out using 50-, 70and IOO% alcohol for 5 min each. The air dried sanrples were then put on "leit-Tabs" (Plano), evaporated with gold-palladium (Sputter coater, Bio-Rad) and examined with JSM, U3 scanning electron microscope. Direkturat Pengkajian lhnu Kehidupan, BPP Teknologi, Jakarta, Indonesia Vol 3, No 4, October-December 1994 Determination of the ATP content of hepatocytes The ATP content of isolated hepatocytes was measured3'4 using Auto-Clinilumina t (LB g52T I 16) from Berthold, Wildbad. A l0O pl aliquot was taken from the cell suspension (l ml) for the viability determination. The remaining vials content 900 pl was put in crushed ice for 5 minutes to enhance the sedimention of the cells. To remove the supernatant, the cell suspension was washed 3 times each with 1 ml physiological saline. After centrifugation for 5 min (200 x g) at 4oC, the supernatant was carefully pipetted out and 900 pl TCA (57o) was added to the cells. The cell suspension was then homogenized using a Branson Sonifier. After centrifugation at 3000 x g for 15 min at 4oC, 50 pl of supernatant was diluted 1:41 (vol/vol) with physiological saline. The measurement was done 5 seconds after 60 pl of the diluted solution was nrixed with ATP-reagent. Figure I. Sccurtritrq1 elcctron nicrogruph of a nonuol,.freshll, isolated hepatocl'te M u gn i.fi ut i o trs : -\U)O Ethanol Effects ott Hepatocytes 2O9 Diluted 5 mmol/l ATP-NazHz .3HzO was used as ATP-standard. During the measurement, the sample were stored at cold temperature.