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TOXICOLOGY AND Al'I'l H O I'llARM ACOI.OGY 102, 80-40 ( 144(1) Interference with Hepatocellular Substrate Uptake by 1,1,1-Trichloroethane and Tetrachloroethylene1 Veerapol Kukongviriyapan,2 Upa Kukongviriyapan,2 and Neill H. Stacey National Institute ofOccupational Health and Safety, The University ofSydney. NSU' 2006. Australia Received April 5, 1989; accepted August 8, 1989 Interference with Hepatocellular Substrate Uptake by 1,1,1-Trichloroethane and Tetrachloroethylenc, Kukongviriyapan, V,, Kukongviriyapan, U,, and Stacey, N. H. (1990). Tox icol. Appl. Pharmacol, 102, 80-90. The effects of chlorinated aliphatic hydrocarbon solvent exposure on hepatocellular transport ofsome model substrates have been investigated. Exposure of isolated hepatocytes to 1,1,1-trichloroethane or tetrachloroethylene resulted in suppression of uptake of taurocholate, ouabain, and 2-aminoisobutyric acid but not CdCL or 3-O-methylD-glucose. The effect was clearly evident at noncytotoxic concentrations, as indicated by the lack ofintracellular enzyme leakage and unaltered intracellular K* ion concentration. Moreover, the ultrastructure of solvent-exposed hepatocytes was similar to that of control cells, except for a reduction in membrane microvilli. The suppression of uptake was reversible provided that sufficient time was allowed for the cells to recover. The mechanism of this inhibition may be associated with energy-linked processes, as uptake of taurocholate. ouabain, and 2-aminoisobu tyric acid is energy requiring while uptake of CdCl- and 3-O-methyI-o-glucose is not. Cellular ATP was reduced in a dose-dependent manner, but a marked depletion occurred only at cyto toxic concentrations. Na+-K+- and Mg^-ATPase activities in hepatocyte plasma membrane preparations were also inhibited by solvent exposure. The data suggest that 1,1,1 -trichloroethane and tetrachloroethylene interfere specifically with energy-dependent hepatic transport functions and that a decrease in ATP levels and/or inhibition of cell membrane ATPases may be the mechanism. t?90 Academic Press, Inc. The hepatotoxicity of organic solvents has been extensively studied, especially the mechanisms by which solvents induce cyto toxicity (Reynolds and Moslen, 1974; Berger et al., 1986). Elevations of serum bile acids have been reported in workers who were oc cupationally exposed to organic solvents (Franco et al., 1986). Interestingly, the in crease in the level of bile acids was not always consistent with increases in serum liver en- 1 Presented, in part, at the 22nd Annual Meeting ofthe Australasian Society ofClinical and Experimental Phar macologists, Adelaide, Australia, 1988; Abstract 075. 2 Permanent address; Departments of Pharmacology and Physiology, Faculty of Medicine, Khon Kaen Uni versity, Kohn Kaen 40002, Thailand. zymes (GPT, GOT, and GGT), which are usually regarded as indices of liver injury (Kaplowitz et al.. 1973; Liss et al.. 1985). In fact, the elevation of serum liver enzymes would primarily reflect the acute disruption of hepatocellular membranes (Zimmerman and Seeff, 1970) rather than liver dysfunction (Editorial, 1982). Therefore, increased serum bile acid levels may be either a very early sign of hepatocellular toxicity or a sign of liver dysfunction, which is not necessarily associ ated with cytotoxicity. Consequently, it is of interest to investigate whether exposure to or ganic solvents leads to the impairment of he patocyte transport function, especially ofbile acids, at a level that does not cause cytotoxic ity. This approach is consistent with the views 0041-008X/90 $3.00 Copynght 1990 by Academic Press, Inc All rights ofreproduction in any form reserved Stacey 1 astralla chloToxIvent isure sion ihyllack r. the for a that iy be >obu- lular ,ytoirane hane \*. 1 | ), which are liver injury ii, 1985). In ver enzymes e disruption Zimmerman dysfunction reased serum ;ry early sign sign of liver sarily associently, it is of posure to orrment ofhexially of bile se cytotoxicith the views CHL.ORINAITD SOI VLNI'S AND CEI.l TRWSPORT 81 expressed by Tamburro and Greenberg (1981). that more appropriate means to mon itor liver function to adequately rellect expo sure to solvents are required. The present experiments were therefore designed to investigate whether organic sol vents interfere with the processes responsible for hepatocellular transport of bile acids and other model substrates transported by the liver. Appearance of cytotoxicity or necrosis was monitored biochemically and by elec tron microscopy. Finally, we explored possi ble mechanisms for observed interferences with hepatic transport. Isolated hepatocyte suspensions were used in these experiments, as this system is well studied, highly repro ducible, and relevant to the in vivo situation with regard to both the hepatic uptake activi ties (Christensen, 1975; Schwarz el al,, 1975; Eaton and Klaassen, 1978; Stacey and Klaassen, 1980) and the production of cytotoxicity (Klaassen and Stacey, 1982; Guillouzo, 1986). 1,1,1-Trichloroethane (TCE) and tetrachloroethylene (TET) were chosen as they have some associations with liver injury and are both widely used in industry (Com mission of the European Communities, 1986a,b). MATERIALS AND METHODS Chemicals. Tauro[carfoy/-'4C]cholic acid, sodium salt, [21,22-3H]ouabain, and carrier-free '"VdCL were purchased from Amersham (Sydney, NSW); 3H20, dextran-carboxyl[carfoxy/-'4C], 2-amino[ l-'4C]isobutyric acid (A1B), and 3-<9-methyl-D-[U-l4C]glucose (3-OMG) were from New England Nuclear (Sydney, NSW). The ATP bioluminescent assay kit and all unlabeled com pounds were from Sigma Chemical Co. (St. Louis, MO). Highest purity 1,1,1-trichloroethane and tetrachloroethylene were obtained from Aldrich Chemical Co. (Mil waukee, WI). Isolation of hepatocytes. Hepatocytes from male Sprague-Dawley rats (300-400 g) were prepared by in situ perfusion of the liver with 0.03% collagenase (type IV, Sigma). The method followed that of Beny and Friend (1969) with some modifications (Kukongviriyapan and Stacey, 1988). Hepatocytes were resuspended in Hanks' buffer supplemented with 20 mM V-2-hydroxy- ethylpipera/iiic-.V-2-ethano sulfonic acid (Hepes) (medium A) at a concentration of 1.4 % 10" cells/ml. Initial viability of hepatocytes always exceeded 90% as deter mined by trypan blue exclusion Solvent exposure and substrate uptake Medium A was used as the incubation butler in all uptake studies, except for the 3-OMG, where glucose was omitted. Hepatocyte suspensions (1.9 ml) were incubated in 25-ml Erlenmeyer flasks at 37"C with continuous shaking. TCE or TET, at various volumes, was applied against the wall of the incubation flask by microsvnnge to allow the solvents to evaporate into the gas phase. The flasks were sealed immediately and incubated for 20 min. Uptake of sub strates (10/iM taurocholate, 125 iim ouabain, l mM AIB, 2 pM CdCL .and 15 mM 3-OMG) commenced upon the addition of 0.1 ml of substrate containing radiolabeled substrate, fn studies with 3-OMG the suspensions were exposed to solvent at 37*C for 20 min and cooled down to 24*C in another water bath for 5 min before the deter mination of the uptake. Aliquots of cell suspension were removed at appropriate times and the radioactivity in the pellet and supernatant was determined by using the sili cone oil centrifugation technique (Eaton and Klaassen, 1978). Samples were also taken at the end ofeach experi ment for determination of protein content (Lowry el ai, 1951) and cell membrane integrity, by the measurement of extracellular lactate dehydrogenase (LDH) and ala nine aminotransferase (ALT) activities as well as intra cellular potassium ion concentration, as previously de scribed (Stacey et ai. 1980). Intracellular xvater space and adherent fluid were estimated using 3HjO and dextrancarboxyl[ca/fw;cW-l*C] according to Eaton and Klaassen (1978). To investigate the reversibility ofthe uptake inhi bition, cell suspensions exposed to the solvents were washed twice by centrifugation at 50; and further incu bated for 20 min in the solvent-free medium prior to de termination of AIB uptake. ATP assays. Cell samples were taken after the expo sure to solvent and extracted with cold 2.5% trichloroace tic acid for 1 hr. The denatured protein was removed by centrifugation and samples were assayed immediately for ATP content by a bioluminescence method (Leach and Webster, 1986). Preparation of plasma membranes and exposure to TCE and TET Plasma membranes from rat livers were prepared according to Loten and Redshaw-Loten (1986) with minor modifications. Briefly, the liver was perfused in situ to remove blood cells with ice cold buffer (250 mM sucrose, 10 mM Tris-HCI, pH 7.5), then excised, minced, and homogenized in a loose-fitting Dounce homogenizer with 8 strokes in the same buffer. The homogenate was diluted to 200 ml and centrifuged at 1500; for 15 min. The pellet was resuspended and diluted to 75 ml and mixed with 10.1 ml of Percoll (Pharmacia, Uppsala, Swe den) and 1.45 ml of 2 m sucrose. The resulting mixture was centrifuged at 35,000; for 20 min. The plasma mem brane band was collected, homogenized with 20 strokes 03630A SL 82 KUKONGVIRIYAPAN. KUKONGVIRIYAI'AN. AND STACEY in a Dounce homogenizes diluted to 75 ml, mixed with 10.1 ml of Percoll, 1.45 mlof2 m sucrose, and 0.28 ml of400 mM CaCI2, and centrifuged at 45,000.1,' for 10 min. The membrane band was harvested and washed twice by centrifugation at 4500# for 10 min. The punty of the preparation was analyzed by intracel lular and plasma membrane marker enzymes. Succi nate-cytochrome c reductase and NADPH-cytochrome c reductase were measured by the methods of Sottocasa et al. (1967), alkaline phosphatase was measured by the procedure of Walter and Schutt (1974), and Na'-K*ATPase and Mg"*-ATPase were measured by the record ing spectrophotometric method of Scharschmidt et al. (1979). Plasma membrane ATPase activity after expo sure to TCE or TET was determined by the following method: Aliquots (40 mO of plasma membrane suspen sions (0.5 mg protein/ml) in 1.89 ml of ATPase assay buffer (to the final concentration of 125 mM Tris, I mM EGTA, 120 mM NaCI, 12.5 mM KCI, 5 mM NaN3, 2.5 mM phosphoenolpyruvate, 0.5 mM NADH, and with or without 2 mM ouabain) were preincubated for 1 min at 37"C. Various amounts ofTCE or TET were applied via microsyringe against the wall of the incubation flask, which was then capped tightly and further incubated for 6 min with continuous shaking. Thereafter, 20 mI of an enzyme mixture containing lactate dehydrogenase and pyruvate kinase (20 units of each) and 50 mI of ATP in MgCl2 solution (to final concentrations of 5 mM) were added and mixed well. The assay mixture was immedi ately transferred to a prewarmed cuvette and sealed with a teflon cap before the rate of NADH oxidation at 340 nm was recorded. In a preliminary study, it was verified that under these experimental conditions solvents did not alter the activities of the coupling enzymes ofthe as say system. This can be seen from the comparable rates of NADH oxidation between the assay mixtures which were exposed or not exposed to solvents where ATP was substituted with 0.2-1 Mmole of ADP in order to initiate the reactions. Quantitation of TCE and TET by gas chromatogra phy. Cell-free medium or plasma membrane-free me dium was obtained by rapid centrifugation of samples in capped microfuge tubes at 10,000# for a few seconds or 20 seconds, respectively. The supernatant was extracted with heptane (Hipersolv, BDH, UK) and samples were kept on ice in glass-stoppered tubes without head space until analyzed. Microliter volumes were injected into a gas chromatograph (Varian, Model 3300 with Integrator Model 4290) equipped with a 2 mm X 6 ft column (0.1% SP-1000 on 80/100 Carbopack) and an electron capture detector. Temperature conditions were: injector, 250*C; detector, 250*C; column, 120*C for TCE and <80*C for TET. Electron microscopy. Hepatocytes exposed to TCE or TET at various concentrations were fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer for 1 hr and then postfixed in 1 % phosphate-buffered osmium tetroxide for I hr. The specimens were dehydrated through graded ac etone solutions, embedded m Spurr's resin, sectioned on an Ultracut E ultramicrotome, and stained with uranyl acetate and Reynolds' lead citrate prior to examination with a Phillips 400-HMG electron microscope. Data analysis. Calculation of intracellular K* concen tration was based upon the !H,0 estimation of an intra cellular water space of 3 rd/mg cell protein. Initial rates of uptake were determined from the slopes of the linear portions ofthe curves which were over 1,2,5.and lOmin for taurocholate, CdCl,, ouabain, and AIB, respectively. Uptake of 3-OMG was determined at 15- and 30-sec in cubations. Data were evaluated by analysis of variance with Duncan's test. Statistical significance was set at p < 0.05. RESULTS Cytotoxic effects of TCE and TET. When TCE (2, 5, or 10 *d per flask) and TET (1,2, and 4 M1 per flask) were vaporized, steady state concentrations of TCE and TET in the medium were established within 5 min (Ta ble 1). The application ofTCE to the flask did not significantly change the cell viability as there was no increase in LDH and ALT re lease into the medium, and no decline of in tracellular K+ concentration (Table 1). TET, on the other hand, at 4 /d/flask caused a re markable cell membrane damage, as there were large increases in LDH and ALT activi ties in the medium and a marked reduction in intracellular K+ concentration. The appli cation of 1 and 2 *d/flask did not consistently alter the cell viability. Ultrastructure of hepatocytes treated with solvents. When control hepatocytes were in cubated in medium A for 20 min, numerous microvilli were apparent on the cell surface membrane and the hepatocellular ultrastruc ture was generally well preserved (Fig. 1A). Exposure of the isolated hepatocytes to 2-10 /d TCE for 20 min did not lead to any signifi cant changes in the ultrastructure, except for a reduction in microvilli on the cell surface, particularly at the highest dose of TCE (Fig. IB). Similarly, exposure to 1-2 pi TET re sulted only in the reduction ofcell surface mi crovilli. Other ultrastructural features were similar to control. However, 4 pi TET (Fig. SL 036305 Experiments groups Control TCE 2 M1 TCE 5 m1 TCE 10 m1 TET l m! TET 2 mI TET 4 mI " Values a * Values a ` Values v 1C) resu1 marked c reticulur mic retit files, and Moreovt observec examina were cot and sugj to TCE. and met cause ai Upta, rochola a dose-< pi caus late up suited i Uptc uptake depres: all con these i ouaba' gree o borne ing lo ATE affect ough graded acresin, sectioned on uiined with uranyl tor to examination eroscope. ellular K+ eoncenmation of an intrarotein. Initial rates slopes of the linear 1.2,5, and 10 min I AIB, respectively, t 15-and 30-sec in* nalysis of variance cance was set at p id TET. When and TET (1, 2, -torized, steady md TET in the nin 5 min (Tato the flask did :ell viability as 3 and ALT rea decline of in- Iblel). TET, "caused a re mage, as there nd ALT activirked reduction ion. The appiiiot consistently es treated with ocytes were innin, numerous .he cell surface ular ultrastrucrved (Fig. 1A). tocytesto 2-10 :i to any signifiture, except for he cell surface, >e of TCE (Fig. -2 id TET re cell surface mi! features were 4 (il TET (Fig. fI > CHLORINATED SOI.VENTS AND CELL TRANSPORT 83 TABLE I Concentrations of TCE and TET in ihe Meoium and Effects on Release ok Liver Enzymes and Intracellular K.' Content Experimental groups Control TCE 2 TCE 5 pi TCE 10 A TET 1 pi TET 2 A TET 4 mI Concn. (mm)" _ 234 17 526 67 1011 91 21+4 38 4 80 16 LDH (units X liter-1)'1 161 26(A)* 180 40 (A) 168 32 (A) 176 31 (A) 150 34 (A) 154 34(A) 364 85(B) ALT (units X liter-1)'1 43.8 3.8(A) 54.3 5.0(A) 51.0 5.0(A) 60.0 4.8(A) 48.3 10.0(A) 53.3 8.9(A) 185.0 8.8(B) K+ (mM)* 126.2 6.2(A) 129.0 1.3(A) 127.2+ 2.0(A) 116.3 5.4(A) 121.6+ 4.1(A) 118.3+ 9.8(A) 35.5+ 18.8(B) " Values are the means + SE of three separate determinations. h Values are the means SE ofduplicate determinations each from four experiments. 1 Values with the same capital letter are not significantly different (p < 0.05). 1C) resulted in the total loss of microvilli, marked dilation and whirling ofendoplasmic reticulum, degranulation of rough endoplas mic reticulum, aberrant mitochondrial pro files, and the formation oflamellar structures. Moreover, cytoplasmic protrusions were also observed in some cells. The results from the examination of hepatocyte ultrastructures were consistent with the biochemical profiles, and suggested that the condition of exposure to TCE at all concentrations studied, and low and medium concentrations of TET, did not cause appreciable cytotoxicity. Uptake oftaurocholate. The uptake of taurocholate was inhibited by TCE and TET in a dose-dependent manner (Fig. 2). TET at 4 fil caused a marked depression of taurocho late uptake, but this concentration also re sulted in marked cytotoxicity. Uptake of some model substrates. Initial uptake of ouabain and AIB was significantly depressed by TCE and TET (Figs. 3 and 4) at all concentrations employed. It appears from these data that AIB is more sensitive than ouabain to inhibition by TCE but that the de gree of inhibition by TET is similar. This is borne out by the subsequent experiments us ing lower concentrations of TCE and TET. A TET concentration of0.25 fd/flask did not affect the uptake of AIB and ouabain. TCE at 0.5 /d/flask still significantly inhibited the uptake of AIB but not ouabain and at 0.25 /d/flask had no detectable effects (data not shown). In contrast, the initial uptake of CdCL or 3-OMG were not affected signifi cantly by the exposure to solvents (data not shown). Reversibility ofthe inhibition by TCE and TET. AIB uptake activity was fully restored in hepatocyte suspensions previously ex posed to TCE or TET at subcytotoxic con centrations following the washing and rein cubation ofcells in open air for 20 min (Table 2). Reincubation times of less than 20 min resulted in only partial recovery ofthe uptake activity (data not shown). ATP content. The solvent exposure re sulted in a dose-related decline in intracellu lar ATP content (Table 3). TET at 4 gzl/flask produced a marked depletion of ATP which was consistent with the cell damage at this concentration. Effects on membrane ATPase activities. The profile of marker enzyme activities and enrichments in purified membrane fractions, compared with starting homogenates, indi cates that the preparations were derived mainly from hepatocyte plasma membrane. Na+-K+-ATPase and Mg2+-ATPase were en riched approximately 8- to 14-fold and alka- SL 036306 84 KUK.ONGVIRIYAPAN, KUKONGVIRIYAl'AN. AND STACEY Fig. 1. Transmission electron micrographs from control and solvent-exposed isolated rat hepatocytes. (A) Control cell incubated for 20 min in medium without solvent Note microvilli on cell surface (inset X3450), ER, and mitochondria (X17,000). (B) Exposed to lOjd/flask ofTCE for 20 min. Surface microvilli are greatly reduced (inset X3450) although mitochondria retain the characteristic coupled configuration and rough ER remains intact (X22,000). (C) Exposed to 4 pl/flask of TET for 20 min. Loss of surface microvilli, cytoplasmic protrusion (inset X3450). Aberrant mitochondrial profiles, degranulation, and dila tation of rough ER (X17,000). SL 036307 line phosph tivities for e cytochrotni nmoU 1 2 prot # O 0.6 Q. 3 o -4to-* 0.4 o O -- 0.0 Fig. 2. Eff of taurochol ous amount the vapor pi of 10fiM tat capital lette = 4 or 5). CHLORINATED SOLVLNIS AND CELL TRANSPORT 85 Fig. I--Continued line phosphatase 10-fold. Marker enzyme ac tivities for endoplasmic reticulum (NADPHcytochrome c reductase) and mitochondria nmol# t 2 mirt-mg prof O 0.8 a 3 O 10 /aM Taurocholate A (succinate-cytochrome c reductase) were ap proximately 0.5- and 0.05-fold that of ho mogenate. Concentrations of TCE and TET in the assay media after incubation for 6 min were 2 S 10 1 2 4 Control TCE (mI) TET (/il) Fig. 2. Effect ofTCE and TET on initial rate of uptake of taurocholate. Hepatocytes were incubated with vari ous amounts f>d) ofTCE or TET which were applied via the vapor phase for 20 min and initial velocity of uptake of 10 /im taurocholate was measured. Bars with the same capital letters are not significantly different (p < 0.05, n = 4 or 5). Fig. 3. Effect ofTCE and TET on initial rate ofuptake of ouabain. Hepatocytes were incubated with various amounts of TCE or TET which were applied via the va por phase for 20 min and initial velocity ofuptake of 125 aM ouabain was measured. Bars with the same capital letters are not significantly different (p < 0.05, n = 3). SL 036308 86 KUKONGVIRIYAPAN. KUKONGVIRIYAPAN, AND STACEY 1 mM AIB TABLE 3 TCE andTET Effect on Intracellular ATP Content Experimental groups ATP (nmole/mg protein)0 Control TCE (/it) TET (d) Fig. 4. Effect of solvents on initial rate of AIB uptake. Initial rate of 1 mM AIB uptake was determined after hepatocytes had been exposed to TCE or TET for 20 min. Bars with the same capital letters are not signifi cantly different (p < 0.05, n = 3). Control TCE 2 d TCE 5 d TCE 10 d TET 1 d TET 2 d TET 4 pi 19.92 0.49 (A)'' 17.48+0.89(B) 16.51 + 0.86(B) 13.09+ 1.10(C) 17.42 0.94(B) 16.53+ 1.14(B) 6.69+ 1.54(D) " Values are the means SE from four experiments. h Values with the same capital letter are not signifi cantly different (p < 0.05). DISCUSSION approximately the same as those found in the cell suspension experiments (data not shown). Exposure of hepatocyte plasma membranes to TCE and TET resulted in a de crease in ATPase activities (Table 4). The in hibition was dose dependent and occurred to about the same degree with both Mg2+- and Na+-K+-ATPases. TABLE 2 Reversibility of TCE and TET Suppression on AIB Uptake Experimental groups Initial rate of uptake (nmole/min/mg protein)" Control'' TCE 5 pld TCE 5 pi and wash out'1 TET 2d' TET 2 d and wash out'' 30.61 + 2.72(A)' 12.90 + 4.27(B) 28.71 +4.05 (A) 12.79 +3.04(B) 25.80+ 3.69 (A) 0 Values are the means SE from four experiments. * Hepatocytes were incubated with or without solvent for 20 min, washed twice by centrifugation, and further incubated in solvent-free buffer for 20 min before deter mination ofAIB uptake. ` Values with the same capital letter are not signifi cantly different (p < 0.05). d Determination ofthe uptake was performed immedi ately after incubation with the solvent. Correlations between in vitro and in vivo hepatotoxicity have been observed with sev eral chemicals tested (Tyson et al,, 1983a; Ty son, 1987; Tyson and Stacey, 1989). Further more, some chemicals which cause impair ment of hepatic transport function in vivo have been reported to suppress the uptake or efflux of bile acids in isolated hepatocytes (Stacey, 1986, 1988; Van Dyke and Scharschmidt, 1987; Stacey and Kotecka, 1988). Consistent in vivo and in vitro results may al low the use of this in vitro system to study the possible mechanism which occurs in the in tact organism. The inhibition oftaurocholate transport is of particular interest as it may ac count for shifts in serum bile acids observed in tetrachloroethylene-treated tats in the ab sence ofother signs ofhepatotoxicity (unpub lished observations). Isolated hepatocytes exposed to TCE or TET at or below 10 /d/flask or 2 /d/flask, re spectively, gave no evidence of cell death by biochemical assays of cell membrane integ rity. Ultrastructural examination is consis tent with the biochemical assays, as there was no appreciable alteration compared to con trols, except for a reduction and shortening of microvilli. In contrast, cells exposed to TET at 4 jd/flask showed evidence of cell death by SL 036309 1 t cellular mole/mg protein)" 72 0.49 (A)'' *8 0.89(B) 11 0.86(B) )9 1.10(C) 12 0.94(B) S3 1.14(B) .9 1.54(D) <ur experiments, er are not signifi- o and in vivo ved with sevil.. l983a;TyJ89). Furthercause impairiction in vivo the uptake or ^hepatocytes ^Rnd Schartecka, 1988). ssults may aln to study the urs in the intaurocholate . as it may ac:ids observed ats in the abicity(unpub- I to TCE or l ul/flask, re cell death by ibrane integ>n is consis. as there was ared to conihortening of ased to TET cell death by CHLORINATED solvents and cell TRANSPORT 87 TABLE 4 Inhibition or ATPasc An l vines by TCE ano TET Experimental group" Control TCE TCE TCE TET TET TET Amount ofsolvent (Ml/flask) _ 2 5 10 1 2 4 Specific activity (jimole Pi/mg protein/hr) Mg'+-ATPase Na*-KC-ATPase 30.16 2.12/'(A)` 26.51 2.12 (A, B) 22.55 + 2.94 (B.C) 16.86 1.18 (C, D) 27.67 2.18 (A) 20.55 1.21 (B, D) 15.12 1.67 (D) 10.74 1.48(A) 9.26 1.81 (A, B) 5.78 1.25(B) 5.72 0.99(B) 7.67 1.23 (A, B) 8.40 1.21 (A, B) 6.80 0.51(B) " Hepatocyte plasma membrane was incubated with assay mixture as described under Materials and Methods. ATPase assay reaction was initiated after mixture containing plasma membrane had been exposed to solvents for 6 nun. h Values are means SE of triplicate determinations from each ofthree separate membrane preparations. ` Values with the same capital letters are not significantly different (p < 0.05). marked release of cytoplasmic enzymes, loss of K.+, and subcellular changes. Dysfunction in transport by hepatocytes is clearly demon strated at solvent concentrations which are not associated with cell death or irreversible cell damage, for which we have used the term cytotoxicity elsewhere in this paper. The loss of cell membrane microvilli has been pre viously reported (Perrissoud et ai, 1981; Sta cey and Fanning, 1981; Tyson et ai, 1983b) in isolated hepatocytes exposed to CC14 and was suggested to be a consequence of the effects which lead to the cell death, because the concentrations of CC14 used also caused cell membrane damage, as indicated by the leakage of intracellular enzymes. In contrast to this previous interpretation, our data sug gest that the reduction of microvilli may well reflect a dysfunction of the hepatocytes but is not necessarily associated with cell death. The actual concentrations of solvents in the buffer determined by gas chromatogra phy were about 10 times higher for TCE than TET (Table 1) despite administration of comparable quantities (TCE, 20, 50, and 100 pmoles/flask; TET, 10, 20, and 40 mmoles/ flask). The lower concentration ofTET in the medium is likely to be due to lower water/air partition coefficients (TCE and TET are 0.93 and 0.43, respectively) and to high lipid solu bility (oil/water partition coefficients for TCE and TET are 383 and 4458, respectively) (Sato and Nakajima, 1979). Thus, more TET would be expected to readily partition into the cells, and this may be the reason why TET is apparently more cytotoxic than TCE in spite of the lower concentrations found in the medium. It is noted that the degree of inhibi tion of taurocholate, ouabain, and AIB trans port by TCE and TET is not the same. A pos sible explanation is that the three substrates are transported by distinct systems (Eaton and Richards, 1986; Shotwell et ai, 1983), al though there is some contention that ouabain may be taken up into the liver cells by the bile acid transport system (Frimmer and Ziegler, 1988). The interaction of solvent with transport mechanisms is not specific for a particular system as we observed interference with at least three probable transport systems. How ever, general perturbation of plasma mem brane lipid resulting in the alteration of fluid ity or mass density of lipid bilayer is rather unlikely (Franks and Ueb, 1978), as the up takes of 3-OMG and CdCl2 are not affected. Nevertheless, we cannot entirely rule out that possibility in the present experiments. Direct SL 036310 88 KUKONCIVIRIYAPAN. KUKONGVIRIYAPAN, AND STACEY S measurement of membrane fluidity by fluo rescence polarization may provide more con clusive evidence. 3-OMG, a hexose sugar, is known to be transported by facilitated diffu sion (Craik and Elliott, 1979). On the other hand, CdCl2 uptake is largely by simple diffusion (Stacey and Klaassen, 1980). How ever, at low concentrations of CdCL, a satu rable component of uptake is evident (un published observation) but this has not yet been proven to be carrier mediated. Neither of these transport systems require energy. In contrast, the transport systems for taurocholate, ouabain, and A IB are all energy depen dent (Schwarz et al., 1975; Le Cam and Freychet, 1977; Eaton and Klaassen, 1978). Berger et al. (1986) have previously re ported that CC14 at concentrations that did not cause intracellular enzyme leakage strongly inhibited hepatocellular oxygen con sumption. The effect was immediate and probably mediated by direct solvent action. This suggests that subcytotoxic concentra tions of solvent may ultimately cause deple tion of cellular ATP, which is consistent with our findings as there is a dose-response rela tionship between concentration of solvent and cellular ATP level. This effect may be dis tinct from the cytotoxic effect, since there was a large decrease in ATP at the clearly cyto toxic concentration of TET (Table 3). How ever, a linear relationship between the cellu lar ATP level and inhibition of uptake does not necessarily indicate a cause and effect re lationship. The inhibition of uptake could re sult from other independent effects or the de pletion of ATP may play a partial or indirect role, as it was observed that the small decrease in cellular ATP occurred concurrently with the marked reduction in uptake, particularly that of AIB. Joseph et al. (1978) have shown that uptake of alanine and serine decreased in direct proportion to the declining level of cellular ATP. Since AIB is an alanine analog and transported largely by the same mecha nism as alanine (Kilberg, 1986), the disparity between the degree of inhibition of AIB up take and ATP depletion suggests that other mechanisms may also contribute to the sup- ,g pression of transport. $ It has been recognized that some halo- genated compounds can inhibit Na+-K+- ATPase in several membrane preparations (Koch et al., 1971; Sharom and Mellors, 1980) including hepatocyte plasma mem branes (Rufeger and Frimmer, 1976). Indeed, the Na+-K+-ATPase pump is thought to be the ultimate driving force for most of the sec ondary active transport systems (Chris tensen, 1975). Our data show that TCE and TET, in the concentration range studied, in hibited membrane Na+-K+-ATPase activity. Our data are also consistent with the report that inhibition by low concentrations of halo- genated compound is reversible (Rufeger and Frimmer, 1976). Therefore, interaction be tween TCE or TET at low level concentra tions with membrane components does not result in denaturation of, or covalent interac tion with, membrane protein. Moreover, the dissipation of transmembrane Na+ gradient by inhibition of the Na+-K+-ATPase may not be directly responsible for the observed inhibition, since uptake of ouabain is sodium independent (Eaton and Klaasson, 1978). It is possible that dissipation of the Na+ gradi ent may result in diminution ofother second ary or tertiary active transports which ulti mately drive the transport of ouabain. In conclusion, it has been shown that TCE and TET inhibit the uptake of three actively transported substances but are without effect on uptake of those not requiring metabolic energy. The presence ofthese chlorinated hy drocarbons caused a small depletion of ATP and an inhibition ofcell membrane ATPases, suggesting that this could be the mechanism ofthe observed inhibition ofthe uptake ofthe actively transported substrates. ACKNOWLEDGMENT The authors are grateful to the staffofthe Electron Mi croscopic Unit, University ofSydney, for the use of their facilities and for their helpful advice. 1 to the sup- some halonit Na4-K+preparattons and Mellors, lasma mem976). Indeed, hought to be ost of the sec erns (Chrishat TCE and e studied, inPase activity, ith the report itionsofhalo(Rufegerand teraction beel concentraents does not alent interacloreover, the Na+ gradient ATPase may the observed ain is sodium fi, 1978). 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