Document 9LrQkr7GvKBer5Z5B03oJDBp3

1,1-Dichloroethylene Hepatotoxicity Time Course of CSH Changes and Biochemical Aberrations Edward S. Reynolds. MO. Mary Traintn Mosten. MS, Paul J. Boer, MO, and Rudolph J. Jaagar, PhD t^Epanae of fated rats to S00 ppm 1,1-dfohforocthylene (1,1-DCE) for 1-4 hours rantlted h> <triUa{ iknifat in htpatfo Na, K, Ca, and CSH Wweis which pracadad and/or ac companied oatestraphic histologic attentions of the liver. Na levels began to rise during Iho Bnt hour, and preceded the morphologically apparent injury. Ca lavals increased Markedly and K leads declined between the noond and fourth hour of expomre, and aeconynfod the eetestrophic morphologic alterations, CSH levels ware rapidly depleted Vet arps to roomer before the end of the raposute to 1,1-DCE. Functions of com ponents of the rafad-hmetion oxidase system of the liver endoplasmic reticulum ware not appreciably affected early in the course of 1,1-DCE exposure; but after injury became manioc, cytochrome P-450 and oxidative N-demsthylase were deactivated Thus effects ' an (he functional eompononts of the endoplasmic reticulum mixed-function naidsat sys tem do not appear to be primary events in 1,1-DCE cytotoxicity. In contrast, thorn were progressive declines in mitochondrial K end marked imbalances in mitochondrial Na, Zn, - and Mg preceding the maoive infiuxof Ca into the cell, indicating that mitochondria are tovoived aarty in the evolution of injurious moltculer events elicited by this potent hopetotexfo. (Am J Pathol 1990,101^31-444) 1, 1,1-DlCHJjOROETHYLENE (1,1-DCE) is an exquisite hepatotoxin. It is more potent, faster acting, and has a far more precipitous dose threshold for injury in the fasted rat than the classic hepatotoxin carbon tetrachloride (CdJ.lJ - 1,1-DCE produces a distinctive morphologic pattern of injury that pref erentially involves mitochondria, spares endoplasmic reticulum, produces chromatin segregation within nuclei, and causes cell borders to retract forming lacunar spaces within hepatic cords.* Thrombosis ensues and ne crosis is manifest within 4 hours after the onset of exposure. This pattern of cell injury differs markedly from that caused by CC1, and by other chloroethylenes, including vinyl chloride and trichloroethylene, each of which primarily involves endoplasmic reticulum in a more gradually evolving process.4 Fromtha Oiemiml hthfa Laboratory. Department of Pathotofy. Uiwvortity of Tram Medical Branch, Galvetan, To , md rite ICimfx Center far Environmental Health. Harvard School of Public Health, Baton. Motadwacttx. Dr. Jacfar's prmoM addrat a InRilutr of Environmental Mrdtcjn*. (tew York Unrvcruty Modical Conttr. New York, ftew York. Supportad by Cram* AM-IMH and ES4000S from the National Intitule* of Hoalth. Accepted for publication May & 1M0. Addict reprint requeM to Edwaid S. Reynohh. MD, Department of h'lmfofy. Unbwnity of Teaaa Medical Branch, Cilraton. TX 77550. 0QQ2-BAO0/B0/lt 10-0331 SOI .00 American AmocIMton of Patootoofote 331 I JSfSi. -V f *' 'x SL 066310 V*. 101, No. 2 NovvnUMMO >.1-OKXLOROrrHYLN6 HEPATOTOXOTY 333 TadmiquM of Analysts Hepatic injury was assessed by light-microscopic study, by the determination of Na, K, M{. Ct, Zn, and Fe content* of wet-sxhed liver sample* with atomic absorption spectrophotometry,'* and by measurement of the activities of three liver-derived enzymes in arum. Hie enzyme activities measured were serum glutamic-oxalacetic transaminase (SCOT) and serum glutamic-pyruvic transaminase (SGPT) (Sigma Reagent Kits) and serum sorbital dehydrogenase (SDH).11 Hie frequency of mitotic figures were estimated in 4*p paraffin sections according to the techniques of Weibel and Elias.'* The functional integrity of the isolated endoplasmic reticulum fraction was --by the assay of microsomal enzymes, primarily mixed-function oxidase components, accord ing to Moslen et al,'3 and expressed in terms of microsomal protein content The protein content was determined by the method of Lowry et al,14 We estimated GSH levels by measuring nonprotein rulfhydryl contents using lman's reagent according to Jaeger et al.* Metal and CSH levels of tissue fractions from individual animals were expressed in terms of the protein content Care was taken in this time course study to compare "experimental" quantitative values obtained from the animals exposed to 1,1-DCE to appropriate "time of day control" val ises obtained from the animals asposed to air only and killed at compatible intervals during the experimental period. The quantitative values obtained from the animals ex posed to sir only remained relatively constant throughout the experimental period Signif icant difference in t test comparisons were interpreted according to Fisher and Yates.1* Change were considered statistically significant if ? < 0.05. Regression correlations were obtained by least-square analysis with the uw of a Wang Programmable Compute. Results Histologic findings are presented in Figure 1. The most striking early histologic changes involved the nuclei. At 1 hour there was a threefold in crease in the number of mitotic figures in parenchymal cells, as compared with their infrequent appearance in the bepatocytes of control rats (685 146 vs 215 23 mitotic figures/cu mm; or approximately 4.0 per 1000 hepatocytes vs 1.3 per 1000 bepatocytes); this increase is statistically signifi cant (P < 0.05, if 6). By 2 hours mitotic figures had all but disappeared, and parenchymal cells in centrolobular and midzonal areas began to show central rarefaction of nuclei with peripheral displacement of chromatin to nuclear margins. Concomitant with changes in chromatin distribution, cell borders of affected parenchymal cells were retracted, and pericellular lacunae formed within hepatic cords. Histologic injury was progressive, and by 4 hours frank hemorrhagic centrolobular necrosis was present At subsequent time points of 6 and 12 hours (not presented in Figure 1) the VoL 101. No. 2 M0WA264F 1MO 1.1-004LOROCTHYVENE HEPATOTQXICtTY 333 Tudmlquw M SwriyiU Hepatic inpay was memd by light-microscopic study, by the determination of Na, X, . Mj, Ca, Zn, aad Fc contents of wot-adiod liver ample* with atomic sbeorption spectro photometry." and by measurement of the activities of three liver-derived enzymes in IVWMl The enzyme activities measured were serum glutxmic-axxjxcetie transaminase (SCOT) and serum ghittmic-pyruvic transaminase (SCFT) (Sigma Reagent Kits) and serum orbital dehydrogenase (SDH)." frequency of mitotic figures were estimeted in 4-a paraffin sections according to the techniques of Weibcl and Elias.1* The functional integrity of the isolated endoplasmic reticulum fraction was assessed by dm assay of microsomal enzymes, primarily mixad-function oxidase components, accord ing to Modes at al." and expressed in term* of microsoma! protein content. The protein content was determined by the method of Lowry at aLM We estimated CSH levels by measuring nonprotein sullhydryl contents using Ellman's fangent according to Jaeger et al.1 Metal and CSH levels of tissue fractions from individual awfmtb were expressed in terms of the protein content 'Clare wet taken in this time course study to compare "experimental" quantitative values obtained from the animals exposed to 1,1-DCE to appropriate "time of day control" vatttat obtained from the animals exposed to air only and killed at compatible intervals wiring the experimental period. The quantitative values obtained from the animals ex- paudio Mr only remained relatively constant throughout the experimental period Signif icant ASaranuas in t test comparison* were interpreted according to Fisher and Yates.14 dings are presented in Figure 1. The most striking early h&toiogic changes involved the nuclei. At 1 hour there was a threefold in crease in the number of mitotic figures in parenchymal cells, as compared with their infrequent appearance in the hepatoeytes of control rats (685 146 vs 215 A 23 mitotic figures/cu mm; or approximately 4.0 per 1000 hepatocytes vs 1J per 1000 hepatoeytes); this increase is statistically signifi cant (F< 0.05, if < 8). By 2 hours mitotic figures had all but disappeared, and parenchymal cells in centrolobular and midzonal areas began to show central rarefaction of nuclei with peripheral displacement of chromatin to nuclear margins. Concomitant with changes in chromatin distribution, cell borders of affected parenchymal cells were retracted, and pericellular lacunae formed within hepatic cords. Histologic injury was progressive, and by 4 hours frank hemorrhagic centrolobular necrosis was present At subsequent time points of 6 and 12 hours (not presented in Figure 1) the Voi. 101, NO. 2 NOfHwV 1VW i .i-wchlorocthyune HEPATcrroxicrtY 335 massive histologic injury was similar in character and extent to that ob served at 4 hours. Serum SDH activity became elevated and Na levels in liver increased by the end of the first hour of 1,1-DCE exposure. As indicated in Text-fig ure 1 (top and center) changes in these two parameters progressed during the second hour, while other serum enzymes and liver metals assayed were not appreciably altered. By the fourth hour, however, serum activi ties of the two transaminases, SCOT and SGPT, were elevated and liver Ca was increased markedly, while K, Mg, and Zn levels had decreased sig nificantly below the values of control animals. Liver K, Mg, and Zn levels plateaued at below normal levels after the fourth hour, while Ca contin ued to accumulate through the twelfth hour. Liver Na peaked at 6 hours. In order to determine how the changes in liver metal levels correspond Liver Homogenate Na (mg/g liver) TBcr-ncuaC S--Exponential uptfon correlonon of th* covananee of Hvor hotnofanaie No Wveh and mram SDH aerivitiet of individual 1.1DCE-expond animalt kilted durinf the 12-hour erpenmenul period. Them lo 4 animal* were kilted at ad) time point. Note the durtennf of adjacent time pointi The correlation it tifmfiant at f .001 level (F m 4SJ8. r-0.3,d/-m v^f, SL 066313 VQI.iOi.Na.2 1.1-OtCHLOROETHYLENE hepatotoxictty 335 massive histologic injury was similar in character and extent to that ob served at 4 hours. Serum SDH activity became elevated and Na levels in liver increased by the end of the first hour of 1,1-DCE exposure. As indicated in Text-fig ure 1 (top and center) changes in these two parameters progressed during the second hour, while other serum enzymes and liver metals assayed were not appreciably altered. By the fourth hour, however, serum activi ties of die two transaminases, SCOT and SGPT, were elevated and liver Ca was increased markedly, while K, Mg, and Zn levels had decreased sig nificantly below the values of control animals. Liver K, Mg, and Zn levels plateaued at below normal levels after the fourth hour, while Ca contin ued to accumulate through the twelfth hour. Liver Na peaked at 6 hours. "In order to determine how the changes in liver metal levels correspond '"V 'U Liver Homogenate Na (rrxj/g liver) TDrmcuiK I Eipowtwttel sgmfatiw of the wnriinee of Hw*r homofanett \* level* ud mrum SDH tetMtm of individual 1.1. DCE-*po**d aramtli killed durtef th* l&hour eiperimental parted Three to 4 animal* ware killed at each tint* point. Note th* dintarinf of adjacent time powiti. Th* correlation i* dgniScant at F .001 level (F 4SJ8. r - 0.83. - 22). *2 SL 066314 Vet 101, Na 2 noww 1 wo 1,1-OCHLOnOCTHYLENE HEPATOTOXICTTV 337 ter panel). The CSH level at each time point from the first to the sixth hour after the onset of 1,1-DCE exposure is statistically distinct (P < 0.025) from that of the time before as well as the zero hour "control" point Liver GSH contents were evidently replenished during the third and fourth hours of 1,1-DCE exposure, when the injury first became mor phologically manifest Mitochondrial CSH levels abruptly decreased to approximately half control values during the first 2 hours of DCE exposure but unlike total liver CSH, failed to rebound. MKodiandrfM Meat Changes in mitochondrial Ca levels paralleled the abrupt increases in total liver calcium. Specifically, between 2 and 4 hours mitochondrial Ca increased 10-fold (Text-figure 1, lower panel). Mitochondrial K levels pro gressively decreased from two-thirds control values at 1 hour to oneeighth by 12 hours. Thus Ca displaced K as the dominant mitochondrial cation; the Ca/K mole ratio rose from 0.03 in controls to 1.9 at 12 hours after 1,1-DCE (Table 1). The mitochondrial Na levels decreased to half oontrol levels by 6 hours, but not as steadily as total liver Na levels. The levels of the other metals fluctuated. The most striking fluctuation was for Zn, which decreased by more than half at 1 hour, and then partially re covered. Mg levels oscillated below control during 1,1-DCE exposure and then declined to approximately half normal. Mitochondrial Fe levels (not illustrated in Text-figure 1) steadily decreased, reaching one-fourth pre exposure values by 12 hours. In contrast to the fluctuations in metal levels, protein contents of mitochondria (per gram wet weight) remained stable until 12 hours. Endaptaantic Reticulum Function The effects of 1,1-DCE exposure on microsomal enzyme components and activities were measured at 2 time points--2 hours, when injury is slight,* and 6 hours, when injury is massive (Table 2). At 2 hours after the Tatta 1---Effacta of 1 .i-OCE Exposure on tha Ratio of Calcium to Potaaaium in Mitochondria TWno* Moia* Ca/mefaa K 0 1 2 4 s 12 0.03 0.04 O.OS 0.70 1.74 1.92 * Hours attar oneat of 1,1-OCE exposure. Vet 101. Mo. 2 Nowwnbf 1--0 1.1-fltCHLQHOeTHYUNE HEPATOTOXX3TY 337 ter panel). The CSH level at each time point from the first to the sixth hour after the onset of 1,1-DCE exposure is statistically distinct (P < 0.025) from that of the time before as well as the zero hour "control" point Liver CSH contents were evidently replenished during the third and fourth hours of 1,1-DCE exposure, when the injury first became mor phologically manifest Mitochondrial CSH levels abruptly decreased to approximately half oontrol values during the first 2 hours of DCE exposure but, unlike total liver CSH, foiled to rebound. ! MNoduirtrM Motab Changes in mitochondrial Ca levels paralleled the abrupt increases in -total liver calcium. Specifically, between 2 and 4 hours mitochondrial Ca Increased 10-fold (Text-figure 1, lower panel). Mitochondrial K levels pro* gressively decreased from two-thirds control values at 1 hour to one* eighth,by 12 hours. Thus Ca displaced K as the dominant mitochondrial OKtion; toe Ca/K mole ratio rose from 0.03 in controls to 1.9 at 12 hours ^Affcer 1*1-DCE (Table 1). The mitochondrial Na levels decreased to half oontrol levels by 6 hours, but not as steadily as total liver Na levels. The leveJl'of the other metals fluctuated. The most striking fluctuation was for Zn, which decreased by more than half at 1 hour, and then partially re* CQvdjrtgi Mg levels oscillated below control during 1,1-DCE exposure and then detained to approximately half normal Mitochondrial Fe levels (not in Text-figure 1) steadily decreased, reaching one-fourth prealues by 12 hours. In contrast to the fluctuations in metal levels, Pintents of mitochondria (per gram wet weight) remained stable mis. Swaphaefc WoOcolinn Function The effects of 1,1-DCE exposure on microsomal enzyme components and activities were measured at 2 time points--2 hours, when injury is slight, and 6 hours, when injury is massive (Table 2). At 2 hours after the TW1 BioctuoM.HICEScpooura on ttortottoWCoiciiwn to Fete-tow to MHocttonOrtc teno* Motoc Ca/motM K 0 0.03 1 0.04 a 0.08 4 0.70 1.74 12 1.82 * * Hows toor onoot of i.l-OCE txpoeurc. SL 066316 voi. 101. no. a NovmbaMMO M-WCHLOROCrHYLENE HEPATOTOXICfTY 33ft ures. Since in ,ndant supply of CSH is reported to be conducive of fast and efficient mitosis,1* the decrease in hepatocyte GSH could have con tributed to an arrest of the normally brief (40-80 minutes) mitotic phase in cycling hepatocytes.17 Alternatively, the influx of Na could have con tributed to a rapid stimulation into mitosis of hepatocytes blocked in the late Gt phase of the cell cycle. An increase in Na ion influx has been found critical to hepatocyte proliferation in vitro,and rodent hepatocytes have been found to contain a subpopulation of Gx-blocked hepatocytes that can rapidly be stimulated into mitosis.'* The progressive Na influx during the first 2 hours of 1,1-DCE exposure should be considered as a potential contributing factor to the segregation and retraction of nuclear chromatin observed after 2 hours of 1,1-DCE ex posure, since alterations in Na levels have been found to affect the organi zation and structure of chromatin preparations.-11 Of course, other bio chemical changes, as yet undetermined, including activation of endonucleases and proteases, could contribute to the observed rapid con version of hepatocyte chromatin from highly integrated nucleic acid-pro tein complexes to segregated pools of DNA, RNA, and protein. Although this time course study indicates a close correspondence be tween the early Na influx and increases in the serum activities of sorbital dehydrogenase above its low background levels (Text-figure 1), appre ciable alterations either in the level of K or Ca in the liver or in the serum activities of liver-derived transaminases were not detected at early times. While it is possible that a generalized "leakiness" of the plasma, mem brane accounts for the marked early entrance of Na, the lack of equiva lent and concomitant changes in other soluble cytoplasmic components indicates a more specific effect -- One obvious kind of specific effect that could explain the early Na in flux is a problem with membrane ion pumps, either directly or indirectly due to ATP deficiency. An indirect effect of 1,1-DCE on the ion pumps due solely to an ATP deficiency appears questionable, since ethionine ad ministration, which rapidly and dramatically depletes liver ATP levels, causes not only a rapid increase in liver Na levels but also a nearly equiva lent decrease in liver K levels. We did measure an early and progressive loss of K from the mito chondria following 1,1-DCE (Text-figure 1, lower panel). This loss of mitochondrial K is suggestive of a deficiency in metabolic energy, since accumulation of K by the mitochondria is an active process dependent on availability of metabolic energy.** The initial loss of K cannot be attrib uted to Ca, for loss of mitochondrial K precedes by several hours the in flux of Ca into the cell. However, at later times the K loss may be aggra- Voc. 101, No. 2 November i960 1.1-ttCHLOnOCTHYLENe HEPATOTOXlCriY 339 ures. Since an abundant supply of CSH is reported to be conducive of fast and efficient mitosis,'* the decrease in hepatocyte CSH could have con tributed to an arrest of the normally brief (40-80 minutes) mitotic phase in cycling hepatocytes.'1 Alternatively, the influx of Na could have con tributed to a rapid stimulation into mitosis of hepatocytes blocked in the late Ct phase of the cell cycle. An increase in Na ion influx has been found critical to hepatocyte proliferation in vitro,'* and rodent hepatocytes have been found to contain a subpopulation of Gt-bk>cked hepatocytes that can rapidly be stimulated into mitosis.'* The progressive Na influx during the first 2 hours of 1,1-DCE exposure should be considered as a potential contributing factor to the segregation and retraction of nuclear chromatin observed after 2 hours of 1,1-DCE ex posure, since alterations in Na levels have been found to affect the organi zation and structure of chromatin preparations.*-21 Of course, other bio chemical changes, as yet undetermined, including activation of endonucleases and proteases, could contribute to the observed rapid con version of hepatocyte chromatin from highly integrated nucleic acid-pro tein complexes to segregated pools of DNA, RNA, and protein. Although this time course study indicates a close correspondence be tween the early Na influx and increases in the serum activities of sorbital dehydrogenase above its low background levels (Text-figure 1), appre ciable alterations either in the level of K or Ca in the liver or in the serum activities of liver-derived transaminases were not detected at early times. While it is possible that a generalized "leakiness" of the plasma mem brane accounts for the marked early entrance of Na, the lack of equiva lent and concomitant changes in other soluble cytoplasmic components indicates a more specific effect One obvious kind of specific effect that could explain the early Na in flux is a problem with membrane ion pumps, either directly or indirectly due to ATP deficiency. An indirect effect of 1,1-DCE on the ion pumps due solely to an ATP deficiency appears questionable, since ethionine ad ministration, which rapidly and dramatically depletes liver ATP levels, causes not only a rapid increase in liver Na levels but also a nearly equiva lent decrease in liver K levels.11* We did measure an ejtrly and progressive loss of K from the mito chondria following 1,1-DCE (Text-figure 1, lower panel). This loss of mitochondrial K is suggestive of a deficiency in metabolic energy, since accumulation of K by the mitochondria is an active process dependent on availability of metabolic energy.* The initial loss of K cannot be attrib uted to Ca, for loss of mitochondrial K precedes by several hours the in flux f Ca into the cell. However, at later times the K loss may be aggra- SL 066318 - - - ,7^.4 VaL 101. Mo. 2 NovemberiMO 1,1-aCMLOBOETMYLENE H6PATOTOXOTY 341 first substrate on the enzyme was found to be the electrophilic one, and for very reactive substrates the substrate-enzyme reaction was irrevers ible, with the enzvitie virtually "committing suicide". Therefore, we sug gest that the catastrophic histologic and biochemical changes observed were a consequence of the continued formation of reactive 1,1-DCE spe cies coupled with abatement of the capacity to detoxify such reactive, spe cies. References 1. JenJtirw LJ Jr, Trabulus MJ. Murphy. SD: Biochemical effects of 1,1-dichloroethylane in rats: Comparison with carbon tetrachloride and 1,2-dichloroethylene. Toaicol AppJ Pharmacol 1972, 23:501-510 2. Jaeger RJ, Trabuius MJ, Murphy SD: Biochemical effects of 1,1-dichloroethylene in rate Diiwciation of its hepatotozicity from a lipoperoxidative mechanism. Toxicol Appl Pharmacol 1973, 24:457-467 3. Reynolds ES. Moslen MT, Ssabo S, Jaeger RJ, Murphy SD: Hepatotoxicity of vinyl chloride and l.l-dichioroethylene: Role of mixed function oxidase system. Am J Pathol 1975,81:219-236 - 4. Reynolds ES. Moden MT: Damage to hepatic cellular membranes by chlorinated olefins with emphasis on synergism and antagonism. Environ Health Petspect 1977, 21:137-147 5. Jaeger RJ, Conolly RB, Murphy SD: Effect of 18 hr fast and glutathione depletion on 1.1-dichloroethylene-induced hepatoxicity and lethality in rats. Exp Mol Pathol 1974, 20:187-196 6. Janet BK, Hsthway DE: The biological fate of vinylidene chloride in rats. Qtem Biol Interact 1978,20:27-41 7. McKenna MJ, Watanabe PC, Gehring PJ: Pharmacokinetics of vinylidene chloride r in the rat Environ Health Penpect 1977, 21:99-105 S. Jaeger RJ, Conolly RB, Murpby SD: Diumal variation of hepatic glutathione con centration and its correlation with 1.1-dichlotoethyiene inhalation toxicity in rats. '< Res Commun Chem Path Pharmacol 1973. 6:465-471 9. Leach LJ: A laboratory test chamber for studying air-bome materials. Atomic En ergy Commission Research and Development Report UR-629, University of Roches ter, 1963, pp 1-12 10. Reynolds ES, Rae HJ. Moslen MT: Uver parenchymal cell injury: IX Hmnobarbital potentiation'of endoplasmic reticulum dmaturation following carbon tetrachloride. Lab Invmt 1972. 9&290-299 1L Carlach U: SarfaMol Dehydrogenase ia Methods of Enzymatic Analysis. Edited by HU Bargmyci. New York, Academic Preta 1965, pp 761-784 12. Weibel ER, Elias H: Introduction to stareology and morphometry, Procnedinp of a Symposium an Quantitative Methods in Morphology. Berlin, Springer-Verlag, 1969, pp3-19 13. Moslen MT, Reynolds ES, Szabo S: Enhancement of the metabolism and hepato toxicity of trichloroethylene and perchloroethylene. Biochem Pharmacol 1977, 26:369-375 14. Lowry OH. Rombrough NJ. Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 1951. 183-265-275 15. Fisher RA, Yates F: Statistical tables for biological, agricultural and medical re search. London, Oliver and Boyd, 1963, p 63 VollOI.No.2 Novao*er 1900 1.1-OCHLOBOeTMYUNE hffiPATOTOXlCmr 341 first substrate on the enzyme was found to be the electrophilic one, and for very reactive substrates the substrate-enzyme reaction was irrevers ible, with the enzyme virtually "committing suicide". Therefore, we sug gest that the catastrophic histologic and biochemical changes observed were a consequence of the continued formation of reactive 1,1-DCE spe cies coupled with abatement of the capacity to detoxify such reactive spe cies. References vV , 1. Jenkins LJ Jr, Trabulus MJ, Murphy-SD: Biochemical effects of 1,1-dichloroethy- lene in rate Comparison with carbon tetrachloride and 1.2-dichloroethylene. Toxicol Appl Pharmacol 1972. 23:501-510 2. Jaeger RJ, Trabulus MJ, Murphy SO: Biochemical effects of 1,1-dichloroethylene in rats: Dissociation of its hcpatotoxiciry from s lipoperoxidative mechanism. Toxicol Appl Pharmacol 1973, 24:457-467 3. Reynolds ES, Mosien MT. Ssabo S, Jaeger RJ, Murphy SD: Hepatotoxicity of vinyl chloride and l,l<3ichloroethyiene: Role of mixed function oxidase system. Am J Pathol 1975.81:219-236 " 4. Reynolds ES, Mosien MT: Damage to hepatic cellular membranes by chlorinated olefins with emphasis on synergism and antagonism. Environ Health Penpect 1977, 21:137-147 5. Jaeger RJ, ConoDy RB, Murphy SD: Effect of 18 hr fast and glutathione depletion on l.l^ichlorocthylene-induced hepatoxicity and lethality in rats. Exp Mol Pathol 1974. 20:187-198 6. Jones BK, Hathway D: The biological fate of vinylidene chloride in rats. Cbem Bio) Interact 1978,20:27-41 7. McKenna MJ, Watanabe PC, Cehring PJ: Pharmacokinetics of rinyiidene chloride in the rat. Environ Health Penpect 1977, 21:99-105 8. Jaeger RJ, Conolly RB, Murphy SD: Diurnal variation of hepatic glutathione con centration and its correlation .with 1.1-dichloroethyiene inhalation toxicity in rats. Res Common Chem Path Pharmacol 1973, 6:465-471 9. Lendl LJ: A laboratory test chamber for studying air-borne materials. Atomic En ergy Commission Research and Development Report UR-829, University of Roches ter, 1963, pp 1-12 10. Reynolds ES. Ree HJ, Mosien MT: Liver parenchymal cell injury: IX PhoobsrbiUi potentiation of endoplasmic reticulum denaturetion following carbon tetrachloride. Ub Invest 1972, 26:290-299 1L Gerlacb U: Sorbitol Dehydrogenase in Methods of Enzymatic Analysis. Edited by HU Bergmyer. New York. Academic Pres, 1965, pp 781-764 12. Weibei ER, Elias H: Introduction to stereology and morphometry, Prooeedinp of a Symposium on Quantitative Methods in Morphology Berlin, Springer-Verlag.. 1969, PP 3-1 13. Mosien MT, Reynolds ES, Scabo S: Enhancement of the metabolism and hepato- toxicity of trichloroethylene and perchloroethylene. Biochem Pharmacol 1977, 26069-375 14. Lowry OH, Reaebrough NJ, Farr AL. Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 1951, 193:265-275 15. Fisher RA, Yates F: Statistical tables for biological, agricultural and medical re search. London, Oliver and Boyd, 1963, p 63 - hi SL 066320 vs