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Environmental Health Perspectives Vol. 21. pp. 137-147. 1977 nainage t0 Hepatic Cellular Membranes chlorinated Olefins with Emphasis 0n Synergism and Antagonism by Edward S. Reynolds* and Mary Tremen Mosien* Tht fteikniMl i --cHUj or tebfllly of the cMorMfeyttav Mtocate ttttc., titer hopomoxic pour tte. EtUMtmd lynteryof ihochhirtwnittetate--. wUekakanttaartm eiloi-iHnUM, eterpo poterlMW, ate tewbUKy, tdact UMo^c mpo--.. TW mi fijMinluny tepotoriite dihroothykte. |.| iib WiinurtiluT (1,1-DCE) it tte non htptmwdc amt atom a MiqiK patun at tepolactUutor PteteT*tehTtet"<VCM)T por- liiimltjli (TEX). Ptiit--nail wtecfc larnm< eywcteM P-4M caaMi, prataitly aaf Mdi otteoMt acttradaa la a waaha iawnaadlat* took at aa rportte, ootetod ar wore tjatt|hUe ia tte btpaMtaak poiiikl of TRI, VCM tad PER bat m pnaatu ar MiptMt to 1,1-DCE tepMMMfcky. Rteoptc rmpmm la 1,1-DCE May ba ixproowd by t difltrtal --rebate pathway. ClaialWtat tpptart la ba tottered la tbt biologic nipoaii to a masymmatrk cbtoreirtj liore aad la ret aa aa MngMtat retetel tywy. Matted dMbrtant ia tte patitm af totery aad tte Mteogk trep-- Mgprel Ibal man tbaa aaa Mtetere it toreivod ia tba pradattka at tapirj by cbtormtbytotei Introduction Our studies of the acute hepatotoxicity of the .hloroethylenes have shown not only certain roianues in their biologic effects, but marked difmcnces in both relative toxicity and in the pattern f hepatocellular injury. These marked differences -ffesi that the chioroethyienes produce iryury by v*t than one mechanism. The relative reactivity :hese molecules is affected by the extent to which vir electron distribution is altered by chlorine sub;mmon. Two effects are involved. First, the more dcctronegative halogens attract electrons. Second, * orbital ofone of the lone pairs of electrons of the .Monne atom overlaps with the pi electrons of the -vbon-carbon double bond. This interaction inohes a partial loss of electrons by the chlorine iMn anda gain by the double bond as illustrated by `V small curved arrows in Figure 1 (/). The result is Jr1: of Pathology, University of Texas Media Texas 77JJ0. Please address reprint re 10 Dr. Edward S. Reynolds at the above address. **ber 1977 that the carbon-chlorine bond acquires some dou ble bond character and the molecule become polarized as shown for vinyl chloride (VCM). When two chlorine atoms are attached to one carbon of the double bond, as with 1,1-dichloroethylene (1,1-DCE). the net electron delocalization is en hanced. A more polarized molecule results. On the other hand, symmetrical attachment of the chlorines to both carbons of the double bond leads to a more uniform electron delocalization, as illus trated by the four resonance forms of perchloroethylene (PER), resulting in a more stable molecule. For trichloroethylene (TRI), electron delocalization would be less extreme than for 1,1DCE but more marked than for PER and some what similar to VCM. Increasing chlorine substitu tion also alters the biologic response to this series of chioroethyienes by increasing the lipid solubility and by depressing the volatility or "exhalabiiity." Our semiquantitative analysis of these factors (molecular reactivity/stability, lipid solubility and volatility) indicates an order of toxicity: 1,1-DCE 137 SL 066295 V:' mixed-function oxidase system is involved in the initial biotransformation of TRI, and that pretreat, ment with phenobarbetal (PBT) causes alterations in the rate and route of TRI metabolism. Carlson ($, reported that pretreatment with PBT 0l! 3-methylcholanthrene (3-MC) exacerbates the hepatotoxicity of TRI. Figure 1. Substitution or chtonnc atoms for hydrogen atoms affects the electron distribution of chloroethylene molecules. Electrons are attracted to the more electro-negative halogen atom. Another factor must also be considered for the orbital (position in space) of one of the lone pairs of electrons of the chlorine atom overlaps with the orbital of the pi electrons of the carbon to carbon double bond. The small curved arrows illustrate this phenomenon as movement of the electrons as sociated with the chlorine to the carbon to chlorine bond coupled with movement of electrons in the double bond to wards the other carbon. This shift of electrons causes a polarization of the molecule as indicated by the partial charges. The chlonnc to carbon bond has some double bond character as indicated by the dashed bonding lines. Sym metry and client of chlorine substitution which alter both the degree of molecular polarization and of electron delocaliza tion affect the molecular stability/reactivity. Principles of the chioroethylcne electron delocalization and description of re sulting molecular polarization from Musgravc (/). >> TRI > VCM > PER. Fortunately, this ranking corresponds to our experimental assessment of the acute hepatotoxicity potential of these four chloroethylenes. We approached the study of the biochemical mechanism(s) responsible for the toxicity of the chlorinated olefins by pretreating animals with chemicals which modify components of the liver mixed-function oxidase system, the enzyme system most likely to be involved in the initial biotransfor mation of lipid-soluble xenobiotics. We hoped that determination of factors which were synergistic or enhancing of the hepatotoxic potential would indi cate the mechanism of activation, while determinati n of factors which were antagonistic or protective against the hepatotoxic course would clarify the nature of the reactive molecular species. Our working hypothesis w hat the hepatotoxic po tential of these chloroc ?nes would be modulated by pretreatments wh nonuniformly enhance and/or uncouple the enzymatic components in volved in biotrantformation of these chloro ethylenes to excretable product' via reactive inter mediates such as epoxides or aldehydes. Powell (2) first suggested an epoxide intermediate for the metabolism ofTRl in 1945. Liebman and colleagues (3-5) had already demonstrated that the liver Methods Our basic experimental procedure utilizes a 7-dav gavage pretreatment of 200 g male Sprague Dawie; rats with one of six agents; 400 ^.mole/kg isomolar doses of PBT, 3-MC. hexachlorobenzene (HCB) spironolactone (SNL) or pregneoolone-l60' carbonitrile (PCN) or 150 wmole/kg of Aroclor 1254. Control animals are given the administrative vehicle (5 ml/kg of 0,1% Tween 80). The animals are then fasted overnight and on the morning of day g sacrificed for determination of microsomal enzyme components or exposed to one of the chloroethylenes. Similarly pretreated "controls" are exposed only to room air. Details of pretreat ment conditions, microsomal enzyme component assays, other chemical analyses, chloroethylene exposure techniques, histologic examination and methods to quantitate liver injury using serum transaminase activities or liver metal contents have been described (7, 8). Chloroethylene exposures were: VCM (5% x 6 hr). l.I-DCE (0.02% x i hrj. TRI (1% x 2 hr) and PER (7.5 mmole/kg. PO). Microsomal enzyme components assayed included; cytochrome P450 and bn. cytochrome c reductase by NADPH and NADH. oxidative N-demethylation of dimethylaminoantipyrine and ethylmorphine. glucose 6-phosphatase, and ary! hydrocarbon hydroxylation of zoxazolamine and 3.4-benzpyrene. The six "agents" chosen to modify the liver mixed function oxidase system gave six different patterns of enzyme induction (7, 5). Results of Parallel Studies Our working hypothesis proved rational. The acute hepatotoxicity of these four chloroethylenes was modulated to different extents in the animals pretreated with the six inducing agents as compared to the control animals given the administrative vehide. To determine if induction of a specific mixed function oxidase system component was associated with modulation of chloroethylene-induced liver injury, we compared mean enzyme component levels (assayed at times compatible with onset of chloroethylene administration) with mean serum transaminase levels of similarly pretreated animals sacrificed after chloroethylene exposure. There was , 1 1 t ' , , ( t 138 Environmental Health Perspectives a strikir p-450 c acetic t TRI F t,v linea ion an* was a It * 5, r - cytocht nadp after T. function axposuv that ant' ... covery urinary concluc liver inj content TRI bin Since molecuU metabol looked only in 1254, tr P-450. 3 nated p greater animals trol" an did we , terms c| cr sis (" As sb which also en fleeted a-ketog' VCM t cant at Pretre had a d of anirr (7). Ess* increase creases appear: sacrifice were co tween i by NA exposun late othc SAKT Decentb< SI 066296 , striking correlation between mean cytochrome pJ50 contents and mean serum glutamic oxalojjetic transaminase (SGOT) activities 2a hr after fRl exposure (significance < 1%, df - 5. r - 0.95) by linear regression analysis or the power regres* 5)0n analysis shown in Figure 2 (bottom) (8). There ^as a less perfect correlation (significance -5%, df ,, 5, r * 0.73) between mean rates of reduction of cytochrome P-450 by NADPH (measured as NiADPH-cytochrome c reductase) and SGOT 24 hr after TRI. No other relationship between a mixed function oxidase component and SGOT after TRI exposure was apparent. In this study we also found that the extent of SGOT elevation in individual animals correlated to prolongation of anesthesia re covery time after TRI exposure and to enhanced urinary excretion of trichlorinated metabolites. We concluded that the relative degree of TRl-induced liver injury related directly to cytochrome P-450 content at the time of exposure and to the extent of TRI biotransformation. Since PER was considered to be a more stable molecule and thus to have a lesser potential for metabolic activation to a reactive species, we looked for augmentation of PER-induced injury only in animals pretreated with PBT or Aroclor 1254. the most potent inducers of cytochrome P-450. While urinary recoveries of total trichlori nated products were approximately 5- and 7-fold greater in the PBT- and Aroclor 1254-pretreated animals compared to the vehicle-pretreated "con trol" animals, only in Aroclor 1254-pretreated rats did we find evidence of PER-induced liver injury in terms of SGOT elevation and focal histologic ne crosis (8). As shown at the top of Figure 2, pretreatments which were potent inducers of cytochrome P-450 also enhanced the hepatotoxicity of VCM as re flected by elevated activities of serum alanine a-ketoglutarate transaminase (SAKT) 24 hr after VCM exposure (7). This relationship was signifi cant at ~\% level. Pretreatment with inducers of cytochrome P-450 had a diametrically opposite effect on the response of animals to 1,1-DCE exposure (Fig. 2, middle) (7). Essentially, as mean cytochrome P-450 content increases, the hepatotoxic effect of 1,1-DCE de creases as reflected by SAKT activities, histologic appearance, and liver metal contents of animals sacrificed 6 hr after onset of 1,1-DCE. Again there were correlations of lesser significance (-5%) be tween mean rates of reduction of cytochrome P-450 by NADPH and the mean SAKT activities after exposure to VCM or 1,1-DCE. Attempts to corre late other mixed function oxidase components with SAKT activities after VCM or 1,1-DCE exposure Figure 2. Correlations between mean liver microsomal cyto chrome P-450 contents at the beginning of chioroethytene exposure and the mean serum enzyme transaminase activities after chloroethx lene exposure in groups of animals preireated with one of six inducers of mixed function oxidase compo nents plus the "control" animals pretreated with the adminis trative vehicle: (Q> control; t*l PBT: (Cl Aroclor 1254: () HCB: tA> 5-MC; (A) SNL: (t.) PCN Top correlation is between increasing cytochrome P-450 contents and elevated SAKT activities 24 hr after onset of VCM exposure signifi cant at the 1% level, df 51. Middle correlation is between decreasing cytochrome P-450 contents and elevated SAKT activities 6 hr after onset of l.l-OCE exposure I significant at the I5t level, df 51. Bottom correlation between increasing cytochrome P-450 contents and elevated SCOT activities 24 hr after onset of TRI exposure is a power relationship such that within the expcnmcniaJ range each 2-foid increase in cytochrome P-450 corresponds approximately to a 10-fold increase in SGOT (significant at < IS* level, df 5). Data for the VCM and l.l-DCE correlations from Reynolds et al (T) Data for the TRI correlation from Motion et al. 0) December 1977 139 Fieunt 3. MidztmN parunchymsl ctUi 2 hr after ths onset of expotore to 0 02** DCE Cell borden are retracted from uihuomUJ wilh at upper left aad center right. 5par- formed contain* cytoplasmic protruwoni. matcnal resembling fibrin and enhyrocytei. Cyto pi.fi of retracted cells focal!y rmw-inT swollen mitochondria and duster* of small vacuoles in the regions of the golf apparatus. Strands of rongb endoplasmic reticulum are iM*n Nuclei show striking segregation of chromatin towards the margins of tht nuclear envelope. Bile canaliculi wpcar intact, x 6,000. resulted in non-significant scattergnuns. It should be pointed outifcgt the amount of 1,1-DCE (0.02% x 4 hr) to which the animais were exposed was much less than that of TR4 (1% x 2 hr), which was in turn much less than the amount of VCM (5% x 6 hr) administered. Our experimental studies with VCM. 1,1-DCE, TRI. and PER as well as the comparative microsomal enzyme component analyses were essentially concurrent using overlap ping series of similarly pretreated animals. Differences in Acute Hepatic Injury Because of the differences in the basic molecular stability/reactivity of the four chloroethylenes as Environmental Health Perspectives SL 066298 De ,fT 'fr VJ * Ita soidal walls ytes. Cytoapparatus. gins of the iverlap- jlecular enes as pectives r~^ 7 \ Ub J V Figure 4. Portion of cemroiobular liver cells of PBT pretreated animals 8 hr after onset of exposure to TRl dSi. 2 hr). Rough endoplasmic reticulum in perinuclear regions of parenchymal cells is vacuolated and smooth is condensed into tangled tubular masses flecked with areas of increased electron opacity ("labyrinthine" tubular aggregates). * 20.000. Inset on bottom. Higher power of tangled tubular masses. Tubular diameters show greatest narrowing in areas of increased electron opacity. Radioopaque material appears applied on outer surfaces of tubular profiles, x 120,000. December 1977 141 SL 066299 illustrated in Figure I, variations were expected in the biologic response to these molecules specifically in the extent of bkttnnsformation, type and stability of intermcdiate(s), and ultimate excreted products. Such factors could contribute to the differences found in the nature of the hepatocellular injury. Hepatic injury following 1,1-DCE (0.02% x 4 hr) occurs abruptly and first appears as a prominent tnidzonal stripe of necrosis which rapidly evolves into hemorrhagic centrolobular necrosis by the end of the 4*hr exposure (7). As shown in Figure 3. parenchymal cell injury is apparent 2 hr after onset of 1,1-DCE exposure and is characterized by retraction of cell borders with the formation of a pericellular "lacunae" which may contain cyto plasmic projections, red blood cells and fibrin. Nu clear changes in such cells are striking with loss of perinucleolar chromatin, and clumping and coales cence of perinuclear chromatin into cresentric de posits of electron-opaque material against the nu clear envelope. Mitochondria in the cytoplasm of such cells appear swollen and outer mitochondrial membranes are ruptured. In contrast, rough and smooth endoplasmic reticulum appear relatively normal. Hepatocellular structural derangement following TRI exposure (1% x 2 hr) in PBT-pretreated ani mals presents in a different form than 1,1 -DCE with increased cytoplasmic disorder, random dispersion of organelles including ergastroplasm and degranu lation and vacuolization of rough endoplasmic re ticulum (9), Smooth endoplasmic reticulum then coalesces into tubular aggregates. By 8 hr after the onset of TRI exposure, coalescent tangles of smooth endoplasmic reticulum membranes contain electron-opaque regions suggestive of membrane collapse (Fig. 4). Vacuolization of rough endoplas mic reticulum is also found in PBT and Aroclor 1254 animals after PER administration. Similar patterns of endoplasmic reticulum denaturation were found in PBT and Aroclor 1254 pretreated rats after VCM exposure, and in animals exposed to other halogenated hydrocarbons including carbon tetrachloride and halothane (10). The enhanced hepatotoxic potential of carbon tetrachloride and of halothane in animals pretreated with inducers of the mixed function oxidase system is considered related to enhanced rates of their activation to reactive in termediates (possibly free radicals) by components of this system (10,11), Carbon tetrachloride causes selective deactiva tion of specific mixed function oxidase system components (12). it is not dear whether this occurs as a direct consequence of molecular attack by the reactive intermediate or as a consequence of lipid peroxidation of the membrane (10). We have found that exposure to VCM or TRI results in sew deactivation of mixed function oxidase comno^* including cytochrome P-450 US, 14). Table]11* pares the effects of exposure to 1,1-DCE. TR|COln' halothane on cytochrome P-450 and bs cont*i the end of exposure. While TRI exposure cauJ/1 loss of both cytochrome P-450 and b halothane exposure caused a loss of cytochnT*1 P-450. 1,1-DCE exposure did not diminish conie"'' of either cytochrome P-450 or bs. Note that combination of pretreatment and halocarbon sure shown in this table resulted in injury t0 ^ . liver. * Table 1. Coatmts of rat liver microaomai cytochrome* Ik at rad of cbloriaated hydrocarbon rxpotun. *** Cytochrome nmole Pretreatmcm Exposure P-130 b, ' None None PBT PBT Aroclor 1254 Aroclor 1234 Air 1,1-DCE* Air TRr Air Halothane' 0 76 a 0.0? 0.73 = 0 011 2.30 = 0.06 1.87 = 0.11" 2.66 = 0 03 2.00 = 0.07" 1-19 -- 0 On i.:: = oas 1.64 = o 10 1.29 = 0.06- 1.29 = 0 06 1.26 = 0 06 "Means = SEM. for three or more animals per group *0.0297 l.l-dichloroethylenc, 4 hr. rltS trichloroethylene. 2 hr. *p < 0.001 compared to similarly pretteated animals export to air. 'p < 0.05 compared to similarly pretreated animals exposed u air. b.85 halothane. 5 hr. i Similarities in Biologic Response Because the chioroethylenes are each members of the same chemical family, certain similarities in biologic response were expected and found. Alt of the chlorinated ethylenes (including cis- and trans-1,2*DCE) have been reported to be metabolized by isolated perfused livers (15). Met abolic studies in vivo, in perfused livers, and/or in vitro have shown that the biotransformation of each chloroethylene results in the production of a relatively stable oxidized metabolite such as an acid or alcohol (S, 15~17). The nature of the oxidized metabolites formed via the biotransformation of each of the chlorinated ethylenes with chlorines) attached to both carbons is compatible with the re arrangement of an epoxide (oxirane) intermediate involving chlorine migration (15, 16). VCM, 1,1DEC, TRI. and PER are all capable of being acti vated by liver preparations (or perhaps are naturally sufficiently reactive) to bind to hepatocellular ma cromolecules (15.18-20). In vitro covalent binding of "radiolabeled VCM or TRI in microsomal activation systems can be minimized by addition of inhibitors of the mixed function oxidase system (20, 21). l ' | ^ i * 142 Environmental Health Perspectives SL 066300 Further evidence that the chlorinated ethylenes can he activated to reactive molecules are the com 1,1-OCt parative mutagenic tests of Gneim ct al. (22). Each of the nonsymmetric chlorinated ethylenes (VCM, 1.1- DCE. and TRi) was found to be activated to a bacteria) mutagen by NADPH-dependent mi crosomal generating systems. In contrast, no mutagenic activity was detected for the sym metrically chlorinated ethylenes, cis- and nans- l.:-DCE. and PER. The mutagenicity of non symmetric chloroethylenes. such as VCM, appears related to electrophilic metabolites, since VCM metabolites, chloroethylene oxide. 2-chloro- acetaldehyde. and 2-chloroethanol--but not chloroacetic acid--are mutagenic 123). VCM, l.l-DCE, and TRI are classified as car cinogens: VCM exposure in man and experimental animals is associated with angiosarcoma (24. 25). 1.1- DCE exposure results in kidney tumors in mice (26). and TRI feeding leads to hepatic tumors in mice (27). Reduced glutathione (GSH) is involved in some way in the biologic response to VCM. l.l-DCE. and TRI. This relationship was first noted for 1.1DCE by Jaeger et al. (28). who found that fasting and other treatments which deplete hepatic GSH enhance the hepatotoxicity of J.J-DCE. TRI also causes more extensive liver injury in fasted-PBT pretreated animals than in fed-PBT pretreated ani mals (14). We have examined the relationship of 1.1- DCE-induced depletion of hepatic GSH to the manifestation of early liver injury by quantifiable compositional parameters such as changes in he Fiei re ? Alterations in liver meial and GSH contents of nun- preireated fasied rats during and after exposure to I. I-DC f(0.029c. 4 hr) expressed as percentage ot control values ob tained from similarly preireated animals sacrificed at equiv alent times during the 12 hr experimental period. (Oi Na. 1A1 K: (a) Mg: (0 I Zn; <) Ca: (.) GSH Dunng the first 2 hr of l.l-DCE exposure GSH contents rapidly plummet con comitant with an increase in Na content Be the end of l.l-DCE exposure Ca. K. Mg. and 7.n contents deviated significantly Ip < 0 O'i from the control values The dramatic influx in Ca continued to 12 hr. Na contents peaked at h hi. and the decrease in 7.n. k. and 7.n reached a plateau in con cert after the a hr GSH contents were clearly being re plenished as ihe iniury. reflected hy the marked metal imbal patic metal contents. As shown in Figure 5. liver ance. became m.inifest. GSH contents rapidly plummet during the first 2 hr of l.l-DCE exposure. Concomitant with this drop. detectable liver injury and does not deplete liver Na contents rise. Striking increases in liver Ca GSH contents. Nevertheless, liver GSH contents content follow' the increases in liver Na at times progressively rise following exposure. GSH deple when GSH contents slowly rebound. In contrast. tion in TRI-exposed, PBT-pretreated rats is par K. Mg. and Zn contents decrease moderately in ticularly marked in the microsomal fraction, the or concert. ganelle which appears profoundly affected in the Interestingly enough, liver metals have also hepatotoxie course of TRI. VCM exposure has also proved to be sensitive indicators of the progressive been associated with progressive depression of hepatocellular derangement caused by TRI. As hepatic contents of GSH (201. shown in Figure 6. the pattern of metal change is In vitro covalent binding of "C-VCM or TRI to different than with l.l-DCE. with an initial loss in cellular macromolecules by microsomal generating liver Ca during TRi exposure, followed at later systems is diminished by the addition of GSH and times by the typical metal imbalances, specifically approximately doubled, by the addition of tn- Na and Ca influx and K loss, found after the ad chloropropene oxide (20. 30). It should be pointed ministration of other hepatotoxins such as CC1.. out that while trichloropropene oxide i* a potent U2l As shown in Figure 7, we have found that in inhibitor of epoxide hydrase in vitro, it also depletes PBT animals which are vulnerable to the hepatic GSH in vitro (31). hepatotoxicity of TRI, liver GSH contents are Figure 8 shows schematically the probable and progressively depleted during TRI exposure, then potential pathways of TRI biotransformaiion. Acti rise above normal levels (14). However. TRI expo vation of TRI via a N ADPH-P-450 system leads to sure to vehicle-pretreated animals does not result in an epoxide which can rearrange to an aldehyde and December 1977 143 SL 066301 TIME (hr) Fiouae 6. AJumiom in liver meuJ contents of PBT-pretreaied animals exposed to TRI (1%. 2 hr) expressed as percentage of control values obtained from similarly pretreated animal* exposed to room air and sacrificed at equivalent times during the 14 hr experimental period. Ca contents are diminished nearly half by the end of exposure The metal imbalance at 8 and 14 hr. influx of Na and Ca coupled with loss of K. is similar to that found after administration of other hepatotoxin* such at CCI> 1/2). Data from Reynolds and Moslen (91. be hydrolyzed and transformed by other enzyme systems to trichloroacetic acid or to trichioroethanol, which is subsequently conjugated with gtucuronide to form the major urinary metabolite. Fernandez et al. (32) concluded from the low recov ery of TRI and its metabolites in the breath and urine of human subjects that "other metabolites exist, or that eventually other means of elimination occur." Our Findings on the alterations of hepatic CSH contents during and after TRI exposure indi cate some involvement of GSH in TRI biotransformation (14), perhaps via GSH transferases. Other members of the chioroethylene family may be biotransformed more extensively by minor metabolic routes. For example, trichloroacetic acid is the major metabolite of PER. presumably formed via epoxidation followed by a chloride shift pro ducing an acid chloride which would be rapidly hydrolyzed to trichloroacetic acid (16). Formation of the mRjor urine-excreted metabolites of VCM; i.e., thiodiglycollic acid and cysteine conjugates, was reported to involve GSH by Green and Hathway (55). Bolt et al. (IS) have suggested that the coupling of reactive VCM metabolites to Figuke 7. Alteration* in liver GSH contents of <) RBT. pretreated and is) vehtcle-pretreated animals during and after TRI exposure <1^. ! hr). The GSH content of the RBT. pretitated animal* i progressively depressed to almost half the pre-exposure levels by the end ofTRI exposure and then rebounds. GSH contents of vehicle pretreated animals art essentially constant during exposure, then rise to above preexposure levels. Asterisks <*) denote statistical!;, significant points (p < 0.05). Data from Moslen et al. (/<). glutathione may be viewed as an alternate metabolic pathway which prevents binding to cellular ma cromolecules. and may have particular significance in preventing the reactions with nucleic acids. Watanabe et al. (J4) have recovered 9-13% of the given dose of ,4C-VCM as exhaled MCO,. It is not clear at what point, or which intermediate, in the biotransformation of chloroethylenes leads to the breakdown of the chioroethylene carbon to carbon bond. Essentially the biologic response to the chloroethylenes must be considered as a series of steps as shown in Figure 9. Biotransformation re quires uptake into the cell and into specific or ganelles, interaction with an enzyme system, metabolic activation and transformation, perhaps subsequent rearrangements or enzymatic conjuga tions. and ultimately excretion. If epoxidation is the essential reaction in the biotransformation of a chioroethylene to a hepatotoxin, then any pretreat ment or condition (such as induction of P-430) which promotes or enhances this epoxidation step (AT,) could be considered "synergistic" to the hepatotoxic effect. Similarly synergistic is any pre- 144 Environmental Health Perspective* FlGt-'l oc lar P- ra del de thr du jug arr Ginv: Or me due Ch. treatri^^ such (fin). less r treatn woulc the c tectiv fend" react it Ob the m VCM. activa expre exten taebo* react of the iron t. organe in the vulnei Decern 066302 SL Figure i Biotrantformxtion of trichloroethylene is assumed to occur chiefly along the metabolic route indicated by the larger arrows. N ADPH dependent oxidation by cytochrome P-4J0 produces an epoxide as the primary metabolite which rearranges by chlonde migration to form tnchloroacetaldehyde as the major secondary metabolite. Trichloroacetaldchyde is converted to a tertiary generation of metabolites through hydration, enzymatic oxidation, and enzymatic re duction to an alcohol. Trichlorocthanol subsequently is con jugated to a glucwonide. the manor urinary metabolite. Smaller arrows indicate possible minor metabolic routes such as G$H epoxide transfer which may account for the apparent involvement of GSH in the btotransformation of TRI i/<l. Other members of the chloroethylene family may be metabolized to a greater extent by minor metabolic routes due to the comparative ttability/iitnability of the respective chloroethylene molecule or its intermediates i/d. JJ. 34). treatment or condition hampering the processes, such as giucuronidation (klt) or GSH conjugation (it,,). which transform reactive intermediate(s) to j less toxic species. In contrast, conditions or preI treatments "antagonistic" to the hepatotoxic effect would be those which increase the concentration (or - the capacity to renew supplies) of endogenous pro tective species such as glutathione c) which "de fend" cellular components against "attack" by reactive molecular species. Obviously the fundamental reactivity/stabiiity of the molecule has an influence on its biologic course. VCM. TRI. and PER apparently require metabolic activation before their hepatotoxic potential can be expressed. 1,1-DCE, in contrast, because of the extensive electron depolarization due to the at tachment of 2 chlorines on one carbon (Fig. 1), may react spontaneously with endogenous electrophiles of the cell such as iron in the cytochromes of elecvon transport systems of mitochondria and other owneiles. GSH may play a vital antagonistic role m the biologic response to 1,1-DCE by protecting vulnerable macromolecules against attack by this Figure 9 Kinetic Dow diagram of ihe cellular response io a xenobionc. Xenobiotics must first pass through the cel! plasma membrane (PM) by a reversible process it,, k,) then interact with functional components of the endoplasmic re ticulum Ik,. k,). with glutathione (GSHi in the cell sap ikt) leading to a glutathione conjugate, or with other organelles Ik,, k-i leading Ik,) to a product XY. Interaction with the mixed function oxidase system of the endoplasmic reticulum Ik.) is assumed to lead to generation of a reactive epoxide capable of interaction with vital cell components <A,. i H ' dration lk,,,). conjugation lk,,. k,,i and rearrangement U,,t transform the epoxide intermediate presumably to less iomc compounds. However compounds formed subsequent!}. such as aldehydes, may also be capable of toxic interaction with cell components (i,,) until further convened to acids Ik,t). alcohols (X,.). or glucuronidc conjugates lkn. k,7). In duction of the pathway <*,) which generates the toxic epoxide species and conditions which reduce the efficiency of path ways which dcioxifv the reactive species ik,,.k,,. k,,. k,,. k,,, would be considered synergistic to hepatoioxicity. since the result is an enhancement of the hepatotoxic action (*,,). Conditions which promote biotransformation by other en zymatic processes, such as it, or maintain adequate GSH U,) contents would be considered antagonistic, since the result is a diminution of hepatotoxic action. highly depolarized molecule. Delocalization of electrons of l.l-DCE away from the chlorines may activate the molecule to wards a nucleophilic attack of.such a nature that a chlorine is replaced by an attacking nucleophile (/). Epoxidation of l.l-DCE may be a relatively unim portant metabolic route or in fact may not occur. Bonse et ai. (15) reported a high uptake of l.l-DCE by isolated perfused rat liver but did not determine the metabolites formed, although they were able to identify di- and trichiorinated acid and/or alcohol metabolites of 1.2-m* and fronr-dichloroethylene, TRI and PER. Bonse et al. (15) were able to syn thesize the epoxide (oxirane) of all chlorinated ethylenes except 1,1-DCE. Leibman and Ortiz (17) have identified monochloroacetic acid as a product of the biotransformation of 1,1-DCE by rat liver 9000g supernatant fractions and an NADPH generating system. We have detected monochlo roacetic acid but not dichloroacetic acid in the urine Dumber 1977 145 t SL 066303 of animals exposed to 1.1-DCE (Reynolds etal.. unpublished observation). is it possible that monochioroacetic acid is pro duced from l.l-DCE not via cytochrome P-450 generated epoxide, but via a dechlonnating system requiring cytochrome P-450. NADPH, Oj and perhaps GSH? Van Dyke and colleagues (33, 36) have demonstrated a microsomal dechlorinating system that is inducible by PBT. preferentially dechlonnates compounds with more than one chlorine attached to a carbon, produces oxidized metabolites including acids and alcohols, and can be reconsti tuted with cytochrome P-450, NADPH cyto chrome c reductase, and lecithin. Multiple cyto chrome P-450 species with varying substrate specificities, markedly different catalytic proper ties. and preferential inducibility are known (37). Pretreatments which induce cytochrome P-450 may be antagonistic to the hepatotoxicity of l.l-DCE (Fig. 2, center) because the essential reaction of the cytochrome P450 system on l.l-DCE is a detox ifying dechlorination, not a toxifying epoxidation as for VCM. TRl. and PER- This report describes experimental studies conducted over sevctal yean which involved at various states the active collab oration of Dr*. Rudolph J, .latter. Senior Szabo. and Paul J. Boor. We would also like to thank Elizabeth A. Gasch. Regina Bean. Rioted J. Trtiittn. and Theresa Paolini Kingsley for their slultftti Thu wort wis supported by Grmntt HL46370, AM-16133 tnd AM-19* U from the National Institutes of Health. REFERENCES 1. Mustrave. W. K. R. Organic halogen compounds. In; En cyclopaedia Britannica Macropaedia. Voi. 13. 1974. p. it2. 2. Powell. J. F. Trichloroethylene; absorption, elimination and metabolism. Urn. J. Ind. Med. 2; M2 (1945). 3. Leibman. 1C. C. 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