Document jBwz3zB2qyeNnR8dzZ1Kym3EO

KtCtIVtD JUL 18 1975 _ R. N. WHEELER, JR. \ MODULATION OF HALOTHANE AND VINYL CHLORIDE INDUCED ACUTE t INJURY TO LIVER ENDOPIASMIC RETICULUM Edward S. Reynolds, Mary Treinen Moslen, Sandor Szabo, Rudolph Jaeger From the Departments of Pathology, Peter Bent Brigham Hospital and Harvard Medical School, Boston, Mass., and Kresge Center for Environmental Health, Harvard School of Public Health, Boston, Mass. Supported by Grants ES-00002, OH-00315, AM-16183 and HL-06370 from the National Institutes of Health. Dr. Reynolds is an NIH Research Career Development Awardee (GM-07309). Address reprints to: Doctor E.S. Reynolds Department of Pathology Peter Bent Brigham Hospital 721 Huntington Avenue - Boston, Mass. 02115 ucc 035474 SUMMARY Acute liver Injury In mature male rats following a single exposure to halothane (0.85% x 5 hr) or to vinyl chloride (5X x 6 hr) can be consistently produced when preceded by pretreatment for 7 days with Aroclor 1254 (15 nmoles/100 g/day) and to a lesser extent phenobarbital (PBT) (40 praoles). Morphologically the Injury Is characterized by primary involvement of the endoplasmic reticulum with denaturation of the smooth membranes. While halothane produces focal damage in PBT animals, centrolobular necrosis following halothane is widespread in Aroclor 1254 animals and is accompanied by increases in serum transaminases. Injury following VCM in PBT animals is characterized by striking vacuolization of hepatic centrolobular parenchyma, while in Aroclor 1254 animals vacuolization is panlobular and focal midzonal necrosis is extensive. Both PBT and Aroclor 1254 induce components of the xenobiotic metabolizing mixed function oxidase system (MFOS) altering rates and pathways of halocarbon metabolism. The- enhanceable hepatotoxicity of halo- * thane and VCM may be related to induction of specific pathways of their activation (toxification) via free radical or epoxide intermediates. ucc 035475 -2- INTRODUCTION Within this half of the 20th century halogenated hydrocarbon products have be come ubiquitous components of our environment. Billions of pounds are produced each year for use In industry, agriculture and medicine. A number of these chemi cals have been found to cause injury to mai\ and to our misfortune, such discoveries usually were made after use had become widespread (1,2,3). While the linkage of occupational vinyl chloride exposure to angiosarcoma of the liver may be viewed as a milestone in occupational hygiene (4), scientists should be challenged to identify the potential hazards of other similar halocarbohs also in use. Screening all industrial chemicals for carcinogenicity by classic .in vivo tech niques is a task of herculean proportions. Mutagenic potential may be detectable by the Ames method or other vitro methods such as those used by Fienta and DlPaolo. Although such screenings may indicate compounds capable of neoplastic potential, the understanding of how xenobiotlcs alone, and in combination, cause both acute and chronic injury requires careful inquiry into the molecular basis of their toxic action. Examination of the chemical-pathology of halocarbon-induced cell damage should provide insights into the toxicological mechanisms involved, and ergo form the basis of new predictive tests. Activation of many halocarbons to proximate toxins involves the enzymes of the mixed function oxidase system (MFOS) which in the liver is localized in the endoplasmic reticulum. A variety of drugs and environmental conditions alter the level and types of MFOS activity and have been used as biochemical tools to modulate response of animals to a variety of halocarbon toxins. Pretreatment with phenobarbital (PBT), a classic inducer of MFOS components has been used to heighten the hepatotoxfc potential of CCl^, bromobenzene, halothane (CFjCHBrCl) and vinyl chloride (ClHCiCI^) (5-8). Aroclor 1254 (1254) a polychlorinated biphenyl, widely used in industry as an insulator or plasticizer, is a more potent inducer of cer tain MFOS components and also a more potent enhancer of the hepatotoxicity of some ucc 0354~6 -3- halocarbons than PBT. This presentation vill describe our recent studies on the acute hepatotoxicity of halothane and vinyl chloride in animals with enhanced MFOS activity and discuss possible mechanisms of halocarbon toxification. MATERIALS & METHODS Male Sprague-Dawley rats (180-200 g) were housed on wire floor cages over pro- allowed cessed clay animal litter and free access to food and water. To induce the components of the hepatic MFOS, the animals were given 40 ^moles/100 g PBT or 15 nmoles/100 g Aroclor 1254 by gavage once daily for seven days. Compounds were solubilized in water with tracers of Tween 80. Controls received vehicle alone. On the eighth day after an overnight fast the animals were exposed to air, halothane (0.857. x 5 hr) or vinyl chloride (57. x 6 hr) in inhalation chambers previously described (7,8). Animals were refed briefly then fasted overnight for sacrifice at 24 hr from commencement of halocarbon exposure. Microsomal enzyme assays were performed concomitantly with th'e experiments to confirm induction of MFOS components. Previously reported procedures for the collection and iso lation of halothane metabolites were followed (7,9). Methods and results of these assays are described fully elsewhere (10). Serum glutamic oxalacetic and glutamic pyruvic transaminase (SGOT and SGPT) were determined with reagent kits from Sigma. Reduced glutathione was determined with Ellman's reagent according to Jaeger et al (11). Model System for Acute Halothane Injury: Interest in the hepatotoxic potential of halothane, currently the most popular and extensively used Inhalation anesthetic, is derived from association between clinical halothane anesthesia and subsequent liver injury in the occasional patient (1). In experimental animals, multiple manipulations of length or frequency of halothane exposure and pretreatment with drugs or surgery or prior depletion of endogenous antioxidants have met with limited success in producing widespread UCC 035477 -4- hepatic damage in the many species studied (7,12-15). In prior studies focal he patic Injury was consistently produced in PBT pretreated animals exposed to 0.65% halothane for 5 hr together with decreased cytochrome P 450 and transient increases in diene conjugation (7). These biochemically determined indices of injury to the endoplasmic reticulum are supported by ultrastructural changes in membranes of smooth endoplasmic reticulum in areas of Injury (Fig, X) consistent with its denaturation (16). Postulating that this injury was related to altered metabolism or binding of the halothane molecule, subsequent experiments were conducted with tracer doses of ^CT^CHBrCl which revealed that less label was exhaled into ex pired gases and more excreted into the urine in PBT pretreated animals as compared to controls (9). In contrast, labeled halothane Incorporation into cell subfractions or chemical constituents other than acid soluble fractions was not enhanced by PBT. pretreated " Aroclor 1254 animals appear to be a better model system in which to study the mechanism of halothane-induced liver injury. Exposure of these animals to 0.85% halothane in air for 5 hr results in elevated serum transaminases apparent as early as 2 hr after cessation of anesthesia (Table I) and widespread centrolobular necrosis by 24 hr (Fig. I). Levels of reduced glutathione were unaffected by anesthesia. In the presence of NADPH halothane is metabolized in vitro by the MFOS (17) but Ihe relationships between injury and the amounts metabolized, specific metabolites produced or pathways of metabolism remain obscure. Somewhat to our surprise, pre liminary experiments.have indicated that the Aroclor 1254 animals excrete no more of a tracer dose of ^CFjCHBrCl in urine by 24 hr and have similar amounts of in comparison to recoverable label in liver fractions other than cell sap H2O pretreated animals (Table II). It is now possible to postulate multiple pathways for the metabolism of halo thane (Fig. Ill)following its initial conversion to a free radical by an electron capture reaction with subsequent displacement of bromine (18). One subsequent ucc 035478 -5- pathway may involve reaction with molecular oxygen, eventually forming an aldehyd , This could be "safely" oxidized to trifluoracetic acid, the primary urinary meta bolite of halothane. Or the aldehyde could add to electrophilic sites in proteins. Generation of an aldehyde within the endoplasmic reticulum may account for the relatively specific labeling of microsomal protein by ^CF^CHBrCl in vivo (9). Chlorine would tend to be lost In the formation of the aldehyde. Alternatively the free radical generated could either add across double bonds of polyunsaturated fatty acids of phospholipids, or abstract labile hydrogen atoms from susceptible molecular species such as methylene bridges of polyunsaturated fatty acids or non protein or protein sulfhydryls. In both addition and abstraction reactions chlorine would remain with the halothane carbons. In vivo investigations in Van Dykes laboratory with or that the metabolite bound to the phospholipid retains the Cl atom while that metabolite Involved in protein binding does not (19). Anaerobiosis in the in vitro systems does not quantitatively alter the rate of NADPH dependent metabolism of halothane but does increase the amount of metabolite bound (17). Hydrogen extraction pathways could lead to injured endoplasmic reticulum since loss of hydrogen atoms from methylene bridges of polyunsaturated fatty acids renders- lipids vunerable to attack by molecular oxygen and subsequent peroxidative composi tions. Increased content of lipid conjugated dienes, an indicator of hydrogen abstration, has been transiently observed after halothane anesthesia only in PBT pretreated animals (20,7). The debrorainated (and/or dechlorinated) reaction pro duct would be exhaled. Thus measurement of Increased halothane metabolism by this pathway would not be reflected in urine, liver, or total exhaled radioactivity, but would require relatively sophisticated chromatographic techniques. Model System for Acute Vinyl Chloride Injury: A single exposure to vinyl chloride monomer (VCM) (57. x 6 hr) produces acute \ '.CC 035479 -6- liver Injury In PBT pretreated rats (8), Involvement of the endoplasmic reticulum Is apparent (Fig, I). Pretreatment with Aroclor 1254 seems to render animals ex quisitely sensitive to VCM (Table III and Fig, II). Exposure to lower levels of' VCM (1.257. or 2.5% x 6 hr) produces both histologic. injury and enhances leakage of hepatic enzymes into serum. Although H2O pretreated, VCM-exposed animals have no apparent hepatic injury either histologically or according to serum transaminase levels (Table III) their livers are heavier and have higher contents of reduced glutathione (GSH) when com pared to H2O-pretreated, air controls. The magnitude of these VCM-induced changes is greater in PBT or Aroclor 1254 pretreated animals (Table TV), In other VCM experimental studies, Hefner, Jaeger and their colleagues (21,22) have reported de creased GSH contents in animals sacrificed immediately after single or multiple VCM exposures. Hefner's group has reported finding a mercapturic acid conjugate of a VCM metabolite in the urine (21). Our finding of elevated GSH levels 24 hr after VCM would be consistent with a rebound phenomenon. Following bromobenzene GSH levels plummet and then rebound to above normal levels by 24 hr (23). If the activation of VCM to a hepatotoxin occurs during its metabolism, an initial reaction would likely occur via the multiraoleeular MFOS, the enzyme system responsible for the conversion of most xenoblotlcs to more readily exeretable metabolites. Meta bolism of xenoblotlcs by this pathway may lead to toxiflcation or detoxification. Correlations between.induction of specific MFOS components and the degree of VCMinduced liver injury as measured by Increased serum transaminases at 24 hr following VCM, reveals significant relationships between injury and both increased NADPH cyto chrome P 450 reductase activity (as measured by reduction of cytochrome C) and total cytochrome P 450 content (24,25). A ijon symetrically chlorinated homologue of VCM, trichloroethylene (TCE), has been studied more extensively. TCE complexes with cytochrome P 450 (26) and is ijCC 035450 - 7- (27) metabolized by components of the MFOS Jri vitro. In FBT and Aroclor 1254 animals, TCE's metabolism is increased and liver injury enhanced (10). Correlations between Induction of MFOS components and degree of TCE-induced liver Injury are similar to that found for VCM, i.e., there are significant correlations between injury and Increased NADPH cytochrome F 450 reductase activity and total cytochrome F 450 content (10). Because of these similarities in the acute hepatotoxieitles of VCM and TCE, a common mechanism for their metabolism (toxification) is a reasonable proposal (Fig. IV). Initial oxidation by the MFOS could produce an epoxide as the primary metabolite. Subsequently the epoxide could rearrange to form a j3 chlorinated acetaldehyde or interact with epoxide hydrase or GSH epoxide transferase to form either a diol or a glutathione conjugate as a secondary metabolite. two latter pathways has not been verified for TCE. The exlstance of these Chlorinated acetaldehydes could be converted to a tertiary generation of metabolites through hydration, or enzymatic reduction or oxidation. Chloral hydrate, trichlorethanol and trichloro acetic acid have all been identified as products of TCE metabolism (27) and mono- chloroacetlc acid has been found in the urine of VCM exposed rats (21). The epoxides, the chlorinated acetaldehyde and J3 chlorinated alcohols must all be considered potential proximate toxins. In conclusion it is hoped that studies such as these will provide insight into the biochemical mechanisms of halocarbon induced liver injury and will establish a framework for the detection of other potentially toxic halocarbons. ucc 03543i -8- FIGURE LEGENDS Figure Ii Masses of smooth endoplasmic reticulum in injured liver parenchymal cells of FBT pretreated rats 24 hr following exposure to (A) 0.857. halothane x 5 hr, and (B) 57. vinyl chloride x 6 hr. Focally, outer surfaces of tubular profiles in these tightly tangled tubular networks are associated with amorphous electron-opaque de posits (black "flecks") similar to those seen in labyrinthine tubular aggregates of denatured endoplasmic reticulum following CCI4 (16) and following peroxidation of microsomes in vitro (28). Contiguous rough endoplasmic reticulum is vacuolated, x 16,000. Figure II: Effect of PBT and Aroclor 1254 pretreatment on the acute toxicity of halothane and vinyl chloride 24 hr following the commencement of exposure. No lesions are seen in control animals exposed to these halocarbons. Focal vacuoli zation of midzonal parenchymal cells (arrows) is seen in FBT animals exposed to halothane. Extensive centrolobular necrosis is present in Aroclor 1254 pretreated animals exposed to halothane. Extensive centrolobular vacuolization following vinyl chloride in FBT animals becomes panlobular in Aroclor 1254 animals, e central vein; x 60. Figure III; Proposed scheme for metabolism of halothane by multiple pathways. The first step is an electron capture reaction with the formation of the trifluroehloroethyl radical and bromide ion. The radical formed can react with oxygen (eventually (not shown) forming an aldehyde) or add onto electrophilic sites in lipid or protein molecules or abstract hydrogen atoms to form a gaseous product. Figure IV; Proposed scheme for the metabolism of chlorinated ethylenes: trichloro ethylene is the example. Oxidation of TCE by MFCS produces an epoxide as the primary metabolite. Subjcoucutly, the epoxide may either rearrangeto form trichlor aceta- ucc 035482 - 9aldehyde (chloral) as the main secondary metabolite or interact with the epoxide hydratase or GSH epoxide transferase to form a diol or a glutathbne conjugate respectively. The existence of these latter two pathways for TCE has not been established (*). Trichloroacetaldehyde is in turn converted to a tertiary generation of metabolites through hydration, or enzymatic reduction or oxidation. Chloral hydrate, trichloroethanol and trichloroacetic acid have all been identi fied as .in vivo products of TCE metabolism^and chloroacetic acid Identified fol lowing vinyl chloride. Scheme adapted from Ikeda and Ohtsufi.(29). ucc 035^03 10 - REFERENCES 1. Bunker JP, Forrest WH Jr, Mosteller F and Vandam LD: The National Halothane Study, U.S. Govt. Printing Office, Washington, D.C. 1969. 2. Marsteller HJ, Lelbach WK, Muller R, Gedlgk P: Ann NY Acad Sci 246:95-134, 1975. 3. Makk L, Creech JL, Whelan JG Jr, Johnson MN: JAMA 230:64-68, 1974. 4. Editorial Lancet June 29, 1974, 1323-1324. 5. Gamer RC, McLean AEM: Blochera Pharmacol 18:645-650, 1969. 6. Brodie BB, Reid WD, Cho AK, Sipes G, Krishna G, Gillette JR: Proc Nat Acad Sci 68:160-164, 1971. 7. Reynolds ES, Moslen KT: Blochera Pharmacol 23:189-195, 1974. 8. Jaeger RJ, Reynolds ES, Conolly RB, Moslen MT, Szabo S, Murphy SD: Nature 252:724-725, 1974. 9. Reynolds ES, Moslen MT: Biochcm Pharmacol (In press). 10. Reynolds ES, Moslen MT, Szabo S; Modulation Of the hepatotoxicity of trichloro ethylene anesthesia by inducers of the liver mixed function oxidase system. (in preparation). 11. Jaeger RJ, Conolly RB, Murphy SD: Exp Mol Pathol 20:187-198, 1974. 12. Gopinath C, Jones RS, Ford EJH: J Path 102:107-114, 1970. 13. Hughes HC Jr, Lang CM: Anesthesiology 36:466 -471, 1972. 14. Almersjo 0; Acta Chirugica Scand (Suppl) 51:135, 1972. 15. Brown BR Jr, Sipes IG, Sagalyn AM: Anesthesiology 41:554-561, 1974. 16. Reynolds ES, Ree HJ: Lab Invest 25:269-278, 1971. 17. Van Dyke RA, Wood CL: Drug Metabolism and Disposition 3:51-57, 1975. 18. Atallah MM, Geddes IC: Brit J Anaesth 45:464-470, 1973. 19. Gandolfi AJ, Van Dyke RA: Society of Toxicology Abstract, Fourteenth Annual Meeting, March 9-13, 1975, p 23-44. 20. Brown BR Jr: Anesthesiology 36:458-465, 1972. ucc 035454 II 21. Hefner RE Jr, Watanabe PG, Gehrlng PJ: Ann NY Acad Sci 246:135-148, 1975. 22. Jaeger RJ, Conolly RB, Reynolds ES, Murphy SD: Environ Health Pars (in press). 23. Jollow DJ, Mitchell JR, Zarapaglione N, Gillette JR: Pharmacology 11:151-169, 1974. 24. MoslenKT, Jaeger RJ, Szabo S, Reynolds ES: Society of Toxicology Abstracts, Fourteenth Annual Meeting, March 9-13, p 23-25. 25. Reynolds ES, Moslen MT, Szabo S, Jaeger RJ, Murphy SD: Am J Path (in press). 26. Kelley JM, Brown BR Jr: Anesthesiol Clin 12(2):85-92, 1974. 27. Liebman KG: Mol Pharmacol 1:239-246, 1965. 28. Arstila AU, Smith MA, Trump BF: Science 175:530-533, 1972. 29. Ikeda M, Ohtsuji H: Brit J Ind Med 29:99-104, 1972. ucc 035455 12 - TABLE I SGOT LEVELS AFTER HALOTHANE1" IN AROCLOR 1254 ANIMALS Time After Exposure hrs 0 2 24 ___________Exposure to Air (control) HalothaneT Karmen units SEM* 153 18 149 t 21 375 t 152 211 21 603 129** 0.857. x 5 hr in air. p<\0.025 compared to respective air (control) group. 4 to 6 animals in each experimental group. IJCC 035486 - 13 - TABLE II RECOVERY AT 24 HR OF 14CF3CHBrCl METABOLITES IN URINE AND LIVER MFOS Inducer First Seriestt H2O (control) (8)** PBT (12) Second Series H2O (control) (4) Aroclor 1254 (6) Urine 8.6 0.6 15.3 1.2 Liver Total Acid Soluble - 7. dose* SEM - 1.1 0.1 ' 1.9 0.1T 0.5 0.1 0.9 0.^ 15.5 2.3 12.7 2.4 2.5 0.2 " 2.6 t 0.3 0.8 0.1 1.5 0.1* * = 7.0 x 106 dpm ; 10 pmoles/halothane/kg. ** (number of animals). t Difference between experimental and control p^O.OS Reynolds and Moslen(9) . ucc 035487 14 - TABLE III EFFECT OF MFOS INDUCEBS ON SGOT 24 HR AFTER EXPOSURE TO VCM1- MFOS Inducer H2O (control) PBT Aroclor 1254 Exposure to Air (control) VCMt Karmen units SEM* 185 11 156 6 196 16 572 72tt 211 20 1162 376tt 57. VCM x 6 hr. * 4 to 9 animals in each experimental group, ft p<0.05 between VCM and respective air (control) group. ucc 035438 15 TABLE IV EFFECT OF MFOS INDUCERS ON LIVER WEIGHT'AND REDUCED GLUTATHIONE CONTENT 24 HR FOLLOWING VCM* MFOS Liver Weight InducerAir (control)VCMAir (control)VCM gm/100 gm animal* H2O (control) PBT Aroclor 1254 4.3 0.2 5.9 t 0.0 6.2 0.2 4.9 0.2 7.7 o.ett ` 8.7 o.itt Glutathione mg/gm liver* 1.2 0.1 1.5 0.2 1.1 0.1 1.5 0.1 3.0 0.2tt 3.4 0.2ft 57. VCM x 6 hr. * 4 to 8 animals in each experimental groups mean SEM. ** p<0.025 between VCM and respective air (control) group. ucc 035489