Document MJOmmaD9bOoE0jVw3NyQxQmYM

ARCHIVES of BlOdEKOTItT AND BIOPHYSICS Vol 218, No. 1. October 1, pp. 2&-, 1982 1,1-Dichloroethylene Inhibition of Liver Microsomal Calcium Pump in Vitro PRABHATI RAY1 and LEON MOORE Dtpartmmt <jT Pkarmacoievv. Uniformtd Sanricm Urooarmty of tko Htolth Sdtnctt, Bathmda, Maryland K>tU Baeeivtd Jusuxry 26, 1982. and in rvvbud forte lisp 10.1982 Rat liver microsomes were preincubated with 1,1-dichloroethylene (1,1-DCE) and NADPH-generating system for 20 min, then ER calcium pump activity determine; Calcium pump activity was inhibited as a function of 1,1-DCE concentration. 1,1-DC did not inhibit in the absence of a NADPH-generating system. Calcium pump activit was inhibited to a significantly greater extent by 1,1-DCE in microsomes isolated froi pbenobarbital-pretreated rata than in microsomes from control rats. These studit suggest that the mixed function oxidase system generates a metabolite that attack the endoplasmic reticulum (ER) calcium pump. The time course of pump inhibition b Ll-DCE and CCL were compared in vitro. 1,1-DCE produced inhibition of the calciur pump at a substantially slower rate. CCL at 1 iditer/ml inhibited calcium pump activit within 1 min. 1,1-DCE did not produce significant inhibition within 1 min, but produce S8 5% inhibition by 20 min. CCL but not 1,1-DCE, produced lipid peroxidations judged by malonic dialdehyde production. It is possible that lipid peroxidation cor tributes to the rapid course of inhibition produced by CCL- These studies suggest ths metabolic activation, but not lipid peroxidation, is'a prerequisite for inhibition of th liver ER calcium pump by chlorinated hydrocarbon hepatotoxins in vitro. 14-Dichloroethylene (1,1-DCE* vinyli- liver endoplasmic reticulum in matur dene chloride) is a potent hepatotoxin en rats (4) and then is no evidence that thi countered in our environment In general, compound increases lipid peroxidation i toxicity of 1,1-DCE may be considered vivo (5). Qualitatively other aspects of th quantitatively comparable to CCL (1). - action of these two chlorinated hydrocar However, qualitatively the actions of these compounds differ in certain aspects. Mor phologically the first evidence of CCL-induced damage occurs at the liver endo plasmic reticulum (2). One of the first biochemical changes noted after CCL administration is a marked increase of lipid peroxides in liver membranes (3). After 1,1-DCE there is little or no mor phological evidence of early damage to the bon hepatotoxins are similar. Previou work from our laboratory shows that hot CCL *nd 1,1-DCE inhibit the liver endc plasmic reticulum (microsomal) calciur pump after in vivo administration, j prompt, dose-dependent inhibition of thi ATP-dependent calcium pump occurs af ter CCL and 1,1-DCE (6, 7). Other labo ratones have demonstrated similar ef fects of other hepatotoxins (8, 9) and an 'Author to whom all corrwpondtnce should be addressed: Department of Pharmaeotety, USUHS, 4301 Jones Bridge Rd* Betheada, Md. 20814. * Abbreviation* need: 1,1-DCE, 1,1-dichloroethy oxia (10). Inhibition of the endoplasmic reticulun calcium pump appears to be ne of th' earliest effects of bepatotoxic chlorinate; lene; TK buffer, 0.1 u KQ, 008 M Trie. pH 1.4; MFO, hydrocarbons in the liver (9). It is possibl mixed function oxidase. that inhibition of this pump by hepato 0003-9861/E2/110026-05*02.00/0 Gftnrrisfcl C ttto by AcBidwir Fmo. knc. AH rtffet* *f ripredertit to My f#n* fiiwii. * M SL 066254 m ( t n v , y / , / a / / / , a ,i a >\ 14-DCE AND MICROSOMAL CALCIUM PUMP IN VJTK) 27 Pic. L Inhibition of liver mkrooome calcium pump in vitro by various concentrations of 1J-DCE. After 20 mis of incubation with tbc indicated concentration it LJ-DCE and is tb pwag of (O) at absence ) of NADPH generating system, calcium pomp ictieity vu determined a* described under Material* ind Method* Each point repreecau the mean SEM 'or the determination with microtome preparation* tolated from three anitnala No SEM is indicated for be experiment! without a NADPH generating syscm because the SEM fell within the area occupied 7 the symbol oxins may disrupt calcium homeostasis n the liver and initiate a series of events hat" lead to the massive calcium influx is ^ins several hours after intoxica te To examine the interaction between the *ct endoplasmic reticulum calcium pump itb chlorinated hydrocarbon hepatotox.s more closely, we have characterized .e in vitro effect of 1,1-DCE on the mi* osomal calcium pump. As part of this aracterization we have compared effects -oduced by 1,1-DCE and CCh- Because l-DCE does not increase lipid peroxides liver membranes this hepatotoxin alvs us to demonstrate that inhibition of e liver endoplasmic reticulum calcium mp, in vitro, does not require peroxilion of membrane lipids. MATERIALS AND METHODS faW-iaL. Mole Sprague-Dawley rats (200-300 g, onic Farm*) were n*d in thi* ttudy. Unlaw othi*c noted, rat* were pretreated with phenohar*1 (80 mg/Vg, ip) daily for 3 succesiivt day*. The malt were used 1 day after the last injection. DP, nicotinamide, ATP, isocitric add, and isocitdehydrogenase (EC 1.1J.42) were purchased from Sigma Chemical Company. (St. Louie, Mo.). [*Ca)Cl, waa porchaaed from New England Nuclear Corpo ration (Boaton. Mesa.). lJ-Dichkuwethylene waa ob tained from the Aldrich Chemical Co. (Milwau kee, Wit.). Microtome preparation. After stunning, liver sam ples were removed and quickly homogenised (0.5 g/ 10 ml) in ice-cold 0.25 M sucrose containing 3 mu EDTA, pH 7. A PoUer-EWchjem homogeniser waa used with the Teflon pestle driven at 750 rpm. The homogenate was centrifuged at 1500? for 10 min and then at 12500? for 20 min. The supernatant waa cen trifuged for 60 min at 105,000?. The microtome pellet ww resuspended by homogenization in ice cold 04 M KQ, 0.05 M Trie buffer, pH 7.4 (T buffer). For most experiment*, the microtome suspension was used immediately after preparation. For some ex periments the microtomes were quickly frozen with a dry ice ethanol bath and stored for up to 10 days at -70*C IvaUxttioH of1,1-DCE or CG, viLh. liver mixedfunc tion aside** system. Microtomes were incubated in a metabolic shaker at room temperature in tightly capped rials Micrusomes at a protein concentration of IX to 12 mg protein/ml were incubated with a NADPH regenerating system (8) containing 100 pM NADP, 25 a* nicotinamide, 5 mM MgG 3 mM SX> iaodtrate, and 0.07 units iaocitric deHydrogenase/ml in TK buffer (final volume 3 ml). 1,1-DCE or CCL dissolved in ethanol, was added to the incubation medium. The maximum volume of ethanol added was 5 eliter/ml and the final concentration of 14-DCE or CQ, ww 10 slitcr to 1 pliter/mL Incubation waa initiated by addition of 14-DCE or CQ, to ice-cold medium. Because of volatility of LI-DCE, special precau tions were necessary to handle this compound. 14DCE ww cooled is an ice bath before use- For preinesbation with the microsomal mixed function oxidase (MFO) system, cold 14-DCE was quickly pipetted into the preincubation mixture which was also at 04*C. The preincubation vial ww filled to capacity with liquid, tightly capped, and finally placed in a metabolic shaker (shaking water bath) at room tem perature (20-22*0). Caleutm pomp activity determination. After incu bation with 14-DCE or CQ, calcium pump activity was determined as previously described (11). Briefly, microsomal (0.015 to 0.05 rog/ml) were incubated in 100 mM KG, 90 mM imidazole-histidine buffer (pH 88), 5 mM MgQ 6 mM ATP (pH adjusted with im idazole to 68), 5 mM ammonium oxalate. 5 mM sodium azide, 20 >M CaQ and [*Ca**] 02 pQ/ml The assay ww initiated by addition of the mierooome suspen sion to prewarmod (37*C) asaay medium. At timed intervals 05-ml samples were filtered through nitrocellulese filters (0.45-pm pore diameter). Filters were dried and "Cad* determined by liquid scintillation spectrophotometry. Microsomal protein was deter- f.l/ . / / ` SL 066255 RAY AND MOORE mined by the Lowery method u modified by Shot* bin (12). Lipid ptraruU determination. At u index of lipid peroxidation, mnlonic dinldehyde wo* determined by thiobarbituric acid method (13). RESULTS When liver microsomes were preincu bated with 1,1-DCE and a NADPH gen erating system for 20 min, calcium uptake activity was inhibited in a concentration- dependent manner. Compared to control, calcium uptake was reduced 38% at 100 ljliter/ml and inhibition reached a maxi mum of 55% at 1 Mliter/ml (Fig. 1). No inhibition of uptake activity occurred if mierosomes were incubated with as much FlC. 2 Time court* of liver microtome ealciu as 1 Mliter/ml 1,1-DCE in the absence of pomp inhibition tn vitro fay XJ-DCE () and CC the NADPH-generating system. Ethanol (a). Liver microtome* wen incubated with the eh' at the maximal concentration used as a rinated hydrocarbon (1 oliter/ml) and a NAD? solvent for the chlorinated hydrocarbons generating tyttem for the time indicated in the 6 did not affect calcium pump activity. Phenobarbital pretreatment of rats has been reported to increase hepatotoxicity of a number of chlorinated hydrocarbons including 1,1-DCE. To examine whether art. Sample* were removed and calcium pump ecti ity determined aa deacribed coder Method* and M ttrial*. Each point repreaento the moan SEM i the determination with miaveome* isolated fro throe animal*. phenobarbital pretreatment of a rat af fected the in vitro response of the calcium different time course of calcium pump h pomp to preincubation with 1,1-DCE we bibition. After preincubati n for 1 mi compared the effect of 1,1-DCE on calcium with T4-DCE, activity f the calciu. pomp activity of liver mierosomes pre pump was reduced no more than 10? pared from normal rats and from pheno- After 5 min, inhibition was statistical barbital-pretreated rats. In this study, significant at 31%. Prolonging preina liver microsomal calcium pump activity bation to 20 min increased calcium punr was inhibited 30 2% in control rats and inhibition to nearly 60%. 42 3% in phenobarbitol-pretreated rats To confirm that 1,1-DCE did not i: when isolated mierosomes were incubated crease lipid peroxidation in this in vit: with 1,1-DCE (0.3 ml/ml) for 20 min. This system the following experiment was pe difference is statistically .significant (P' formed. Mierosomes were incubated wit < 0.05 Student's t test). The current study 1,1-DCE, CCU (1 Mliter/ml) or vehicle coi and previous work (16) Bhowed no differ trol in the presence of the NADPH gei ence is calcium pump activity in control erating system for 20 min at room ten and phenobarbitol-pretreated rats. perature. MDA produced during this tin Figure 2 shows the time course of in period was determined. Control and 1,' hibition of the calcium pump after prein DCE-treated mierosomes did not produ< cubation with 1,1-DCE and CCU at 1 Mli detectable MDA during this incubatio ter/ml in vitro. After only 1 min of prein- At the same concentration, incubation : eubation with CCU, calcium pump activity the presence of CCU resulted in the gei is inhibited almost 50%. After 5 min of eration of 0.28 0.02 mS MDA/mg protei preincubation, inhibition by CCU reaches In our hands the assay could detect as li near maximal values of 80%. Proineuba- tie as the equivalent of 0.05 mE MDA/n tion for as long as 20 mis produced only protein measured with 1,1,3,3-tetraetho: slightly greater inhibition. Preineubation ypropane as standard. Under identical e: with 1,1-DCE produced a substantially perimental condition, CCU produced ! SL 066256 U-DCE AND MICROSOMAL CALCIUM PUMP IN VITRO 29 1 s and 1,1-DCE produced 41 6% in hibition of calcium pump activity. DISCUSSION Disruption of calcium homeostasis has been implicated as a mediator of cell death after hepatic injury produced by either toxic chemicals or ischemia. A number of workers suggest that bepatotoxins may disrupt intracellular calcium homeostasis and there is reemphasis of the role of dis rupted calcium homeostasis in cell death (14,15). A calcium pump activity has been described in liver endoplasmic reticulum (16). This calcium pump may be analogous to the sarcoplasmic reticulum calcium pump of skeletal muscle and may partic ipate in regulation of cytoplasmic calcium levels in liver (11,16). This calcium pump is promptly inhibited after in vivo admin istration of a number of hepatotoxins in cluding CCU (6. 8,9,11), BrCCU (8,9), UDCE (6,7), CHC1, (6), carbon disulfide (17). and after hepatic ischemia (10). These studies suggest that inhibition of this cal cium pump may disrupt calcium homeoste"! in the liver. )CE is used in the manufacture of vinyl chloride products and is a potent tepatotoxin (1, 5). 14-DCE induced liver damage is thought to depend upon meta bolic activation to a reactive metabolite by the MFO system of liver ER (18-20). Morphological studies of the early effects >f 1,1-DCE demonstrate damage to the tueleus, mitochondria, and plasma memorane of hepatic cells (4). Again, in eon-, .rest to agents such as CCU, liver ER does iot exhibit morphological changes at early times after 1,1-DCE administration (4). however, a recent study from our labo ratory shows that biochemical changes xxur in the liver ER at very early times ifter 1,1-DCE administration. Within 20 min after intraperitoneal doses of 1,13CE, liver ER calcium pump activity is inhibited 45% (7). This biochemical change may lead to loss of an ER calcium pool and thus represent a change of ER func tion (7). If inhibition of the ER calcium pump 'lays a role in cell death after 1,1-DCE dministration; inhibition of the pump hould depend upon activation of 1,1-DCE to a reactive intermediate by the MFO sys tem of liver ER. To demonstrate that me tabolism of 1,1-DCE is essential for inhi bition of the ER calcium pump, an in vitro system has been employed. The data pre sented in this report suggest that metab olism of 1,1-DCE by the MFO system is essential for calcium pump inhibition. In cubation of 1,1-DCE, an NADPH gener ating system and liver microsomes inhib its ER calcium pump activity. 1,1-DCE does not affect ER calcium pump activity in the absence of the NADPH generating system. This, of course, suggests that the MFO system generates a metabolite that attacks the calcium pump in liver ER. Fur ther evidence that 1,1-DCE is metabolized before it inhibits the calcium pump is pro vided by enhancement of 1,1-DCE inhibi tion in liver microsomes from phenobarbital-pretreated rats. Phenobarbetal pre treatment induces activity of the MFO system but does not affect ER calcium pump activity (11). Presumably phenob&rbital pretreatment of animals results in a mierosome fraction that produces reac tive metabolites of 1,1-DCE to a greater extent or at a more rapid rate. Some in formation is available from tn vivo studies that support these findings. 1,1-DCE treat ment depletes liver glutathione and deple tion of glutathione prior to 1,1-DCE ad ministration enhances hepatotoxicity (21). Presumably glutathione serves as an en dogenous substrate for conjugation of re active 1,1-DCE metabolites. Depletion of glutathione by diethylmaleate pretreat ment enhances both hepatotoxicity and calcium pump inhibition (7). Phenobarbe tal pretreatment has been found to pro duce conflicting results, such as protection of animals in some reports (4,18) and en hancement of hepatotoxicity in others (7, 22). We have observed that both bepatotoxicity and calcium pump inhibition are enhanced in vivo (7). The time course of calcium pump inhi bition by CC1, and 1,1-DCE tn vitro ob viously differs (Fig. 2). A number of ex planations can be considered. It is possible that incubation conditions are favorable for activation of CCU but less so for 1,1DCE. On the other hand, it is also possible that reactive metabolites of 1,1-DCE are A V* * ,\ -- - -: - " --..* J -O^.,<>.-.7 RAY AND MOORE inactivated more quickly than those of REFERENCES CC14. These two possibilities have not been 1. JENXINS,JR,L.J.,TRaBULUS,M.J.,ANDMUX. explored. One possible explanation has S- D. (1972) Toxicol Appl Pharmacol 23,. been studied. CCL produces lipid peroxi dation in ER membranes while 1,1-DCE does not (5, 6). Lipid peroxidation inhib its liver ER calcium pump activity (P. Ray and L. Moore, unpublished). EDTA is included in the homogenization buf 510. 2. Rewolds, E. S. (1963) J. Call Biel l*. 139-1 1 ReckmacEL R, 0. (1967) Pharmacol Rea. 19,) 202 t Rewolds, E. S. Moslen, m. T, Szabo. S. . Jaeger, R. J. (1975) Amor. J. Pathol SI, I 231. fer to reduce iron contamination of the 6. Jaeger, r. j,, Trabulus. m. j. add Muju microsome preparation. This minimizes NADPH'Fe+-induced lipid peroxidation (23, 24). During the 20-min incubation with the NADPH generating system, lipid peroxidation is not detected in control or 1,1-DCE experiments. Incubation of these S. S. (1973) Toxical Appl Pharmacol 24,4 467. 6. MOORE. L. (1980) Biechem. Pharmacol 29, 21 2511. 7. MOORE, L (1982) Biechem. Pharmacol 8L 14 1465. 1 Lowrey, K., Glende, Jr., El A., and Recxnac microsomes with CCL and the NADPH R 0. (1981) Btoehcm. Pharmacol 30,135--: generating system results in lipid perox 9. Lowrey, 1C, Glende, Jr.. E. A, and Recxnac idation. This is the result of metabolism of CCL to the CCli free radical which in turn initiates lipid peroxidation (S, 25). The results of Waller *t aL suggest that lipid peroxidation is not required for CCL inhibition of the calcium pump (26). How R. O. (1981) Toxicol Appl Pharmacol 59,2 394. 10. CK1EN, K. R. AND FaRBER, J. L (1977) Arch. 2 them Biopkyi ISO, 191*198. . 11. Moore. L. Davenport, G. r. and Land E. J. (1976) J. Biel Chent 251,1197-1201. 12. Shatkdv A. J. (1969) m Fundamental Teehnigi ever, it is probable that when lipid per in Viroloe (Mabel, K. and Salzman, N. oxidation occurs it contributes an addi tional factor to calcium pump inhibition. Thus it is possible that CCL inhibits the liver ER calcium pump more promptly than 1,1-DCE because lipid peroxidation contributes to pump inhibition. The dif it ), p. 231, Academic Preti, New York, ^ 12 GhoshaL A. K. and Recknagel, R. O. (19 Life Sd 4,1521-1530. 14. Sckanne, F. A. X., Kane, A. B,, Young, E. and FaRBER, J. L (1979) Sooner 206,700-7 15. FaRBER, J. L. (1931) Uft Set 29,1289-1295. 16. Moore, L, Chen, T, Knapp, Jil, H. r., a; ference in the ability of CCL and 1,1-DCE LONDON, E. J. (1975) J. Biol Cham 250, 4* to induce lipid peroxidation may also ex 4568. plain why ER damage is observed as ah early morphological change after CCL but not after 1,1-DCE action. Both compounds 17. MOORE, L (1982) Biechem. Pharmacol 31,146 1467. 12 Andersen, M. E. and Jenkins, Jr, L J. (197 Environ. Health Pratptct 21,157-163. damage the ER calcium pump early after, 19^Andersen, M. E., Jones, R. A_ and Jenkins, J administration to an animal (7,9,11). But I- J. (1978) Tadcol Appl Pharmacol 44,22 the morphological changes CCL produces may be secondary to lipid peroxidation. TTiis study shows that 1,1-DCE inhibits the liver ER calcium pump t vitro. It dem onstrates that m vitro, as well as in vivo (6, 7), inhibition of this pump by 1,1-DCE is not dependent upon lipid peroxidation. 234. . Andersen, m. . French, J. ., Gargas, m. l Jones, R. a, and Jenkins, jr. L. J. (197 Tadool Appl Pharmacol 47, 385-392 21. Jaeger, R. J, Conolly. R. B,, and Murph S D. (1974) Exp Mot Pathol 20,187-192 22 Harris, R. N, and anders (1980) Pharmacol put 22, 223. ' work presented suggests that 1,1-DCE , ctivated by the MFO system to a com pound that interacts with and inhibits the calcium pump. 23. Kornbrust, D. J, and Mavis, R. D. (1980) Mi Pharmacol 17.400-407. 24. Kornbrust, D. J. and Mavis. R. D. (i960) hh Pharmacol 17.408-414. 25. Foyer, J. L, McKay, P. B, Lai, . K, Janzet E. G, and Davis, E R. (1980) Biechem. Bit ACKNOWLEDGMENT phyt Bat. Common. 94,1154. 26. Waller. R. L, Lowrey, K., Glende Jr. E. a Supported in pert by 1 grant from the United and Recknagel R 0. 0981) Fed. Prop 4< State* Public Health Service (ES 02691-01). 1782 SL 066258 --w.1-