Document wrn4q9Xn9ZyjGaJ3401OGEnVd

ii Umlwaiul r!vimia'.tiliifi. Voi 26. |>p pCTS*roon Prat 1977 Printed in Urtjt Br.utn ENHANCEMENT OF THE METABOLISM AND HEPATOTOkjCITY OF TRICHLOROETHYLENE AND PERCHLOROETHYLENE Mary Treinen Moslem*, Edwaro S. Reynolds* and Sandor Szabo Departments of Pathology, Peter Bent Brigham Hospital and Harvard Medical School, Boston, MA 02115, U S A. {Received 19 January 1976; accepted 12 May 1976) Abstract--Trichloroethylene anesthesia (1% for 2 hr) caused acute hepatic injury in rats pretreated with live different inducers of the hepatic mixed function oxidase system, phenobarbital, Aroclor 1254, hexachlorobcnzene, T-mcihylcholamhrene and pregnenolone-16-x-carbonitrile. Injury did not occur after trichloroethylene in rats pretreated with spironalactone or controls given vehicle alone. Morpho logic liver injury was most severe in the phenobarbital- and Aroclor 1254-pretrcated animals and was accompanied by marked perturbations in liver electrolyte content and more than 20-fold elevations in serum transaminase. Extent of serum transaminase elevation appears to relate directly to prolonga tion of anesthesia recovery time and the enhanced urinary excretion of trichlorinated metabolites. Metabolism of perchloroethylene (7,5 m-moles/kg, p.o.) was increased 5- and 7-fold, respectively, in phenobarbital- and Aroclor 1254-pretreated animals, but liver injury after perchloroethylene appeared only in Aroclor 1254 animals. Differential induction of various components of the microsomal mixed function oxidase system was quantified in parallel experiments using animals similarly pretreated with isomolar doses of the six inducers and the vehicle control and sacrificed at times corresponding to onset of chloroethyfcne exposure. Magnitude of induction of cytochrome P-450 among these seven groups of animals correlates with the mean extent of trichloroethylene-induced liver injury as quanti tated by serum transaminases level (r = 0.95). with prolongation of anesthesia recovery time (r = 0.95) and with enhanced urinary excretion of trichlorinated metabolites (r = 0.88),- <v'-' ' Trichloroethylene (TRI) and perchloroethylene (PER) are versatile solvents with numerous commercial, in dustrial and household applications. TRI is especially valuable as a degreaser and PER as a dry cleaning solvent. In medicine, 1R1 is occasionally used for ob stetric anesthesia [1] and one of its metabolites, chloralhvdrate. if a valuable sedative [2]. Neither of these unsaiurared chloroethylenes is considered a potent hepatotoxin [3-5]. However, preliminary evaluations of National Cancer Institute (U.S.A.) studies indicate that TRI exposure may have a lumorigenic potential analogous to that of vinyl chloride [6]. Although little is known of the biochemical effects of TRI and PER or their metabolites on the livercells, the mechanism of their metabolism has been the focus of numerous investigations. In 1945, the demonstration by Powell fT} of oxidized metabolites of TRI in human urine led her to suggest that TRI was metabolized via an unstable epoxide interme diate, arid to comment, "It would be remarkable if this channnel of metabolism could be followed with so little evidence of ill-effect.'' In 1961, YUner [8] con ducted metabolic studies with `*C-labeled TRI which indicated the formation of an epoxide intermediate. In 1963, Daniel [9], after measuring the specific acti vity of 4<'CI-labeled TRI metabolites isolated from urine, proposed that intramolecular rearrangement of chlorines after epoxidatioo would account for both the lack of loss of 3fiG to the chloride pool and the 'Prevent address; Department of Pathology. University of Texas Medical Branch at Galveston, TX 77550. known metabolites, trichloroacetaldehyde, trichloroethanol and trichloroacetic acid Daniel [9] con ducted similar studies with 3f,Cl-labeled PER, and concluded that epoxidation, followed by a chloride shift, would lead to an acid chloride which would be rapidly hydrolyzed to trichloroacetic acid, the major metabolite of PER. Further metabolic investigations [10-12] have identified the liver mixed function oxidase system (MFOS) as the stystem primarily responsible for the oxidation of TRI and PER. Other enzyme systems, including those in the adjacent cytoplasm, participate in subsequent hydration, oxidation and/or reduction and conjugation of secondary metabolites [13]. Leibman and McAllister [14] found that pretreat ment of animals with phenobarbital (PBT), classic MFOS inducer, caused alterations in the rate and route of TRI metabolism. Carlson [15] reported that pretreatment with PBT or 3-methylcholanihrene (3-MC) exacerbated the hepatotoxicity of TRI, We have found that potentiation of the acute hepatotoxi city of TRI or PER in the rat, produced by pretreat ing animals with chemicals that induce components of the MFOS, appears related to enhanced metabo lism of these chloroethylenes. experimental Treatment of animals. A series of 200 g male Sprague-Dawley rats from Charles River were given isomolar doses (400 ^molcs/kg) of PBT, 3-MC, hexachlorobenzene (HCB), spironolactone (SN'Lt. pregnenolone-16-x-carbonitrile (PCN) or 150/jmoleskg 369 SL 032785 370 M. T. Moslen. E. S. Reynolds and S. Szabo or 300 ^unoles/kg of Aroclor 1254 (A-1254) by savage once daily for 7 days. All compounds were solubilized in water with traces of Tween 80. "Control'' animals received this vehicle. Throughout the induction period animals were housed in wire floored cages over processed clay animal litter and allowed free access to food and drinking water. On the morning of day S after a 16-hr fast, animals were sacrificed for deter mination of microsomal enzymes, or exposed to TRI, or PER, or room air. Animals pretreated with 3u0;rmoles kg of A-1254 failed to survive TRI anes thesia and this dose level of A-1254 was not used in further studies. Chloroethylene administration. Four groups of eight animals (one animal from each pretreatment regimen) were exposed to 1% TRI in air for 2 hr in an inhala tion chamber previously described [16]. Based on the data of Guyton [17] the total dose of TRI inhaled was approximately 20-30m-moles. Immediately after cessation of the anesthetic, animals were placed in individual metabolism cages to collect urine for deter mination of trichlorinated excretion products. PER (0.75 ml/kg diluted with mineral oil) was given by gavage to vehicle, PBT- and A-1254-pretreated rats (four of each) and these animals were placed in meta bolism cages. All animals were sacrificed 24 hr after administration of the chloroethylene. Assessment of metabolism and liver injury. Anesthe sia recovery time, the interval between removal from the inhalation chamber and return of the righting reflex, was noted for each animal. Serum glutamic oxalacetic transaminase (SGOT) and serum glutamic pyruvic transaminase activities were assayed with Sigma reagent kits. Transaminase changes were con sistently measured by both assays. Samples of each liver were rapidly frozen on dry ice for histochemical detection of increased calcium and to provide mater ial for determination of metal contents [18], or fixed in formalin for histologic examination by standard techniques. The alkali-pyridine-toluene method of Tanaka and Ikeda [19] was followed to quantitate urinary tri chloroacetic acid and total trichlorinated products. This method was selected because its vigourous oxi dizing conditions were reported to give good recov eries of total trichlorinated metabolites in rat urine. In preliminary studies, we found that added trichloroethanol gave minimal response without the oxidizing procedure and that neither 2,2-dichloroethanol nor 2,2-dichloroacetic acid was detected by this method. These observations suggest that this procedure detects only 2,2,2-trichlorinated metabolites of chlorinated ethylenes. Twenty-four-hr urine collections from vehicle-air, PBT-air, or A-1254-air animals gave vir tually no color response even after the oxidation step. Microsomal enzyme assays. Differential induction of MFOS components by the six inducers was verified in a series of parallel experiments each of which con tained at least one animal from each pretreatment regimen. At times compatible with the onset of chloroethylene exposure, the animals were sacrificed, and the livers were perfused and homogenized with cold 0.25 M sucrose; the microsomal fraction was iso lated by differential centrifugation essentially as pre viously described [16]. Activities of the microsomal enzymes were assayed on the day of sacrifice. Protein contents were determined by the method of Lowry et al. [20], Difference spectra of cytochrome P-450 and bf were charted after Dallner et al. [21] using a Gilford 2400-2 in the split beam mode. Rates of cytochrome c reduction by NADPH and 0-NADH were recorded spectrophotometrically using the res pective reaction systems of Wilson [22] and Hogeboom [23]. Oxidative A'-cfemethylation of dimethylaminoantipyrine was assayed by the method of Orrenius [24J. Glucose 6-phosphatase activity was deter mined according to Sell and Reynolds [25], Activities of zoxazolamine and 3,4-benzpyrene hydroxylase were measured by the methods of Nebert and colleagues [26,27], Statistical analysis. Student's t-test and regression correlations were calculated using a Wang 600 pro grammable calculator and assessed after Snedecor and Cochran [28,29]. Chloroethylene-induced changes in SGOT and liver metal contents were ana lyzed for statistical significance by comparison with values obtained from similarly pretreated animals exposed to room air. RESULTS f t Trichloroethylene injury. Effects of the seven chemi cal pretreatment regimens on the quantitative par ameters examined after TRI anesthesia are detailed in Tables 1 and 2. In vehide-jxetreated (control) ani mals, TRI exposure was without effect on SGOT acti vity. liver Na, K, Mg, Ca, Zn and Fe contents or the microscopic morphology of the liver (Fig. 1) as compared to similarly pretreated animals exposed to air. In striking contrast, animals pretreated with PBT or A-1254 had at least doubled anesthesia recovery times, marked increases in urinary excretion of tri chlorinated metabolites of TRI, more than 20-fold increases in SGOT, marked perturbations of liver Na, K and Ca contents, and hank and extensive hepato cellular necrosis. In PBT animals, hepatic necrosis Table 1. Effects of MFOS inducers on the metabolism of trichloroethylene*1! Anesthesia Trichlorinated urinary metabolites bimoles/24 hr/animal) PretreatmentJ time (min) Total TCA Vehicle PBT A-1254 HCB 3-MC SNL PCN (4) 81 10 (4) 196 12$ (4) 244 17 (4) 94 6 (4) 79 5 (4) 71 3 (4) 82 14 314 6 823 38 829 655 371 45 401 17|| 282 22 268 33 30.0 3.0 62.1 32 71.8 92 33.1 7.0 60.4 7.7 22.5 5.0 29.6 4.1 * Mean S-E.M. t Trichloroethylene: 1% x 2 hr in air. J Chemicals were given by gavage once daily for 7 days; the number in parentheses mdicates the number of ani mals. J Statistically different from vehicle-TRl group (line 1) P < 0.001. ;| Statistically different from vehicle-TRI group (line 1) P < 0.01. SL 032786 Metabolism and hepatoxicity of chloroethylenes Table 2. Effects of MFOS inducers on chemical indicators of liver injury 24 hr after trichloroethylene* + PretreatmentJ SGOT (Karmen units) Liver metal contents (mg metal/g liver) Na K Ca Vehicle PBT A-1254 HCB 3-MC SNL PCN (4) 185 + 16 0.43 + 0.03 (4) 4418 + 8435 1.33 0.10 (4) 7497 + 1066 0.92 + 0.065 (4) 739 1215 0.72 0.06 (4) 253 + 41 0.46 0.02 (4) 183 26 0.47 0.03 (4) 1015 502J 0.82 + 0.18 3.5 + 0.1 1.9 0.25 27 0.15 4.0 + 3.0 3.7 + 0,3 3.3 0.3 3.0 + 0.4 0.032 0.003 0.213 + 0.0285 0.131 + 0.017|| 0.084 + 0.01911 0.026 + 0.002 0.034 0.004 0.087 + 0.046 * Mean S.E.M. t Trichloroethylene 1% x 2 hr in air. i Chemicals were given by gavage once daily for 7 days: the number in parentheses indicates the number of animals. Statistically different from similarly pretreated animals exposed to air P < 0.001. || Statistically different from similarly pretreated animals exposed to air P < 0.01. 371 rasas M. CONTROL && PBT Fig. 1. Effect of PBT or A-1254 pretreatment on hepatocellular injury 24hr after TR1 exposure (1% for 2 hr). Top row shows hematoxylin and eosin stained paraffin sections, and bottom row the corre sponding cryostat sections stained for calcium with Alizarin red S. In each panel the central vein is at the left with portal areas to the right. No necrosis or intracellular calcium deposits are seen in the TRl-exposed, vehicle-pretrcated "conlror at left. In the PBT (center) and A-1254 (right) livers, necrotic bands of pyknotic hepatocytes are present in the centrolobular and midzonal regions respectitely (arrows). Alizarin red S stains calcium-rich necrotic cells in corresponding regions. (magnification: x 150). sc 32781 372 M. T. MosLis. E. S. Reynolds and S. Szabo Fig. 2. Correlation between anesthesia recovery times and serum SGOT levels 24 hr after exposure to trichloroethy lene (1% x 2 hr) in the seven pretreatment groups. Each point represents the data for an individual animal. Symbols represent the different pretreatments: (O) vehicle, f*) PBT, () A-1254. (!) IICB, (A) 3-MC, (A) SXL and (<>) PCN. The power relationship is such that each 2-fold increase in anesthesia recovery time corresponds approximately to a 10-fold increase in SGOT (significance <S 1 per cent, d/ = 26, r = 0.89). Enhancement of the rate of metabolism of TRI should lead to a more rapid clearing of TRI from the blood after termination of anesthetic exposure. However, the anesthesia recovery time may not necessarily be shor tened if enhanced TRI clearance is coupled to in creased production of chloral hydrate and trichloro ethanol. In this experiment, we did find a highly signi ficant linear association (4 1 per cent) between ele vated urinary excretion of total trichlonnated metab olites and prolongation of anesthesia recovery time (df = 26, r *= 0.92). Therefore, prolongation of anes thesia recovery time appears a valid indicator of enhanced TRI metabolism. The extent of hepatic injury 24 hr after TRI as quantitated by SGOT in individual animals appears directly related to enhancement of TRI metabolism in terms of both prolongation of anesthesia recovery times (Fig. 2) and increased urinary excretion of tri- chlorinated metabolites (Fig. 3). Both of these relation ships are significant at the 1 per cent level by linear regression analysis as well as the power function analyses illustrated (Figs. 2 and 3). The ratio of tri chloroacetic acid to total trichlorinared metabolites was similar in all the animals except for the animals pretreated with 3-MC. -. Enzyme induction. The varied effects of the pretreat- ment regimens on components of the MFOS are detailed in Tables 3 and 4. PCN, PBT and A-1254, and histochemically stainable calcium were centrolobular. while in A-1254 animals stainable calcium was shifted periportally and necrotic zones appeared as prominent midzonal or periportal stripes (Fig. 1). In both groups, liver injury was extensive although TRI caused greater changes in liver Na, K and Ca con tents of PBT animals, while the SGOT values at 24 hr were higher in the A-1254 animals. Liver contents of Mg, Zn and Fe were not altered in any group of animals 24 hr after TRI exposure. Increases in SGOT of a lesser magnitude were also measured 24 hr after TRI in 3-MC-, HCB- and PCNpretreated animals (Table 2). Elevated transaminases in HCB and PCN animals were associated with in creased liver Na and Ca contents. Morphologic hepa tic injury appeared mild with focal necrotic and/or vacuolated cells ir. both centrolobular anti midzonal regions. Anesthesia recovery times and urinary excre tion of trichlonnated products were slightly if at all increased in these animals (Table 1), All parameters examined in the SNL-pretreated animals after TRI were unaltered (Tables 1 and 2). Correlations between anesthesia recovery time, uri nary excretion of trichlorinatcil metabolites anil hepatic injury. Interpretation of anesthesia recovery times after TRI anesthesia is not straightforward. At least two metabolites of TRI, chloral hydrate and trichloroethanol, also have anesthetic properties [2]. Fig. 3. Correlation between total urinary' excretion of trichlonnated metabolites and serum SGOT levels 24 hr after exposure to trichloroethylene (1% x 2 hr) in the seven pretreatment groups. Each point represents the data for an individual animal. Symbols represent the different pretreatments: (o) vehicle. () PBT, () A-1254, IB) HCB, (A) 3-MC. (A) SNL and (O) PCN. The power relationship is such that each 2-fold increase in urinary trichlorinated metabolites corresponds approximately to a 10-fold in crease in SGOT (significance < 1 percent. d/= 26. r = 0.77). Metabolism and hepatoxicity of chloroethylenes Table 3. Differential induction of electron transport components of the liver MFOS* Cytochrome (nmoles/mg protein) Cytochrome c reductase (nmoles product mg protein-min) Pretreatmentt P-450 NADPH NADH Vehicle (13) 0.77 0.03 PBT (11) 1.83 + 0.1 It A-1254 (ID 2.52 0.151 HCB (9) 1.05 0.09$ 3-MC (9) 1.18 0.10$ SNL (9) 0.76 0.05 PCN (11) 1.38 + 0.06$ 0.71 + 0.04 0.75 + 0,03 0.84 0.04 0.76 0.04 0.84 0.06 0.73 0.04 0.88 0.04|1 48 + 6 89 + 155 83 + 6$ 59 + li 62+11 57 + 8 78 + 8 211 + 16 130 + 11$ 143 + 85 160 13 169 + 6 144 + I2 98 11$ * Mean S.E.M. t Chemicals were given by gavage once daily for 7 days; the number m parentheses indicates the number of animals. $ Statistically different from vehicle group (line 1) P < 0.005, Statistically different from vehicle group (line 1) P < 0,05. 373 in that order, markedly increase cytochrome P-450 content. Peaks of the cytochromes induced by A-1254 or 3-MC show hype'-bromic shifts ("P-448"). Cyto chrome hs is increased significantly only by PCN. NADPH-cytochrome c reductase activity is enhanced by those agents which increase cytochrome P450, while NADH-cytochrome c reductase is usually pro portionally decreased. PBT, HCB, SNL and to a lesser extent PCN increase oxidative A-demethylation of dimethylaminoantipyrine, while glucose 6-phospliatase, an enzyme localized in the endoplasmic retic ulum but not associated with the MFOS is decreased by PBT, A-1254 and PCN. Both A-1254 and 3-MC Strikingly increase arene hydroxylase activities. Correlations between differential induction of MFOS components and trichloroethylene toxicity. To deter mine if induction of specific MFOS components by chemical pretreatment regimens was associated with potentiation of TRI-induced liver injury, mean MFOS activities (assayed at times compatible with onset of TRI exposure) were compared with mean SCOT levels of similarly pretreated animals sacrificed 24 hr after TRI exposure. The correlation between cytochrome P-450 contents and SCOT levels was sig nificant at the 1 per cent level by both linear and power (Fig. 4) analysis. The relationship between the rates of reduction of cytochrome P-45Q by NADPH (measured as NADPH-cytochrome c reductase) and SGOT levels was less perfect, and no other relation ship between the MFOS components and SGOT levels was apparent. When we looked for similar cor relations between the corresponding induction of spe cific MFOS components and the enhancement of trichloroethylene metabolism, highly significant linear correlations were found between mean cytochrome P-450 content and both the mean anesthesia recovery time (d/ = 5, r = 0.95) and the mean urinary excre tion of total trichlorinated metabolites (dIf = 5, r = 0.88). Perchloroethylene injury. PER was metabolized pri marily to TCA. Urinary recoveries of total trichlor inated products were approximately 5- and 7-fold greater in the PBT- and A-1254-pretreated animals (Table 5). Pretreatment with A-1254 enhanced the hepatotoxicity of PER as manifest both by doubling of SGOT and the appearance of focal areas of vacuo lar degeneration and necrosis along the posterior aspect of the liver at 24 hr. Cornish et al. [30] also found slight elevations of SGOT in non-induced and PBT-induced rats after oral administration of PER. Table 4. Differential induction of microsomal enzyme activities* Arene hydrocarbon hydroxylase Oxidative Glucose Pretreatmentt .V-demcthylase 6-phosphatase Zoxazolamine (nmoles product/mg protein min) Benzyprene (O.D.^j/mg protein min) Vehicle (13) 10.5 0.8 PBT (H) 16.4 0.8$ A-1254 (ID 9.3 0.6 HCB (9) 15.6 + 0-5$ 3-MC (9) 11.0 1.4 SNL (9) 15.1 + 0.8$ PCN (11) 13.8 + I.0 0.23 + 0.01 0.14 0.01$ 0.11 0.01$ 0.20 + 0.01 0.23 + 0.02 0.22 0.02 0.19 0.01$ 0,50 + 0.09 0.61 + 0.02 14.5 + 1.30$ 1.05 + 0.01 6.0 + 0.60$ 0.60 + 0.07 0.85 + 0.20 22 2 26 + 3 66 6$ 23 5 38 + 1 17 + 2 15 + 2 * Mean S.E.M. t Chemicals were given by gavage once daily for 7 days; the number in parentheses indicates the number of animals. $ Statistically different from vehicle group (line 1) P < 0,005. Statistically different from vehicle group (line 1) P < 0.05. SL 032789 374 M. T. Moslem, E S. Reynolds and S. Szaro Fig. 4. Correlation between mean contents of microsomal cytochrome P-450 (nmoles/mg of protein) for the seven groups of chemically pretreated animals using data from Table 3 and the mean serum SGOT levels 24 hr after expo sure to TRI using data from Table 2. Symbols represent the different pretreatments: (O) vehicle, () PBT, () A-i:5.t, () HCB, (A) j-MC, (A) SNL and (O) PCN. The power relationship is such that, within the experimental range, each 2-fold' increase in cytochrome P-450 corre sponds approximately to a 10-fold increase in SGOT (sig nificance < 1 per cent, d/ = 5, r = 0.95). DISCUSSION Our working hypothesis is that the chlorinated ethylenes are activated to reactive molecular species by an enzyme system localized in the liver endoplas mic reticulum, and that induction of components of this system can alter the rates and/or routes of chloroethylene biotransformation. In this and prior studies [31,32], we have demonstrated that pretreat ment of rats with chemicals capable of inducing com ponents of the liver mixed function oxidase system potentiates the acute hepatotoxicity of monochloroethylene (vinyl chloride), trichloroethylene and perchloroethylene. Oxidized metabolites of di-(l,l- and 1,2-), tri-, and tetra-(per) chlorinated ethylenes have been identified in the perfusate of isolated perfused liver prep aration [34], Hepatic cytochrome P-450 appears to have a central function in the biotransformation of these chloroethylenes. Microsomal enzymes, presum ably the mixed function oxidases, have been reported to oxidize vinyl chloride [34], 1,1- and 1,2-dichloroethylene [12], TRI [10,11] and PER [12]. and to transform vinyl chloride and 1,1-dichIoroethylene into mutagens [35]. The metabolism of TRI by microsomes is competitively inhibited by carbon monoxide, hexobarbital or aniline, each of which binds to cyto chrome P-450 [11]. Covalent binding of l4C-labelcd vinyl chloride is suppressed in vitro by l-naphthyl-4(5)-imidazole, an inhibitor of microsomal cyto chrome P-450 dependent oxidation [34], Exposure in vivo to vinyl chloride (5% x 6 hr) resulted in a pattern of microsomal enzyme deactivation suggestive of a relatively toxic action or reaction centered about cytochrome P-450 [36]. Contents of cytochrome P-450 and the oxidative A'-demethylation of dimethylaminoantipyrene and cthylmorphine were markedly decreased, while glucose 6-phosphatase, cytochrome bs and NADPH- and jJ-NADH-cytochrome c reduc tase were scarcely affected in both induced and noninduced animals sacrificed 24 hr after vinyl chloride. In parallel in vivo studies-using animals from-seven pretreatment regimens, we found a direct correlation between mean cytochrome P-450 content (at the time of chloroethylene exposure) and the respective mean extent of liver injury (SGOT level) 24 hr after expo sure to either TRI or vinyl chloride [32]. Paradoxi cally, the hepatotoxicity of 1,1-dichloroethylene (vinylidene chloride), a very reactive molecule [37], was reduced in animals pretreated with most effective in ducers of cytochrome P-450 [32]. However, the hepa totoxicity of the relatively stable fully chlorinated ethylene, PER, was potentiated in animals pretreated with Aroclor 1254. In this study, the metabolism of TRI and PER was enhanced in animals pretreated with inducers of cyto chrome P-450. In fact, the trichloroethylene data indi cate direct relationships between the mean magnitude of cytochrome P-450 induction and both the prolon gation of anesthesia recovery time and the enhanced urinary excretion of trichlorinated metabolites. Ad ditional support for a relationship between cyto chrome P-450 content and extent of trichloroethylene metabolism can be inferred from the data of Bartonicek and Teisinger [38] and Stripp et at. [39]. Bartonicek and Teisinger [38] reported decreased urinary excretion of TRI metabolites coupled with increased recovery of TRI from the expired air of subjects pre viously given disulfuram. They attributed this shift to a disulfuram-induoed suppression of TRI oxi dation. Stripp et al [39] have shown in the rat that disulfuram produces prolonged impairment of mixed Table 5. Effects of PBT or Aroclor 1254 pretreatment on the metabolism and hepatotoxicity of perchloroethylene*') Pretreatment] Urinary metabolites (jtmoles/24 hr/animal) Total TCA EGOT (Karmen units) Vehicle PBT A-1254 (4) 4.1 0-5 2.3 0.4 211 +9 (4) 19.4 1.15 15.8 0.9 212 36 (4) 29.5 2.5$ 24.8 2.5$ 336 311| * Mean S.E.M. f PcrchloroethyJene; 0.75 ml/kg (7.5 m-mo!es/kg). | Chemicals were given by gavage once daily for 7 days; the number in parentheses indicates the number of ani mals. Statistically different from vehicle-PER group (line 1) p < aooi. | Statistically different from similarly pretreated animals exposed to air P < 0.001. Metabolism and hepatoxicity of chloroeih\lenes 375 function oxidase components including decreased cytochrome P-450 content It is far from clear which of the metabolites of TRI is the ultimate toxin. Trichloroethylene is metabolized in a multi-stage process. The postulated epoxide inter mediate rearranges to form an aldehyde (chloral); subsequent metabolism involves aldehyde and alcohol dehydrogenase and/or glucuronidases [13]. The com ponents of this multimolecular process may vary in response to chemical induction. Non-uniform enzy matic induction could account for the changes Leibman and McAllister [14] described in the rate and route of TCE metabolism in PBT-induced animals. Alternatively, factors other than enzyme induction could influence the hepatotoxicity of chloroethylenes, for example, changes in the redox state of the hepatocyte, or depletion of cofactors required for specific metabolic steps. Cornish and Adefuin[40] found striking elevations in SGOT levels in rats given eth anol 16 hr prior to TRI exposure (0.5% x 4 hr), while TRl-exposed rats not receiving ethanol had normal SGOT levels. Alcohol metabolism increases the NADH/NAD ratio in the liver and thus can slow reactions which require NAD or enhance reactions requiring NADH or NADPH [41], Hepatic microso mal oxidation of TRI is NADPH dependent [10], and reducing equivalents for the reduction of chloral hyd rate can be supplied by NADH or NADPH [42], Acknowledgements--Research was supported by U.S. Pub lic Health Service Grants HL-06370 and AM-16183. E. S. 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