Document 9132JMbgOgYaM3veBmn9odopL

xr content and kidney 31-138. ka, S., and Okahara, led metallothionein in 33,258-266. , C. D. (1985). Concenajor organs ofrats after Is. Fundam. Appl. Toxifcadmium-thionein. In v, and Biology of Cad423-431. Elsevier/North am. hioneins. In TheChemlogy of Cadmium (M. sevier/North Holland, onein and the nephroiturn Symposium (M. ), pp. 101-107. Freid- i i l i ! I TOXICOLOGY AND APPLIED PHARMACOLOGY 92, 103-112(1988) The Role of Trichloroacetic Acid and Peroxisome Proliferation in the Differences in Carcinogenicity of Perchloroethylene in the Mouse and Rat J. Odum, T. Green,' J. R. Foster, and P. M. Hext Imperial Chemical Industries PLC, Central Toxicology Laboratory, Alderley Park, Nr Macclesfield, Cheshire SKI04TJ, United Kingdom Received June 8,1987; accepted September 21,1987 The Role ofTrichloroacetic Acid and Peroxisome Proliferation in the Differences in Carcino genicity ofPerchloroethylene in the Mouse and Rat. Odum, J,, Green, T., Foster, J. R., and Hext, P.M.( 1988). Toxicol. Appl. Pharmacol. 92, 103-112. Fischer 344 rats and B6C3F1 mice ofboth sexes were exposed to 400 ppm perchloroethylene (PER) by inhalation, 6 hr/day for 14, 21, or 28 days or to 200 ppm for 28 days. Increased numbers of peroxisomes were seen under the electron microscope and increased peroxisomal cyanide-insensitive palmitoyl CoA oxida tion was measured (3.6-fold increase in males and 2.1-fold increase in females) in the livers of mice exposed to PER. Hepatic catalase was not increased. Peroxisome proliferation was not observed in rat liver or in the kidneys of either species. Trichloroacetic acid (TCA), a known carcinogen and hepatic peroxisome proliferating agent, was found to be a major metabolite of PER. Blood levels of this metabolite measured in mice and rats during and for 48 hr after a single 6-hr exposure to 400 ppm PER showed that peak blood levels in mice were 13 times higher than those seen in rats. Comparison ofareas under the curves over the time course of the experiment showed that mice were exposed to 6.7 times more TCA than rats. The difference in metabolism ofPER to TCA in mice and rats leads to the species difference in hepatic peroxisome proliferation which is believed to be the basis ofthe species difference in hepatocarcinogeriicity. Peroxisome proliferation does not appear to play a role in the apparent carcinogenicity of PER in the rat kidney, e toss Academic Ptw, ioc. Perchloroethylene (PER) (1,1,2,2-tetrachloroethylene) is a volatile liquid which is used extensively in the dry cleaning industry and as a general degreasant in manufacturing in dustry. A significant increase in hepatocellular car cinoma has been observed in male and fe male mice but not rats in two carcinogenicity bioassays of PER. In the first study (NCI, 1977) Osbome-Mendel rats and B6C3F1 mice received PER by gavage in com oil at doses of approximately 500 or 1000 mg/kg. Both dose groups showed about a 50% inci dence of hepatocellular carcinoma in mice. In the second study (Mennear el al., 1986) Fi- 1 To whom correspondence should be addressed. scher 344 rats and B6C3F1 mice were ex posed to PER by inhalation (mice 100 or 200 ppm and rats 200 or 400 ppm 6 hr/day). In creased hepatic tumor incidence was again observed in mice, 50% in low and high dose males and 26 and 72% in low and high dose females. In the latter study a low incidence of kidney tubular adenocarcinoma was ob served in male rats at the highest dose. The species difference in hepatocarcinogenicity is similar to that seen with trichloro ethylene (TRI) (NCI, 1976; NTP, 1983). TRI has been shown to induce peroxisome prolif eration in mouse liver but not rat liver, after oral administration (Elcombe et al., 1985). A causal relationship has been suggested be tween hepatic peroxisome proliferation and 103 0041-008X/88 S3.00 Copynfhl by Academic Ptcm. Ioc. AU fighti ofreproduction in any form inerved 0355^8 104 ODUM ET AL. hepatocellular carcinoma in rodents (Reddy et al, 1980) although no such relationship has yet been shown between renal peroxi some proliferation and renal tubular adeno carcinoma (Reddy et al., 1975, 1982). The species difference in hepatic peroxisome pro liferation elicited by TRI is believed to be the basis ofthe species difference in carcinogenic ity (Elcombe et al., 1985). Trichloroacetic acid (TCA), a major me tabolite of TRI (Green and Prout, 1985) has recently been shown to be carcinogenic in the B6C3F1 mouse (Herren-Freund et al., 1986). TCA has also been shown to be responsible for peroxisome proliferation in TRI-dosed mice (Elcombe, 1985). Quantitative differ ences in the metabolism of TRI in rats and mice and hence in circulating levels of TCA (Prout et al., 1985) may lead to the species difference in peroxisome proliferation and consequent carcinogenicity. TCA is reported to be a major metabolite of PER in mice and rats (Yllner, 1961; Daniel, 1963; Dekant et al, 1985) and may therefore elicit the same response when animals are exposed to PER. In view of the lack of mutagenicity of PER (Greim et al., 1975; Bartsch et al.. 1979) this may be the basis for the species difference in carcinogenicity. In this paper the pathological and bio chemical changes in rat and mouse liver and kidney, with particular regard to peroxisome proliferation, were determined after inhala tion exposure to PER. Blood levels of TCA in rats and mice exposed to PER were also determined. The animal strains and dose lev els used in the 1985 PER bioassay (Mennear et al., 1986) were adopted in order to assess the relevance of our results to the develop ment of tumors in these animals. METHODS Materials 1,1,2,2-Tetrachioroethylene (Analar grade, 99.9%) and trichloroacetic acid (Analar grade, 99% pure) were obtained from BDH Chemicals PLC (Poole, Dorset, UK). Biochemicals were obtained from Sigma Chemical Co. (Poole, Dorset, UK). Animats Male and female Fischer 344 rats (160-180 g) and * imale and female B6C3F1 mice (23-28 g) were supplied by Charles River (Margate, Kent, UK). Animals were multiply housed in suspended stainless steel wire mesh cages in long-term inhalation exposure chambers, equipped with a 12-hr light cycle, prior to, during, and after exposure. They received food (PCD diet. Special Diets Services Ltd., Witham, Essex, UK) and water ad libitum before and after, but not during exposure. it- Exposure to PERfor up to 28 Days I Exposure. Male and female rats and mice (5 per group) were exposed to concentrations of 200 or 400 ppm of PER for 6 hr/day for 14,21, or 28 consecutive days. Con trol animals were exposed to air only, but otherwise were treated in a manner similar to that ofthe test animals. Exposures were whole body in stainless steel chambers (Doe and Tinston, 1981) having an internal volume of approximately 3.4 m3. The chambers were air condi tioned to have a nominal temperature of 22'C and rela tive humidity of40-60%. The air flow through the cham bers was 300 liters/min. Atmospheres were generated by passing vaporized PER into the input air of the cham bers. Atmospheres in the test chambers were analyzed for PER by gas chromatography (GC) on a Hewlett-Packard 5880A GC (flame ionization detector) fitted with a Porapak PS column (1.8 m x 4 mm). The column tempera ture was 195*C, helium carrier gas 50 ml/min. Eighteen hours after the last exposure period, animals were killed by overexposure to halothane (Fluothane, Imperial Chemical Industries PLC, Pharmaceuticals Di vision) and exsanguinated. The livers and kidneys were rapidly removed, weighed, and then divided to provide tissue for light microscopy, electron microscopy, and biochemical analysis. Light microscopy. Slices of liver and kidney were fixed in 10% neutral buffered formol saline, dehydrated through an ascending ethanol series, and embedded in paraffin wax. Sections (5 m) were cut and stained with hematoxylin and eosin. Electron microscopy. Tissues were fixed in 3% glutaraldehyde in 0.1 m sodium phosphate buffer, dehydrated, and embedded in epoxy resin. Sections (1 *im) were cut and stained with 1% toluidine blue in 1% borax for light microscopy. Areas were selected from the centrilobular regions of the livers and the S3 regions of the proximal tubules of the kidney for electron microscopy. Ultrathin sections of these areas were stained with uranyl acetate 4 i 5 :* I i l SL 035559 am Sigma Chemical ts (160-180 g) and -28 g) were supplied . UK). Animals were mless steel wire mesh exposure chambers, prior to, during, and d (PCD diet. Special . UK) and water ad ng exposure. 1 mice (5 per group) 00 or 400 ppm of secutue days. Con* b^^Mrwise were th^^rcanimals, less steel chambers internal volume of rs were air condit of 22`C and relathrough the chamwere generated by t air of the cham- were analyzed for i Hewlett-Packard fitted with a Poracolumn temperanl/min. re period, animals thane (Fluothane, armaceuticals Diand kidneys were livided to provide microscopy, and kidney were fixed line, dehydrated and embedded in and stained with t 4 i i T * PERCHLOROETHYLENE AND PEROXISOMES 105 TABLE 1 Peroxisomal Cyanide-Insensitive Palmitoyl Coenzyme a Oxidation in Rat and Mouse Liver and Kidney after Exposure to PER CN-insensitive palmitoyl CoA oxidation (nmol/min/mg protein) Concentration (ppm) Duration (days) Mouse Liver ; Rat Male 0 200 400 400 400 Female 0 200 400 400 400 14-28 28 14 21 28 14-28 28 14 21 28 5.16 1.06" 11.19 4.46** 11.98 2.86** 13.90 3.27** 18.64 5.61** 9.01 1.62 16.68 3.52** 14.40 2.27** 18.74 1.68** 17.99 2.35** 10.26 0.51 12.95 0.93* 13.68 1,68** 12.94 0.81** 13.61 0.89** 12.62 0.77 15.76 1.06** 14.90 1.91* 15.31 2.31* 14.14 1.90 Note. Control animals were exposed to air only for 14,21, or 28 days. " Values are x SD, n - 5 except for controls, where = 15. 4 Mouse kidneys were pooled according to group; values are x. * Statistically significant, p < 0.05. ** Statistically significant, p < 0.01. Kidney Mouse Rat 5.574 6.92 7.69 8.70 8.18 2.48 2.85 2.59 2.28 2.49 2.37 0.29 2.98 0.21** 2.97 0.65 2.44 0.49 2.76 0.33 1.98 0.21 3.11 0.45** 2.56 0.28** 2.68 0.26** 2.41 0.19** and lead citrate and viewed and photographed in a JOEL JEM 100CX electron microscope. Morphometric analy sis of peroxisomes was performed according to the gen eral principles ofWeibel el al. (1964) on electron micro graphs ofareas ofcytoplasm at a magnification of25,000. Biochemical analysis. Sections of liver and kidney re maining after tissue had been taken for light and electron microscopy were placed in ice-cold sucrose (250 ism) EDTA (5.4 mM) Tris-HCl (20 dim) bulfer, pH 7.4. Mouse kidneys were pooled according to group. Homog enates (25% w/v approximately) were prepared using a Teflon glass homogenizer at 4*C. Homogenates were centrifuged at 3000# for 5 min at 4*C. The supernatants from the kidney homogenates were stored at -70*C until used. Supernatants from the liver homogenates were fur ther centrifuged at 15,000g for 15 min at 4'C, as de scribed by Elcombe et al. (1985). The supernatants were discarded and the pellets (containing peroxisomes) were resuspended in the above buffer and stored at -70*C. The protein content of the liver and kidney fractions was determined by the method of Lowry et al. (1951). The activities of the peroxisomal enzymes catalase and cyanide-insensitive palmitoyl coenzyme A oxidase were determined by the methods ofBeers and Stzer (1952) and Bronfman et al. (1979), respectively. TCA Concentrations in Blood after Exposure to PER Rats and mice were exposed to 400 ppm PER for up to 6 hr. Animals killed at time points of less than 6 hr were exposed in glass desiccators at a flow rate of 5-10 liters/min. Those exposed for the full 6 hr were housed in the long-term chambers described above. Atmospheres were generated by vaporizing PER into the air stream and were monitored by gas chromatogra phy. Groups of three rats or three mice were killed by exposure to CO? and bled by cardiac puncture at inter vals from the start of exposure until 48 hr postexposure (see Fig. 2). TCA was extracted from blood as described by Prout et al. (1985) and the methylated samples were analyzed on a Hewlett-Packard 5890A gas chromato graph fitted with an electron capture detector. A glass col umn (2 m X 2 mm), packed with Porapak PS and oper ated at 180*C with a nitrogen carrier gas flow of 25 ml/ min, was used for the analysis. Under these conditions TCA had a retention time of 5.7 min. The limit ofdetec tion for TCA in blood was 0.2 /ig/ml. Statistics Values were tested for statistical significance using the two-sided Student t test. SL 035560 106 ODUM ET AL. RESULTS Effects ofExposure to PER The mean analyzed concentrations ofPER for the 28 days of exposure were 193 and 389 ppm for the rats and 196 and 395 ppm for the mice. These were close to the target levels of 200 and 400 ppm. No significant clinical ab normalities were seen in rats or mice exposed to either concentration of PER. Liver TABLE 2 Peroxisomal Catalase Activity in Rat and Mouse Liver after Exposure to PER Concen tration (ppm) Duration (days) Catalase (ksec-1 mg protein-1) Mouse Rat Male 0 400 400 400 Female 0 400 400 400 14-28 14 21 28 14-28 14 21 28 1.05 1.17* 1.12 0.19 1.30 0.17* 1.44 0.34* 1.79 0.12 1.56 0.09 1.85 0.05 1.76 0.28 1.62 0.26 1.88 0.57 1.62 0.21 1.90 0.13 1.56 0.23 1.46 0.15 1.77 0.30 1.59 0,24 Exposure of B6C3F1 mice to 400 ppm PER for either 14, 21, or 28 days resulted in small but statistically significant increases in liver/body weight ratios up to 1.2- and 1.3fold in males and females, respectively. F344 rats exposed to PER showed no changes in liver/body weight ratios. Cyanide (CN)-insensitive palmitoyl CoA oxidase, a marker for peroxisomal (8-oxida tion was significantly increased in mouse liver after exposure to PER (Table 1). This enzyme increased to a similar level in males and females but the control rate ofCN-insensitive palmitoyl CoA oxidation was higher in females. Therefore the increase over control rates was lower in females than males and maximum response (seen after 28 days expo sure to 400 ppm) was a 3.6-fold increase in males and a 2.1-fold increase in females. In contrast, only small increases in CN-insensitive palmitoyl CoA oxidation were ob served following treatment of F344 rats with PER (Table 1) although these were some times statistically significant. The maximum increase (1.3-fold) was in males exposed to 400 ppm for 28 days. The basal activity of CN-insensitive palmitoyl CoA oxidation was noted to be approximately 2-fold greater in F344 rats than in B6C3F1 mice (Table 1). Catalase, another peroxisomal enzyme, was largely unaffected in mice and rats ex- Note. Control animals were exposed to air only 14,21, or 28 days. * Values are x SD, n 5 except for controls, where n = 15. * Statistically significant, p < 0.05. posed to PER (Table 2). The only increases (up to 1.4-fold) were observed in male mice exposed to 400 ppm. By light microscopy the livers of mice ex posed to 400 ppm PER showed centrilobular eosinophilia and centrilobular fatty vacuolation. Both effects were seen to a similar extent in males and females and the numbers ofani mals affected increased from 14 to 28 days. Similar effects on lipid were seen at the elec tron microscope level in mice exposed to 200 ppm for 28 days or 400 ppm for 14,21, or 28 days. Extensive lipid accumulation was ob served in centrilobular hepatocytes. The lipid was present in the form of large droplets, 2to 5-ttm diameter, lying free in the cytoplasm of the cells (macrovesicles), and small drop lets, 0.1- to 0.5-trm diameter, contained within the cistema of the endoplasmic reticu lum (microvesicles). Figure 1 shows the ultrastructural appearance of a centrilobular hepatocyte from (a) an untreated male mouse and (b) a male mouse exposed to 400 ppm PER for 28 days. Electron microscopy showed proliferation of peroxisomes in the SL 035561 4? TTY IN Rat AND URE TO PER Catalase mg protein-1) ,e Rat 4 1.17" 0.19 0.17* '34* 12 )9 15 28 1.62 0.26 1.88 0.57 1.62 0.21 1.90 0.13 1.56 0.23 l.460.15 1.77 0.30 1.59 0.24 d to air only 14,21, Is, where * V 4 i i,- only increases d in male mice ers of mice exd centrilobular r fatty vacuolaa similar extent lumbers ofani14 to 28 days. Jen at the elecexposed to 200 3r 14,21, or 28 lation was ob:ytes. The lipid ge droplets, 2the cytoplasm ad small dropter, contained plasmic reticuhows the ultraatrilobular hed male mouse d Jfl^lO ppm a ^Beroscopy isomes in the & F1g. 1. (a) Ultrastructural appearance of a centrilobular hepatocyte from an untreated male B6C3F1 mouse showing nucleus (N) and peroxisomes (%). X6300. (b) Ultrastructural appearance ofa centrilobular hepatocyte from a male mouse exposed to 400 ppm PER 6 hr/day for 28 days. The cell shows an accumula tion of lipid in the form of large droplets (L) and small vesicles and a proliferation of peroxisomes (IK ). The nucleus is seen at (N). X6300. Insert: higher magnification ofperoxisome showing electron dense core, XI 9,800. SL 035562 107 r! IU I: i i i 108 ODUM ET AL. TABLE 3 Morphometric Analysis of Hepatic Peroxisomes in Mice and Rats Exposed to PER Exposure Concentration (ppm) Duration (days) 0 28 200 28 400 14 400 21 400 28 Peroxisome volume (% cytoplasm) Mouse Male Female 2.5 0.6* 5.2 1.5* 4.9 0.7* 5.4 1.3* 6.0 1.4* 2.5 0.8 4.4 0.6* 4.9 1.5* 4.8 0.9* 4.8 1.2* Rat Male 3.1 0.1 3.7 1.3 2.3 0.6 2.8 0.7 3.4 1.4 Female 3.3 0.7 4.7 1.9 2.7 0.5 3.2 1.1 3.4 1.0 i * Values (x SD, n = 5) are calculated from three micrographs per animal, with 375 points applied to each micro graph. 'Statistically significant,/) <0.01. sj centrilobular region of the mouse liver (Fig. lb, Table 3). The proliferated peroxisomes were small (<0.5 ftm) and the majority re tained the central nucleoid (Fig. lb, insert). Exposure to 400 or 200 ppm resulted in sta tistically significant increases in the volume of cytoplasm occupied by peroxisomes (Ta ble 3). Exposure of male mice to 200 or 400 ppm PER also resulted in a decrease in mitochon dria after 14 days but this was followed by mi tochondrial proliferation in those animals subsequently exposed to 400 ppm. The effect was not seen in females. Concomitant with these changes, exposure at either level for any of the time periods investigated resulted in a decrease in the amount of normal rough en doplasmic reticulum in the cells. Light microscopic examination of livers from rats exposed to PER showed centrilobu lar hypertrophy in both sexes with a concom itant loss of glycogen. The effects in males were of similar intensity in both the 200- and 400-ppm dose groups and there was little evi dence of progression of the lesion from 14 to 28 days in the 400-ppm group. Results sug gest that the males were more sensitive to the liver hypertrophic effects of PER since no effect was seen in females exposed to 200 ppm for 28 days. Electron microscopy showed a time-dependent proliferation of smooth endoplas mic reticulum in the liver in both sexes which correlated well with the centrilobular hyper trophy. The males were more susceptible than the females. There was no dose- or timedependant increase in peroxisomes in the liv ers of either sex (Table 3). Kidney No increases in kidney/body weight ratios were seen in rats and mice exposed to PER. The effect of PER on peroxisomal cyanideinsensitive palmitoyl CoA oxidation in rats and mice is shown in Table 1. Insufficient mouse kidney tissue precluded the measure ment of this marker in individual animals. Consequently values could not be tested for statistical significance. Slight increases were seen in /3-oxidation in male mouse kidney, the maximum being a 1,6-fold increase after 21 days exposure to 400 ppm. Small increases in this marker were also observed in female rat kidneys after exposure to PER (Table 1) up to a maximum of 1,6-fold. There was no effect of PER on renal cata lase activity in rats or mice of either sex (data not shown). Fig. 2. Blood leve to PER (400 ppm) means with three ar No compou served in the k light or electro; Blood Levels v Blood level: ing and after ppm) are sho' of TCA (appi were reached sure period ; half-life of 7levels persist blood levels approximate declining to exposure. Ct of TCA to posed, by c curves, sho posed to 6.7 The hep mouse has satisfactor epigenetic step in the be oxidati SL 035563 3 TO PER 171) Rat Female I 3.3 0.7 3 4.7 1.9 6 2.7 0.5 7 3.2 1.1 4 3.4 1.0 'plied to each micro- ' t wed a time-detoaflkndoplastoffies which rilobular hyperore susceptible o dose- or timetomes in the liv- Tf ^ ^ ^ ' iy weight ratios nosed to PER. isomal cyanideridation in rats 1. Insufficient d the measureridual animals, ot be tested for increases were mouse kidney, d increase after Small increases irved in female PER (Table 1) . o nal cataei x (data PERCHLOROETHYLENE AND PEROXISOMES 109 FIG. 2. Blood levels of TCA in mice and rats exposed to PER (400 ppm) for 6 hr and recovery. Values are means with three animals to each time point. No compound-related changes were ob served in the kidneys of either species at the light or electron microscope level. Blood Levels ofTCA after Exposure to PER Blood levels of TCA in rats and mice dur ing and after a 6-hr exposure to PER (400 ppm) are shown in Fig. 2. Peak blood levels of TCA (approximately 130 /tg/ml) in mice were reached 3-4 hr after the end ofthe expo sure period and thereafter declined with a half-life of 7-8 hr. Forty hours after exposure, levels persisted at 8-10 pg/ml. In contrast blood levels in the rat reached a plateau of approximately 7 Mg/ml after 3 hr of exposure declining to 4 Mg/ml 48 hr after the end of exposure. Comparison of the concentrations of TCA to which the two species were ex posed, by calculation of the area under the curves, shows the mouse to have been ex posed to 6.7-fold more TCA than the rat. DISCUSSION The hepatocarcinogenicity of PER in the mouse has been known for some years but no satisfactory mechanism, either genotoxic or epigenetic, has so far been proposed. The first step in the metabolism of PER is believed to be oxidation to an epoxide (Reichert, 1983) and the metabolites which have been identi fied support this assumption (Yllner, 1961; Daniel, 1963; Bonse et ai, 1975; Sakamoto, 1976). Alkylation of nucleotides by reactive epoxides has been described for other chlori nated alkenes such as vinyl chloride (Laib and Bolt, 1977) and vinylidene chloride (Re itz et al., 1980). DNA binding has not how ever been demonstrated after treatment of rats and mice with PER (Schumann et al., 1980) nor does PER induce gene mutations in bacteria (Bartsch et ai, 1979; Greim et ai, 1975; Bronzetti et ai, 1983). These observa tions led to proposals (Schumann et ai, 1980) that PER-induced liver tumors in B6C3F1 mice are a result ofrecurrent cytotoxicity and enhancement of the high spontaneous inci dence of liver tumors found in this strain of mouse. The results of the present study suggest an alternative hypothesis for the mechanism of PER-induced carcinogenicity, that of peroxi some proliferation leading to cancer forma tion via an epigenetic mechanism (Reddy et ai, 1980). Exposure of male and female mice to PER for up to 28 days resulted in a signifi cant proliferation of peroxisomes in the liver as measured by peroxisomal /3-oxidation (Ta ble 1) and morphometric analysis of electron micrographs (Table 3). The induction of per oxisomal /3-oxidation in this study was not accompanied by increases in catalase (Table 2). This phenomenon has been observed after administration of other peroxisome proliferators (Cohen and Grasso, 1981; Reddy and Lalwani, 1983, for review) and is believed to lead to increased levels of hydrogen peroxide in the cell, causing oxidative damage, cyto toxicity, and possibly DNA damage. How ever, a definite link between such changes and the eventual development of cancer re mains to be established. Although increases in the activity of per oxisomal enzymes were observed in the livers of rats after treatment with PER (Table 1), these were slight compared to the changes seen in the mouse and could not be corrobo rated by electron microscopy (Table 3). Sim- SL 035564 no ODUM ET AL, ilar results after treatment of rats and mice with PER have recently been reported by Goldsworthy and Popp (1987). A sixfold in crease in hepatic CN-insensitive palmitoyl CoA oxidase was observed in B6C3F1 mice dosed PER by gavage at 1000 mg/kg for 10 days. No such increase was seen in F344 rats. This species difference in peroxisome prolif eration is identical to that found by Elcombe et al. (1985) after TRI was dosed by gavage to mice and rats. There were however differ ences in the pathology of treated livers be tween the present study and that reported by Elcombe et al. (1985) for TRI. PER-induced peroxisome proliferation was observed in the centrilobular region of mouse liver. The in duced peroxisomes were small and retained the nucleoid core, whereas after TRI treat ment they generally lacked the nucleoid core. Exposure to PER also resulted in a concomi tant accumulation of lipid in centrilobular cells with periportal cells unaffected. The rea sons for these differences are unknown but may be due to the effects of other metabolites or the parent chemical. TCA, the major metabolite of PER (Dekant et al., 1985), is a known hepatic peroxi some proliferator in both rats and mice (El combe, 1985) and is the metabolite responsi ble for increased peroxisomes in mice exposed to TRI in previous studies (Elcombe et al., 1985). In the present study, TCA aris ing from metabolism of PER only induced hepatic peroxisome proliferation in mice be cause of the much higher concentrations of this metabolite in mouse blood. The lack of a response in rats indicates that a threshold concentration of TCA has to be reached in order to induce peroxisome proliferation in rodent liver. The low blood levels of TCA ob served in rats compared with mice correlates with the lower rate of oxidative metabolism of PER in rats than mice (Schumann et al., 1980; Ikeda and Ohtsuji, 1972). Recent studies have confirmed that TCA is in fact a carcinogen in B6C3F1 mice (HerrenFreund el al., 1986). TCA dosed to male mice in drinking water at 5 g/liter for 61 weeks pro duced a 50% tumor incidence compared to a 5% incidence in the control group. Peroxi- ' some proliferation was observed in the livers of treated animals. Thus the species differ ence in the carcinogenicity of PER between rats and mice may be explained by the vmarked difference in blood levels ofTCA and a mechanism which induces peroxisome pro liferation. The effect of PER on the kidney in mice and rats was minimal (Table 1). No com pound-related changes were seen at the light or electron microscope level in regions of the nephron where peroxisomes are known to be most prevalent (Beard and Novikoff, 1969). Similarly the increases observed in peroxi- $ somal enzymes were slight and not related to dose or exposure. Peroxisome proliferation is therefore unlikely to play a role in the carci nogenicity of PER in the rat kidney and fur ther investigations are needed to establish an alternative mechanism. Metabolism ofPER in man is known to oc cur at a very slow rate (Fernandez etal., 1976; Monster et al., 1979). It is also a saturable process, saturation occurring at the low inhalational exposure level of 100 ppm (Ikeda et al., 1972;Ohtsuki etal., 1983). Consequently man is exposed to lower concentrations of ^ TCA than mice or rats. Furthermore, TCA does not induce peroxisome proliferation in vitro in human hepatocytes (Elcombe, 1985); indeed the response of primates to the induc tion of peroxisome proliferation by other agents is generally much lower than that of . rodent species (Cohen and Grasso, 1981; Reddy and Lalwani, 1983). In conclusion this study demonstrates that quantitative differences in the metabolism of PER to TCA in mice and rats lead to prolifer ation of peroxisomes in the livers of mice but not rats. The known carcinogenicity of TCA in B6C3F1 mice and the correlation between hepatic peroxisome proliferation and cancer in rodents strongly suggests that TCA-induced peroxisome proliferation is the basis of the species difference in hepatocarcinogenicity of PER. The limited capacity of humans to metabolize PI deficiency in rc oxisome prolife unlikely to cam in man. ACKN The authors than! for carrying out ttu Gowans and Mr. V> TCA blood level stu Bartsch, H., M PlanCHE, G. (1 tabolitesofhalo-' orobutenesprodi Evidence for oxii somal mono-ox> Beard, M. E., an tion of peroxiso the rat. A cytoc 518. Beers, R- F., and metric method i gen peroxide by Bonse, G., Urba ler,D.(1975).` formation and 1 ylenes in the is Biochem. Phan BRONFMAN, M., I 11979). Fatty a somes. Biochei 1036. Bronzetti, G,, tore. R., Gai (1983). Genetic ethylene "in > 323-331. Cohen, A. 3., a hepatic respor and assessmen Food Cosmet- Daniel, l. W. ( trichloroethyli Biochem. Pha Dekant, W., H Absorption, e roethylene. N, (Suppl.) 329,1 Si "35565 ence compared to a trol group. Peroxijserved in the livers the species differty of PER between explained by the d levels ofTCA and .ces peroxisome pro- i the kidney in mice (Table 1). No comvere seen at the light vel in regions of the es are known to be d Novikoff, 1969). bserved in peroxi: and not related to me proliferation is a role in the carciatJjfciey and furde^pestablish an I I 4 * i # $ i * \an is known to octandez etai, 1976; is also a saturable ng at the low inha100 ppm (Ikeda et 83). Consequently concentrations of urthermore, TCA ie proliferation in ;(Elcombe, 1985); lates to the inducferation by other ower than that of id Grasso, 1981; demonstrates that he metabolism of ts lead to proliferlivers of mice but ogenicity of TCA Delation between 'ation and cancer its that TCA-intioius the basis of ia(^^cinogenicia^^)f humans I 4 $ I PERCHLOROETHYLENE AND PEROXISOMES 111 to metabolize PER coupled with an intrinsic deficiency in response to TCA as a per oxisome proliferator indicates that PER is unlikely to cause hepatocellular carcinoma in man. ACKNOWLEDGMENTS The authors thank Mr. S. Miliward and Mr. I. Bennett for carrying out the inhalation exposures and Mr. N. Gowans and Mr. W. M. Provan for their help with the TCA blood level study. REFERENCES Bartsch, H., Malaveille, C., Barbin, a., and Planche, G. (1979). Mutagenic and alkylating me tabolites ofhalo-ethylenes, chlorobutadiene and dichlorobutenes produced by rodent or human liver tissues. 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