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> Weissman, G. (1982). Leusecretagogue in human neusis. Biochem. Biophys. Re.i. 2. and, W. (1969). Solubility of 0221,265-267. rie, J. H. (1980). Principles ics: A Biometrical Approach, 85-186,336-347. McGraw- JE, K., AND Minakami, S. tuction of human polymorimulated by leukotriene B4. 103,271-277. in kinase C and the activation NADPH-oxidase. Blood 69, ! Hepatotoxicity: The Adverse r Chemicals on the Liver, pp. 7-Crofts, New York. M4., th* ' 1/003 L ( T Pi \IiaJ VL TOXICOLOGY AND APPLIED PHARMACOLOGY 95,93-103 (1988) Potentiation of 1,1-Dichloroethylene Hepatotoxicity: Comparative Effects of Hyperthyroidism and Fasting1 Mary F. Kanz, Robert F. Whitehead, Ann E. Ferguson, and Mary Treinen Moslen Chemical Pathology Laboratory, University ofTexas Medical Branch, Galveston, Texas 77550-2774 Received November 23,1987: acceptedApril 11.1988 Potentiation of 1,1-Dichloroethylene Hepatotoxicity: Comparative Effects of Hyperthyroid ism and Fasting. Kanz, M. F., Whitehead, R. F., Ferguson, A. E., and Moslen, M. T. (1988). Toxicol. Appl. Pharmacol. 95, 93-103. The responses of fed, fasted, and hyperthyroid (T4) Sprague-Dawley male rats to 50 mg 1,1-dichloroethylene (l,l-DCE)/kg were compared. Hyperthyroid rats received three sc injections of thyroxine (100 ug/100 g) at 48-hr intervals; all other rats were sham-injected. 1,1 -DCE was given po in mineral oil 24 hr after the last T4 dose; controls received only mineral oil. Animals were killed at 2, 4, and 8 hr. Liver GSH contents were lowered about 55% by both fasting and T4 while GSH transferase activities were lowered about 20% by fasting and 35% by T4. Only T4 pretreatment lowered alcohol dehydrogenase activities. Liver injury (i.e., serum glutamate pyruvate transaminase, histology) after 1,1-DCE was minimal in fed rats, moderate in fasted rats, and intermediate in T4 rats. Fasted rats showed a more pronounced depletion of liver GSH after 1,1-DCE than T4 rats and only in fasted rats did the toxicant decrease activities ofthe detoxification enzymes. Hypoglycemia after 1,1-DCE occurred in fed rats, but more rapidly in T4 rats. In contrast, fasted rats unexpectedly became hyperglycemic after the toxicant. Patterns ofbody temperature change after the toxicant, which might be due to its metabolites, were dissimilar. Hypothermia was not observed in fed rats, was only transiently evident in T4 rats, but occurred rapidly within 1 hr in fasted rats and steadily became more severe. The dissimilar patterns of liver enzyme and body temperature and serum glucose change after the toxicant in the three groups are indicative ofdifferent pathways ofinjury potentiation by fasting and hyperthyroidism, e 1988 Academic Fra, tnc. 1,1-Dichloroethylene (1,1-DCE) is an inter the cause-effect sequence ofevents leading to esting model hepatotoxicant because multi cell injury. 1,1-DCE is used for the synthesis ple different conditions or pretreatments of plastic film and has been identified in in which are known to affect cell constituents in dustrial sewage treatment effluents, well wa volved in the metabolism of 1,1 -DCE (Fig. 1) ter, and treated drinking water (Coleman et modulate its acute hepatotoxicity (Reynolds al, 1976). el al,, 1975; Szabo el al., 1977; Anderson et Fasting and excess thyroxine are two preal,, 1980; Masuda and Nakayama, 1983). treatments which markedly enhance the hep These pretreatments provide a way to probe1 atotoxicity of 1,1-DCE (Jaeger el al, 1974; Szabo et al, 1977; Jaeger el al, 1977). Both 1 Portions ofthis paper were presented at the 1986 An nual Meeting of the American Society of Pharmacology and Experimental Therapeutics and the Society ofToxi cology, Baltimore, Maryland, 1986 (Pharmacologist 28, 191. 1986). of these pretreatments lower liver contents of GSH (Jaeger et al., 1977) which, as illustrated in Fig. 1, is needed for the formation ofconju gates with reactive metabolites of 1,1-DCE (Liebler. et al, 1985). However, the mecha- >3 0041-008X/88 $3.00 Copyright & 1988 by Academic Press, Inc. AH rights of reproduction in any form reserved SL 040442 ) Weissman, G. (1982). Leusecretagogue in human neusis. Biochem. Biophys. Res. 2. \nd, W. (1969). Solubility of ') 221,265-267. RiE, J. H. (1980). Principles ics; A Biometrical Approach. S5-186, 336-347. McGraw- ;e, K., and Minakami, S. uction of human polymorimulated by leukotriene B. 03,271-277. m kinase C and the activation NADPH-oxidase. Blood 69, Hepatotoxicity: The Adverse r Chemicals on the Liver, pp. y-Crofts, New York. -h$JL t CLu\aM Potentiation of 1,1-Dichloroethylene Hepatotoxicity: Comparative Effects of Hyperthyroidism and Fasting1 Mary F. Kanz, Robert F. Whitehead, Ann E. Ferguson, and Mary Treinen Moslen Chemical Pathology Laboratory, University ofTexas Medical Branch. Galveston, Texas 77550-2774 Received November 23,1987: acceptedApril 11.1988 Potentiation of 1.1-Dichloroethylene Hepatotoxicity: Comparative Effects of Hyperthyroid ism and Fasting. Kanz. M. F., Whitehead, R. F., Ferguson, A. E., and Moslen, M. T. (1988). Toxicol. Appl. Pharmacol. 95, 93-103. The responses of fed, fasted, and hyperthyroid (T4) Sprague-Dawley male rats to 50 mg 1,1-dichloroethylene (!,l-DCE)/kg were compared. Hyperthyroid rats received three sc injections of thyroxine (100 fig/100 g) at 48-hr intervals; all other rats were sham-injected. 1,1-DCE was given po in mineral oil 24 hr after the last T4 dose; controls received only mineral oil. Animals were killed at 2, 4, and 8 hr. Liver GSH contents were lowered about 55% by both fasting and T4 while GSH transferase activities were lowered about 20% by fasting and 35% by T4. Only T4 pretreatment lowered alcohol dehydrogenase activities. Liver injury (i.e., serum glutamate pyruvate transaminase, histology) after 1,1-DCE was minimal in fed rats, moderate in fasted rats, and intermediate in T4 rats. Fasted rats showed a more pronounced depletion of liver GSH after 1,1-DCE than T4 rats and only in fasted rats did the toxicant decrease activities of the detoxification enzymes. Hypoglycemia after 1,1-DCE occurred in fed rats, but more rapidly in T4 rats. In contrast, fasted rats unexpectedly became hyperglycemic after the toxicant. Patterns ofbody temperature change after the toxicant, which might be due to its metabolites, were dissimilar. Hypothermia was not observed in fed rats, was only transiently evident in T4 rats, but occurred rapidly within 1 hr in fasted rats and steadily became more severe. The dissimilar patterns of liver enzyme and body temperature and serum glucose change after the toxicant in the three groups are indicative ofdifferent pathways ofinjury potentiation by fasting and hyperthyroidism. toss Aodemic Press, inc. 1,1-Dichloroethylene (1,1-DCE) is an inter esting model hepatotoxicant because multi ple different conditions or pretreatments which are known to affect cell constituents in volved in the metabolism of 1,1 -DCE (Fig. 1) modulate its acute hepatotoxicity (Reynolds el al., 1975; Szabo el al., 1977; Anderson el al, 1980; Masuda and Nakayama, 1983). These pretreatments provide a way to probe 1 Portions ofthis paper were presented at the 1986 An nual Meeting of the American Society of Pharmacology and Experimental Therapeutics and the Society of Toxi cology, Baltimore, Maryland, 1986 (Pharmacologist 28, 191. 1986). the cause-effect sequence ofevents leading to cell injury. 1,1-DCE is used for the synthesis of plastic film and has been identified in in dustrial sewage treatment effluents, well wa ter, and treated drinking water (Coleman et al, 1976). Fasting and excess thyroxine are two pre treatments which markedly enhance the hep atotoxicity of 1,1-DCE (Jaeger et al., 1974; Szabo et al, 1977; Jaeger et al, 1977). Both of these pretreatments lower liver contents of GSH (Jaeger et al, 1977) which, as illustrated in Fig. 1, is needed for the formation ofconju gates with reactive metabolites of 1,1-DCE (Liebler, et al, 1985). However, the mecha- SL 040443 93 004I-008X/88 $3.00 Copyright 1988 by Academic Press, Inc. All rights of reproduction in soy form reserved. 94 KANZ ET AL. A M4tH - CIbC-CH, --*>[0C-Oif-P450 I--* ClgC^Ol --* wm Hiutim [transition states! ______ ri1 cijiecHO* I ciHjCCOCi*----------- ciHgCcogH CljHCO^OH I nno anil ms GSH con legates I Fig. 1. Pathways for metabolism of 1,1 -DCE by a cyto chrome P-450-dependent Phase l reaction to reactive in termediates (a, b and c) that can be further converted by hydrolysis or phase II oxidation, reduction (to intermedi ates d, e and f) and finally conjugation with one or two molecules of GSH. Alcohol and aldehyde dehydroge nases presumably convert the dichloroacetaldehyde in termediate (b) to alcohol (e) and acid (0 intermediates. Glutathione transferases may facilitate the conjugation with GSH. Intermediates capable of binding to cell con stituents are marked with an *. Modified from Costa and Ivanetich(1984). 1.1- DCE on cytochrome P-450 content were not evaluated in this in vivo study. Isozymes of cytochrome P-450 differ widely in their rates of oxidizing 1,1-DCE, particularly with regard to formation of the very reactive di chloroacetaldehyde (metabolite b in Fig. 1) (Liebler and Guengerich, 1983). Therefore, an in depth in vitro study would be required to determine the effects of these pretreat ments and 1,1 -DCE on oxidation of 1,1 -DCE to reactive metabolites. However, we did in vestigate effects of the pretreatments and 1,1DCE on other liver constituents (including glutathione transferases, alcohol and alde hyde dehydrogenases) potentially involved in the detoxification ofreactive intermediates of 1.1- DCE. Liver injury was monitored by measuring serum activities of liver-derived enzymes and bilirubin content. MATERIALS AND METHODS nisms underlying the injury enhancement are poorly understood. In the two studies of thy roxine enhancement (Szabo et al., 1977; Jae ger et al., 1977), 1,1-DCE was administered at a very toxic dose (2000 ppm for 4 hr) that was lethal to 60% of the fasted rats within 3 days, whereas 75% ofthe hyperthyroid, fasted rats died--most during the 4-hr 1,1-DCE ex posure period. This study was designed to compare the re sponses offed, fasted, and thyroxine (T4)-pretreated rats to 50 mg 1,1-DCE/kg which pro duces moderate liver injury in fasted rats and is not lethal (Moslen and Reynolds, 1985). Body temperature changes were monitored because 1,1-DCE was reported to produce a rapid and dose-dependent hypothermia in mice (Masuda and Nakayama, 1983). Pat terns of GSH depletion in liver and other tis sues were examined because GSH conjugates are the major metabolites of 1,1-DCE (Liebler et ai, 1985), and because covalent bind ing of 1,1-[14C]DCE in mice has been re ported highest in kidney, liver, and lung with smaller amounts in heart and gut (Okine et al., 1985), Effects of the pretreatments and Chemicals. 1,1-DCE of99% purity was obtained from Aldrich Chemical Co. (Milwaukee, WI). Thyroxine, NAD, glutathione, and propionaldehyde were obtained from Sigma Chemical Co. (St. Louis, MO). The glutathi one transferase substrates, chloro-2,4-dinitrobenzene (CDNB), 3,4-dichloronitrobenzene (DCNB), and 1,2epoxy 3-(p-nitrophenoxy)-propane (ENPP), were ob tained from Aldrich or Sigma. CDNB and DCNB were recrystallized in ethanol-water twice before use. Animals. Male Sprague-Dawley rats (Timco, India napolis, IN) weighing 230-300 g were used. The rats were housed in wire-floored cages suspended over absorbent paper in a 12-hr light/dark cycle animal room for at least 1 week prior to beginning treatments. Purina Lab Chow was provided ad libitum. Experimental protocol. Animals were randomly as signed to the fed group, the fasted group, or the thyroxine group. Animals in the thyroxine group received three sc injections of thyroxine (100 Mg/100 g) dissolved in alka line saline at 48-hr intervals with the last injection 24 hr prior to the experiment. Animals in both the fed and fasted groups received three injections of alkaline saline. Measurements of serum T4 levels by radioimmunoassay (Mallinckrodt, SPAC T4 RIA kit, Mallinckrodt, Inc., St. Louis, MO) verified that this T4 pretreatment regimen produced a fourfold elevation ofserum T4 level (i.e., 16.4 Mg T4/dl compared to 4.0 Mg T4/dl in fed controls). Ani mals in the fasted group had their food removed at 4 pm on the day prior to the experiment. At 8 am on the morning ofthe experiment, all animals were weighed and randomly assigned to 1,1-DCE treat- SL OAQAAA -450 content were o study. Isozymes er widely in their , particularly with t very reactive dibolite b in Fig. 1) 1983). Therefore, would be required of these pretreatdation of 1,1-DCE owever, we did inreatmentsand 1,1tituents (including alcohol and aldentially involved in ve intermediates of /as monitored by _`s of liver-derived tent. mMETHODS ;y was obtained from ;ee, Wl). Thyroxine. ildehyde were obtained iuis, MO). The glutathioro-2,4-dimtrobenzene ne (DCNB), and 1.2ine (ENPP), were obDNB and DCNB were ice before use. ley rats (Timco, Indiavere used. The rats were pended over absorbent .inimal room for at least lents. Purina Lab Chow als were randomly asgroup, or the thyroxine group received three sc 00 g) dissolved in alkathe last injection 24 hr Is in both the fed and lions of alkaline saline. < by radioimmunoassay . Mallinckrodt. Inc.. St. pretreatment regimen crum T4 level (i.e.. 16.4 11 in fed controls), Anir food removed at 4 pm it. xperiment, all animals ied to I.I-DCE treat POTENTIATION OF 1,1-DCE hepatotoxicity 95 ment or control groups, and food was removed from the ed and T4 groups. Between 9 am and 11 am, 1,1-DCE as administered at a dose of 50 mg/kg by gavage in 2 ml nineral oil/kg. Treatment controls received mineral oil. Body temperatures were measured with an electronic ,'Ctal thermometer (Harvard Bioscience, South Natick, 1A) at 30-min intervals for the first 2 hr and at 60-min nervals for the next 6 hr. Fasted and T4 animals were tiled at 2, 4, or 8 hr after treatment with 1,1-DCE. Fed its were killed only at 4 and 8 hr, since other studies ave indicated little difference between 2 and 4 hr in the iochemical parameters of interest such as serum gluose. glutathione, and the cytoplasmic enzyme levels Vloslen el al.. 1987). Control animals were killed at the eginning and end ofthe experimental period to provide early" control values and "late" control values for the - and 4-hr experimental animals and the 8-hr experi'tental animals, respectively. Diurnal variation of liver lutathione is less than 5% in fed animals and less than 5d in fasted animals between 9 am and 1 pm (Jaeger et :i, 1973; Reynolds el al., 1980). Animals were anesthelzed with ether. Liver, kidney, and testis were homogenized in 5 vol ind heart, lung, and small intestine in 10 vol ofbuffered ).25 M sucrose, 0.01 mM KP04 (pH 7.4), and cytosol was irepared by differential centrifugation as previously de scribed (Moslen and Reynolds, 1985). Liver cytosol con fining 0.33 mM dithiothreitol was stored at -80*C for biochemical assays'. Preliminary studies indicated that dithiothreitol addition provided excellent preservation of enzyme activities and had no effect on toxicant-induced alterations. Biochemical analyses. Serum glucose and bilirubin concentrations and glutamate pyruvate transaminase (GPT) activities were measured with Sigma reagent kits Nos. 16,605, and 57, respectively. Protein was measured by the Lowry et al. (1951) method with bovine serum albumin as the standard. Reduced glutathione content was estimated with Ellman's reagent as previously de scribed (Moslen and Reynolds, 1985). Glutathione trans ferase activities towards CDNB and DCNB were mea sured spectrophotometrically at 25`C, and ENPP at 30"C, according to Habig et al. (1974) and Younes et al. (1980a), respectively. Alcohol dehydrogenase was as sayed in the direction ethanol -* acetaldehyde according to Crow et al. (1977). Aldehyde dehydrogenase was as sayed according to Lindahl (1979) using propionaldehyde as substrate and NAD as coenzyme. Statistical analysis. The data obtained were analyzed by one-way analysis of variance using a statistical pack age (STATSOFT) for personal computers; when differ ences were found at p < 0.05, individual comparisons between a treatment group and its respective "early" or "late" control were evaluated by the t test. RESULTS As illustrated in Fig. 2, body temperatures of fed rats were not altered by 1,1-DCE. Fig. 2. Time course of body temperature changes in fed, fasted, and thyroxine (T4)-pretzeated rats given 50 mg 1,1 -DCE in mineral oil (filled symbols) or mineral oil (open symbols). Values are means SE of four rats per group. Where error bars are not shown, the SE lies within the symbol. Asterisks near symbols of 1,1-DCE-treated rats indicate values that were significantly lower than their respective controls at p < 0.05. Fasted rats showed a decline in body temper ature within 1 hr after 1,1-DCE administra tion; the hypothermia was persistent and pro gressive. In contrast, 1,1-DCE had a slight and transient hypothermic effect in T4 rats that was statistically significant only at 2 and 3 hr. Figures 3 and 4 compare the relative mag nitudes of liver injury in the animals given 1,1-DCE as quantitated by the elevation in SGPT activities and serum bilirubin con tents. The change in SGPT was slight in the fed group, most severe in the fasted group, and intermediate in the T4 group. Only the fasted group showed statistically significant increases in bilirubin. Histological changes in the centrilobular regions oflivers of fasted and T4 rats after 1,1DCE are shown in Fig, 5. The pretreatments of fasting and T4 administration did not alter liver histology in control animals (i.e., see Fig. 5A). At 8 hr after 1,1-DCE, morphological changes were confined to an area of three to SL 040445 i;'i [ 96 KANZ FT AL. Hrs. after 1.1-OCE Hrs. after 1,1-DCE Figs. 3 and 4, Time course ofchanges in serum GPT activities and bilirubin contents of fed (Fd), fasted (Fs), and thyroxine (T4)-pretreated rats given JO mg t_ 1-DCE/kg in mineral oil (filled symbols) or'mineral oil (open symbols). Values are means SE of four ran, per group. Where error bars are not shown, the SE lies within the symbols. Asterisks near symbols of 1.1-DCE-treated rats indicate values that were signifi cantly higher (p < 0.05) than their early pretreatment control at 2 and 4 hr or late pretreatment control at 8 hr. five cell layers surrounding the central vein in livers of T4 pretreated animals (Fig. 5B) while livers of fasted rats showed extensive centrilobular necrosis (Fig. 5C). Morphological changes were not consistently observed in liver sections from fed rats 8 hr after 1,1 -DCF (Fig. 5D), even though serum GPT activities were increased by approximately 19-fold. Patterns of serum glucose change were markedly different in the fed and T4 animals compared to the fasted animals, as indicated in Fig. 6. Fed rats showed no appreciable change by 4 hr after 1,1 -DCE and then serum glucose decreased to 81 % of the fed late con trols by 8 hr. T4 rats showed a much more abrupt decrease in serum glucose values after toxicant administration; values at 2 hr were 66% of the T4 early control values. In con trast. the fasted rats became hyperglycemic after 1,1-DCE; values at 4 hr were 150% of fasted controls. Figure 7 compares the different patterns of change in liver GSH. Note that the livers of both the fasted and T4 control rats had less than 60% as much GSH per kilogram body weight as did the livers of the fed controls. The lesser amounts of GSH in the livers of the fasted and T4 controls reflect, in part, their smaller average liver weights of 30.2 and 34.0 g/kg body wt, respectively (compared to 38.9 for the fed control). However, their smaller livers did not fully account for the Fig. 5. Livers of rats given mineral oil (A) or 50 mg l.]-DCE/kg (B, C, D) 8 hr previously, (c, central vein; p, portal). H&E, X325. (A) Pretreatment b> T4 administration (or fasting, not shown) did not produce morphological alterations in liver parenchyma. iB> Pale, enlarged vacuolated cells occur up to five cell layers away from the central vein (arrow) in T4-pretreated animals. (C) Extensive liver necrosis and conges tion surround the central vein and extend toward tbe midzonal region in fasted animals. (D) Appreciable alterations to centrilobular liver parenchyma were not observed in fed animals. SL 040446 POTENTIATION OF 1,1-DCE HEPATOTOXICITY 97 i ----- Fd 6 7a l-DCE of fed (Fd), fasted nbols) or mineral lot shown, the SE that were signifiatment control at $ I 3i dues at 2 hr were ol values. In conne hyperglycemic hr were 150% of ~ : fferent patterns of that the livers of itrol rats had less er kilogram body the fed controls. H in the livers of s reflect, in part, eights of 30,2 and ely (compared to However, their account for the * ; >usly. (c, central 1 did not produce ir up to five cell osis and conges(D) Appreciable SL 040447 98 KANZ ET AL. Hrs. after 1.1-DCE Fig. 6. Time course of changes in blood glucose con tents of fed (Fd), fasted (Fs), and thyroxine (T4)-pretreated rats given 50 mg 1,1 -DCE/kg in mineral oil (filled symbols) or mineral oil (open symbols). Values are means SE of four rats per group. Star near the symbol for the fasted early control value indicates difference at p < 0.05 from the fed early control value. Asterisks near symbols of 1,1-DCE-treated rats indicates values that were significantly different (p < 0.05) from their early pretreatment control at 2 and 4 hr or late pretreatment control at 8 hr. difference in hepatic GSH, since when he patic GSH content is evaluated in terms of micromoles GSH/gram liver, values of liver GSH in the fasted and T4 rats were still about 70% that of fed rats. 1,1 -DCE caused an early depletion of he patic GSH to about 50-60% control values in both the fed and T4 groups and a relatively greater magnitude of decrease to about 30% control values in fasted rats. The patterns of hepatic GSH change at 8 hr after 1,1-DCE were quite different, with a small but signifi cant rebound above late control values in the fed group compared to sharper rebounds above the nadir in the fasted and T4 groups. Another difference in the response of the three groups to the toxicant was that average amounts of hepatic GSH depletion at the early time point in the fed and fasted groups were similar with decreases of 107 and 97 mmol/kg, respectively, while the amount of average early decrease in the T4 group was less at 67 mmol/kg. Kidney and lung patterns of GSH change are shown in Fig. 8. Fasting did not alter kid ney or lung GSH content, unlike the marked effect of fasting on liver content of this nu cleophile. T4 administration led to a statisti cally significant increase in kidney GSH con tent, but a decrease in lung GSH content compared to fed controls, 1.1-DCE had no evident effect on kidney GSH in fed rats. However, the toxicant had opposite effects on kidney GSH in the other two groups; these were an increase to 123% of control values by 8 hr in the fasted rats but a decrease to 82% by 4 hr in T4 rats. Lung GSH contents were diminished after 1,1-DCE in. fed and fasted rats, but were not appreciable altered in T4 rats. Patterns of GSH change in testis and heart and in small intestine are presented in Figs. 9 and 10, respectively. Fasting produced sig nificant increases in GSH in both testis and heart compared to fed controls while T4 pre treatment increased GSH content only in the heart. In contrast to the effects of 1,1 -DCE on liver, kidney, and lung GSH levels, testis and heart GSH were not appreciably altered by 1.1- DCE. Small intestine GSH contents were similar in the pretreated and fed controls with 1.1- DCE administration producing a GSH decrease only in the fasted group, to 76% of fasted control value by 4 hr with a subsequent rebound by 8 hr. Tables 1 and 2 detail the changes measured in liver cytosolic enzymes that potentially have a role in the detoxification of reactive epoxide, alcohol, aldehyde, or acid metabo lites of 1,1-DCE. Glutathione transferase ac tivities toward the three substrates were lower in the fasted controls than the fed controls, and even lower in the T4 controls. 1,1-DCE administration to fed rats led to a small de crease in this enzyme activity toward one substrate (ENPP), while marked decreases were consistently found after toxicant admin istration toward all substrates in fasted rats. SL 040448 while the amount of in the T4 group was terns of GSH change ting did not alter kidnt, unlike the marked r content of this nuation led to a statistis in kidney GSH coni lung GSH content ols. 1,1-DCH had no ey GSH in fed rats, ad opposite effects on ler two groups; these l ofcontrol values by ut a decrease to 82% l GSH contents were CE in fed and fasted eciable altered in T4 ige in testis and heart presented in Figs. 9 f:ing produced sigin both testis and ontrols while T4 preH content only in the effects of 1,1 -DCE on jSH levels, testis and 7 ipreciably altered by e GSH contents were and fed controls with i producing a GSH :ed group, to 76% of hr with a subsequent T j, } / ^ ^ le changes measured nes that potentially ification of reactive de, or acid metabotione transferase acubstrates were lower an the fed controls, 4 controls. 1,1 -DCE is led to a small deictivity toward one marked decreases fter toxicant adminiiates in fasted rats. POTENTIATION OF 1,1-DCE HEPATOTOXICITY 99 Hrs. after 1.i-DCE Hrs. after 1. t-QCE Figs. 7 and 8. Time course of changes in liver, and kidney and lung GSH of fed (Fd), fasted (Fs), and thyroxine (T4)-pretreated rats given 50 mg 1,1-DCE/kg in mineral oil (filled symbols) or mineral oil (open symbols). Values are means SE of four rats per group. Where error bars are not shown, the SE lies within the symbol. Stars near symbols of early control values indicate values that were different at p < 0.05 from the fed early controls. Asterisks near symbols of 1.1 -DCE-treated rats indicate values that were significantly different (p < 0.05) from their early pretreatment control at 2 and 4 hr or late pretreatment control at 8 hr. In contrast, 1,1-DCE had no appreciable effect on glutathione transferases of T4 rats. The most striking change observed in the al cohol and aldehyde dehydrogenase activity w as that the T4 control animals had just 39% as much hepatic alcohol dehydrogenase ac tivities as the fed controls. 1,1 -DCE adminis tration consistently led to decreases in both dehydrogenase activities in the fasted rats, but had no evident effect in either the fed or T4rats. DISCUSSION This study demonstrated that pretreatraent of rats with sufficient T4 to produce hyperthy roidism enhanced the hepatotoxicity of a nonlethal dose of 1,1-DCE. The enhancing effect ofT4 pretreatment on 1,1-DCE hepato toxicity was less than that of fasting pretreat ment. We examined the effects of fasting and T4 pretreatment on several liver constituents whose alteration could affect the detoxification of 1,1-DCE reactive metabolites. Liver GSH contents were lowered to a similar extent by fasting and T4. Fasting slightly decreased alde hyde dehydrogenase activities, and in accord with the study of Mezey and Potter (1981), T4 diminished alcohol dehydrogenase activities more than 50%. In addition, we found for the first time that T4 produced a substantial de crease in cytosolic glutathione transferase activ ities. The T4 effect on the glutathione transfer ases was evident with the universal substrate, CDNB, and also with the substrates, DCNB and ENPP, reported to be more specific for cer tain isozymes of the enzyme (Habig el al, 1974). A prior study in Wistar rats had reported an increase in epoxide transferase activity and no change in aryl transferase activity following a 3-day triiodothyronine pretreatment (Younes etal, 1980b). SL 040449 4 100 KANZ ET AL. Hrs. after 1.1-DCE FIgs. 9 and 10. Time course of changes in testis and heart, and small intestine GSH of fed (Fd), fasted (F$), and thyroxine (T4)-pretreated rats given 50 mg 1,1-DCE/kg in mineral oil (filled symbols) or mineral oil (open symbols). Values are means SE of four rats per group. Where error bars are not shown, the SE lies within the symbol. Stars near symbols of early control values indicate values that were different at p < 0.05 from the fed early controls. Asterisks near symbols of 1,1-DCE-treated rats indicate values that were significantly different (p < 0.05) from their early pretreatment control at 2 and 4 hr or late pretreat ment control at 8 hr. Our observation of more severe 1,1-DCEinduced liver injury in the fasted than the T4pretreated rats, despite their similar GSH content and the lesser effect of fasting on the detoxification enzymes (glutathione transfer ase and alcohol dehydrogenase), suggests that fasting potentiates the hepatotoxicity of 1,1DCE by different pathways than T4. This difference in injury potentiation might be due to differences in 1,1-DCE metabolism, i.e., in the rate or extent of 1,1 -DCE activation to re active intermediates or the subsequent detox ification of these reactive species. Several findings of our study are consistent with possible differences in 1,1 -DCE metabo lism between the fasted and T4 groups. First, 1,1 -DCE had a more pronounced hypother mic effect in fasted rats than in T4 rats. Masuda and Nakayama (1983) proposed that the dose-dependent hypothermic effect of 1,1DCE was due to its metabolites because they found that administration of cytochrome P-450 inhibitors shortly before 1,1-DCE poisoning prevented hypothermia. In addi tion, they found that two of 1,1-DCE's oxi dized metabolites, chloroacetyl chloride and monochloroacetic acid (c and d in Fig. 1). rapidly caused hypothermia. Second 1,1DCE caused a more pronounced depletion of hepatic GSH in fasted than T4 rats which sug gests that metabolite conjugation with GSH occurs at a different rate or extent in fasted than T4 rats. Third, only in the fasted rats did 1.1-DCE cause a decrease in liver glutathione transfer ase activities. Moslen and Reynolds (1985) proposed that the decrease in hepatic gluta thione transferases of fasted animals given 1,1-DCE could be due to the "scavenger" function of the enzyme. Kinetics studies of glutathione transferase isozymes indicate that, under normal physiologic conditions. GSH binds to the enzyme before the electro philic substrates (Pabst et at., 1974). Low GSH concentrations facilitate its scavenging function in vitro, possibly by allowing the re- SL 040450 1 i-DCE I of fed (Fd), fasted vmbols) or mineral not shown, the SE were different at p dicate values that ^ or late pretreat- othermia. In addiof 1,1-DCE's oxiicetyl chloride and and d in Fig. 1), mia. Second, 1,1>unced depletion of i T4 rats which sugugation with GSH or extent in fasted 1 rats did 1,1 -DCE utathione transferI Reynolds (1985) - in hepatic glutated animals given ) the "scavenger" nineties studies of sozymes indicate logic conditions, before the electro' al., 1974). Low late its scavenging tallowing the re- I POTENTIATION OF 1,1-DCE HEPATOTOX1CITY 101 TABLE 1 Effect of Fasting. Thyroxine and 1,1 -DCE on Liver Cytosolic Glutathione Transferase Activities Glutathione transferase Pretreatment Treatment Time (hr) CDNB DCNB Oimol-liver/kg-min) ENPP Fd Fd Fs Fs Fs FdT4 FdT* FdT4 oil 1,1-DCE oil 1. I-DCE 1,1-DCE oil 1,1-DCE 1,1-DCE 2 3307 120 4 2990 226 (91%)" 143 11 135 9(94%)- 65.6 2.0 53.8 0.9 (82ctta+ 2 2654 108 (80%r* 115 4(80%)*-* 48.5 2.8 (74^1+" 2 1836+ 99 (69%)4-tt 67 3 (58%)4tt 30.1 1.6 (62^ F++ 4 1793 86 (68%)4,tt 70 4(61%)4tt 30.6 0.8 (63Tr++ 2 2147 19(65%r** 2 1984 76(92%Y 4 2091 80 (97%)' 82 1 (57%)- 77+ 5(94%F 80 2(97%F 43.3 1.3(66^1" 40.0 3.1 (92% F 39.6 5.4 (9I%F Note, Values are means SE of four animals per group. " Percentage of Fd-oil control group. 4 Percentage of Fs-oil control group. ' Percentage ofT,,-oil control group. * p < 0.05 compared to Fd control group. ** p < 0.01 compared to Fd control group, t P < 0.05 compared to pretreatment control group, tt P< 0.01 compared to pretreatment control group. TABLE 2 Effect of Fasting, Thyroxine and 1,1 -DCE on Liver Cytosolic Enzyme Activities Alcohol dehydrogenase Aldehyde dehydrogenase Pretreatment Treatment Time (hr) Ethanol Propionitrile Oimol - liver/kg min) Fd Fd Fs Fs Fs FdT,, FdT4 FdT4 oil i,i-ix:e oil 1,1-DCE 1,1-DCE oil 1,1-DCE 1,1-DCE 2 4 2 2 4 2 2 4 45.5 3.2 44.7 2.2 (98%r 39.6 3.5 (87%) 32.1 3.5(81%)4t 32.1 1.9 (81%)4t 17.7 0.9 (39%)"* 16.4 1.0(93%)' 20.5 3.1 (116%)' 32.1 1.3 32.6 1.7 (I02%F 25.4 2.4 (79%F21.6 0.1 (85%F+ 18.8 2.1 (74%F-H 30.7 1.0 (96%F 33.1 2.9 (108%F 30.6 2.6(100%F Note. Values are means SE of four animals per group. " Percentage of Fd-oil control group. 4 Percentage of Fs-oil control group. ' Percentage ofTj-oil control group. * p < 0.05 compared to Fd control group. ** p < 0.01 compared to Fd control group, t p < 0.05 compared to pretreatment control group, tt P < 0.01 compared to pretreatment control group. SL 040451 102 KANZ ET AL. active electrophilic substrate to bind first to enzyme not "protected" by a bound GSH (Wolkoff et al., 1979). The lesser magnitude of GSH depletion in the T4 compared to the fasted rats after 1,1-DCE may account for the absence of 1,1 -DCE-induced decrease of glu tathione transferase activities in the T4 rats. Blood glucose was not monitored in prior studies of 1,1-DCE toxicity. We expected a hypoglycemic effect in all groups because of the need to use hepatic glycogen stores for metabolism of 1,1 -DCE--specifically, to pro vide reducing equivalents for the regenera tion of the NADPH required for the first oxi dation of 1,1-DCE by a NADPH-cytochrome .P-450 reaction (Costa and Ivanetich, 1984). Since hyperthyroidism is associated with low hepatic glycogen stores, it is logical that hypoglycemia occurred more rapidly af ter 1,1-DCE in the T4 group than the fed group. Unexpected was the hyperglycemia in the fasted group after 1,1-DCE. It is possible that 1,1-DCE damages liver enzymes which regulate hepatic gluconeogenesis or the reten tion of glucose by the hepatocyte. Alterna tively, 1,1-DCE could cause the hyperglyce mia by an injurious effect on the pancreatic /3 cell as has been observed by Hinson et al. (1983, 1984) in mice poisoned with acet aminophen, However, we have not observed appreciable light microscopic alterations in the pancreas of 1,1-DCE-treated fasted rats (Kanz and Moslen, unpublished observa tion). In summary, the lack of concordance in the responses of the fasted and T4 rats to a moderately injurious dose of 1,1-DCE--spe cifically, the dissimilarities in the magnitude of GSH depletion, and in the onset and dura tion of hypothermia, as well as the opposite changes in serum glucose--indicates that these two pretreatments enhance 1,1-DCE hepatotoxicity by different mechanisms. ACKNOWLEDGMENTS The authors thank Avis D. Morgan for assistance in preparing the manuscript and Carlos Vanoye-Trevino for preparation of the figures. This research was sup ported by NIEHS Young Investigator Award ES 03368 and NIH Grant AM 34806 to M.T.M. and NIEHS FIRST Award ES 04273 to M.F.K.. REFERENCES Anderson, M. E., Thomas. O. E., Gargas, M. L., Jones, R. A., and Jenkins, L. J., Jr. (1980). The sig nificance of multiple detoxification pathways for reac tive metabolites in the toxicity of 1,1-dichloroethylene. Toxicol. Appl. Pharmacol. 52,422-432, Coleman, W. E., Lingg, R. D., Melton, R. G,, and Kopfler, F. E. (1976). The occurrence of volatile or ganics in five drinking water supplies using gas chro matography/mass spectrometry. In Identification and Analysis ofOrganic Pollutants in Water (L. H, Keith, Ed.), pp. 305-327. Ann Arbor Science Publishers, Ann Arbor, MI. Costa, a. K., and Ivanetich, K. M. (1984). Chlori nated ethylenes: Their metabolism and effect on DNA repair in rat hepatocytes. Carcinogenesis 5, 16291636. 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