Document QZjnx9VawzEyj51RerNy7dXo

0 copper content and kidney tt. 5,131-138. Jnosaka. S.. and Okahara, d-labeled metallothionein in macol. 33,2J 8-266. assen. C. D. (1985). Concenn in major organs of rats after s metals. Fundam. Appi Tax logy of cadmmm-thionein. In mistry. and Biology of Cadpp. 423-431. Elsevier/North sterdam. .`tallothioneins. In TheChem1 Biology of Cadmium (M. 66. Elsevier/North Holland. um-thionein and the nephro* i Kadmium Symposium <M. :r. Eds.), pp. 101-107. FreidJena. TOXICOLOGY AND aTFLIED PHARMACOLOGY 92, 103-112(1988) The Rote of Trichloroacetic Acid and Peroxisome Proliferation in the Differences in Carcinogenicity of Perchloroethyiene in the Mouse and Rat J. Odum, T. Green,' J. R. Foster, and P. M. Hext Imperial Chemical Industries PLC. Central Toxicology Laboratory. Aideriey Park. Nr Macclesfield. Cheshire SKIO 4TJ. United Kingdom Received June 8.1987: accepted September 21.1987 The Role ofTrichloroacetic Acid and Peroxisome Proliferation in the Differences in Carcino genicity ofPerchloroethyiene in the Mouse and Rat. Odum, J., Green, T- Foster, J. R,, and Hext, P.M. (1988). Toxicol. Appi. Pharmacol. 92,103-112- Fischer 344 nts and B6C3F1 mice ofboth sexes were exposed to 400 ppm perchloroethyiene (PER) by inhalation, 6 hr/day for 14, 21, or 28 days or to 200 ppm for 28 days. Increased numbers ofperoxisomes were seen under the electron microscope and increased peroxisomal cyanide-insensitive paimitoyl CoA oxida tion was measured (3.6-fold increase in males and 2. l-(bid 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 Mood levels in mice were 13 times higher than those seen in rats. Comparison ofareas under the curves over the time coune ofthe experiment showed that mice were exposed to 6.7 rimes more TCA than ms. The difference in metabolism ofPER to TCa in mice and rats leads to thespeciesdifference in hepatic peroxisome proliferation which is believed to be the basis ofthe species difference in bepatocarcinogenicity. Peroxisome proliferation does not appear to play a role in the apparent carcinogenicity of PER in the rat kidney, e ires Academic po. lac. Perchloroethyiene (PER) (1,1,2,2-tetrachIoroethylene) 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/lcg. Both dose groups showed about a 50% inci dence of hepatocellular carcinoma in mice. In the second study (Mennear et 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 hepatocarcinogenidty is similar to that seen with trichloro ethylene (TR1)(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 CawnwMO Iret by Acadmnc Pn me. Al hereofreproduction many form rer*d. 035538 SL 104 ODUM ET AL. hepatocellular carcinoma in rodents (Reddy et ai, 1980) although no such relationship has yet been shown between renal peroxi some proliferation and renal tubular adeno carcinoma (Reddy et ai, 1975. 1982). The species difference in hepatic peroxisome pro liferation elicited by TR1 is believed to be the basis ofthe species difference in carcinogenic ity (Elcombe etai. 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 etai, 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 ai, 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 ai. 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 ai, 1975; Bartsch etai., 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 animate. METHODS Materials 1,1,2.2-Tetrachloroethylene (Anafar grade. 99.9%) and trichloroacetic add (Analar grade. 99% pure) were obtained from BDH Chemicals PLC (Poole, Dorset, UK). Biochemicals were obtained from Sigma Chemical Co. (Poole. Dorset UK). Amma/s Male and female Fischer 344 rats (160-180 g) and male 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.. Witbam. Essex. UK) and water ad libitum before and after, but not during exposure. Exposure to PERfor up to 28 Daps Exposure. Male and female msand mice (5 per group) were exposed to concentrations of 200 or 400 ppm of PERfor6hr/dayfor 14.21. or2tconsecutive days. Con trol animals were exposed toronly, but otherwise were treated in a manner smiiartothmofthe test animals. Exposures were whole body ia nainlcss steel chambers (Doe and Tinston, 1981) having an internal volume of approximately 3.4 m5. The cbembers were air condi tioned to have a nominal tempenture of 22*C and rela tive humidity of40-60%. Theairflow through the cham bers was 300 liters/min. Almn^heres were generated by passing vaporized PER into the input air of the cham bers. Atmospheres in the ten chambers were analyzed for PER by gas chromatography (GOona Hewlett-Packard 5880a GC (flame ionization detector) fitted with a Porapak PS column (1.8 in x 4 em) The column tempera ture was 195*C, helium carrier gas 50 ml/min. Eighteen hours after the ten exposure period, animals were killed by overexposure to hnlothane (Fluothane, Imperial Chemical Industries PLC, Pharmaceuticals Di vision) and exsanguinated. The liven and kidneys were rapidly removed, weighed, and then divided to provide tissue for light microscopy, eketron microscopy, and biochemical analysis. Light microscopy. Slices ofliver and kidney were fixed in 10% neutral buffered formol saline, dehydrated through an ascending -u miss, and embedded in paraffin wax. Sections (5 pm) were cat and stained with hematoxylin and aosio. Electron microscopy. Taeumwere fixed in 3% glutaraldehyde in 0.1 m sodium phosphasr buffer, dehydrated, and embedded in epoxy rain. Sections (1 n) were cut and stained with 1% toiuidine Uue ia 1% borax for light microscopy. Areas were rekesed from the cenuilobular regions of the livers end the S3 regions of the proximal tubules ofthe kidney for iltx'iiun microscopy. Ultrathin sections of these areas were teemed with uranyl acetate 035539 PERCHLOROETHYLENE AND PEROXISOMES 105 TABLE I 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 Kidney Mouse 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. 4 Values are x - SD, n m 5 except for controls, where n 15. * Mouse kidneys were pooled according to group; values are * Statistically significant, p < 0.05. " Statistically significant p < 0.01. 5.57* 6.92 7.69 8.70 8.18 2.48 2.85 2.59 128 2.49 137 0.29 2.98 0.21" 197 0.65 144 0.49 176 0.33 1.98 0.21 3.11 0.45" 156 0.28" 2,68 0.26" 141 0.19" and lead citrate and viewed and photographed in a JOEL JEM 100CX electron microscope. MorpfiuRKDic analy sis of peroxisomes was performed according to the gen eral principles ofWeibel et 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 dm) EDTA (5.4 nM) Tns-HCl (20 dm) buffer, pH 7.4. Mouse kidneys were pooled according to group. Homog enates (25% w/v approximately) were prepared unag 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 homogenises were fur ther centrifuged at 15,000; for 15 min at 4*C, a* de scribed by Elcombe er 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 *>-- and cyanide-insensitive palmitoyl coenzyme A oxidase were determined by the methods ofBeers and Sizer(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. Croups 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 poatexposure (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 withan electron capturedetector. 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 them conditions TCA had a retention time of5.7 min. The limit ofdetec tion for TCA in blood was 0.2 jig/mL Statistics Values were tested for statistical significance using the two-sided Student t test. 0355* si* 106 ODUM ET AL. RESULTS Effects ofExposure to PER The mean analyzed concentrations of PER 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: Peroxisomal Catalase Activity in Rat and Mouse Liver after Exposure to PER Conceitration (ppm) Duration (days) Catalase (ksec'1 mg protein*') 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 1.460.15 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/bodv weight ratios. Cyanide (CN)-insensitive palmitoyl CoA oxidase, a marker for peroxisomal /3-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 m only 14,21. or 28 days. * Values are x + SD. n - 5 except for controls, where n " IS. * 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 cemrilobular 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-/im diameter, lying free in the cytoplasm of the cells (macrovesicles), and small drop lets, 0.1- to 0.5-um diameter, contained within the cistema ofthe 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 035541 Fig. 1. (a) Ultrastructural appearance of a centrilobular hcpatocyte from an untreated male B6C3F1 mouse showing nudeus(N)and peroxisomes! \). x6300.(b) Ultrastructural appearanceofa centrilobular hcpatocyte from a male mouse exposed to 400 ppm PER 6 hr/day for 28 days. Thccdl shows an accumula tion of lipid in the form of large droplets (L) and small vesicles and a proiiferatioa of peroxisomes (H). The nucleus is seen at (N), X6300, Insert: higher magnification ofperoxisome showingelectron dense core. XI 9.800. 107 035542 SL 108 ODUM ET AL. TABLE 3 Morphometric analysis of Hepatic Peroxisomes in Mice and Rats Exposed to PER Exposure Concentration (ppm) Duration (days) Peroxisome volume (% cytoplasm) Mouse Rat Male Female Male Female 0 28 2.5 ; 0.64 15*0.8 3.1 +0.1 3.3+0.7 200 28 3.2 * 1.5' 4.4 + 0.6' 3.7+ 1.3 4.7 + 1.9 400 14 4.9 * 0.7' 4.9+ 1.5* 2.3=0.6 17+0.5 400 21 5.4* 1.3* 4.8 0-9* 18=0.7 3.2+ l.l 400 28 6.0= 1.4' 4.8 + 1.2' 3.4+ 1.4 3.4+ 1.0 ` Values (x + SD. n 5) are calculated from three micrographs per animal, with 373 points applied to each micro* graph. * Statistically significant, p < 0.01. centrilobuiar region of the mouse liver (Fig. lb. Table 3). The proliferated peroxisomes were small (<0.5 pm) and the majority re tained the central nucleoid (Fig. lb, insen). 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 ieen 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 centrilobular 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 centrilobuiar hyper trophy. The males were more susceptible than the females. There was no dose- or timedependant increase in peroxisomes in the liv ers ofeither sex (Table 3). Kidney No increases in kidney/body weight ratios were seen in rats and mice exposed to PER. The effect ofPER 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 l) up to a maximum of 1.6-fold. There was no effect of PER on renal cata lase activity in rats or mice ofeither sex (data not shown). SL 0355^3 PERCHLOROETHYLENE AND PEROXISOMES 109 140* Fic. 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 of TCA 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 Mg/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 Mg/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 yean 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 al.. 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 al., 1979; Greim et al.. 1975; Bronzetti et al.. 1983). These observa tions led to proposals (Schumann et al., 1980) that PER-induced liver tumors in B6C3F1 mice are a result of recurrent 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 al., 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 I) 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 035544 110 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 at. (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 ofTCA 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 et 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 marked 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 etai. 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;Ohtsukia/., 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 Lahrani, 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 SL 035545 PERCHLOROETHYLENE AND PEROXISOMES 111 to metabolize PER coupled with an intrinsic deficiency in response to TCA as a per oxisome proliferator inacates that PER is unlikely to cause hepatocellular carcinoma in man. ACKNOWLEDGMENTS The authors thank Mr. S. MUlward and Mr. 1. 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, chlorobutadienc and dichlorobutenes produced by rodent or human liver tissues. Evidence for oxirane formation by P450 linked micro somal mono-oxygenases. Arch. Toxicol, 41.249-277. Beard, M. E,, and Novikoff, a. B. (1969), Distribu tion of peroxisomes (microbodies in the nephron of the rat. A cytochemical study. J. Cell Biol. 41, 501518. Beers. R. F., and Sizer, J. w. (1952). A spectrophotometric method of measuring the breakdown ofhydro gen peroxide by catalase. J. Biol. Chem, 195,133-140. Bonse, G., Urban, Th.. Reichert, D.. and Henschler. D. (1975). Chemical reactivity, metabolic oxirane formation and biological reactivity ofchlorinated ethylenes in the isolated perfused rat liver preparation. Biochem. Pharmacol. 24, 1829-1834. Bronfman, M.. inestrosa, N. C,, and Leighton, F. (1979). Fatty acid oxidation by human liver peroxi somes. Biochem. Biophys. Res. Common. 88, 10301036. bronzetti, G,, Bauer, C.. Corsl C., Del CarraTORE, R., GaLLI, A., NlERI, R,, AND PaOLINI, M. (1983). Genetic and biochemical studies on perchloroethylene "in vitro" and "in vivo." Mutat. Res. 116, 323-331. Cohen, a. J., and GraSIO, P. (1981). Review of the hepatic response to hypolipidaemic drugs in rodents and assessment ofits toxicological significance to man. Food Cosmet. Toxicol. 19,585-605. Daniel, J. W. (1963). The metabolism of "CMabeled trichloroethylene and tetrachloroethylene in the rat. Biochem. Pharmacol. 12,795-802. Dekant, W,, Hang. R., and Henschler, D. (1985). Absorption, elimination and metabolism of tetrachlo roethylene. Naunyn-Schmtedeberg's Arch. Pharmacol. (Suppl.) 329, R24. Doe, J. E.. and Tinston, D. J. (1981). Novel chambers for long term inhalation studies. In Proceedings ofthe Inhalation Toxicology and Technology Symposium (B. K, J. Leong. Ed-), pp. 77-88. Ann Arbor Science Pub., Ann Arbor. Ml. Elcombe. C. R. (1985). Species differences in carcinoge nicity and peroxisome proliferation due to trichloro ethylene: A biochemical human hazard assessment. Arch. Toxicol. (Suppl.) 8,6-17. Elcombe. C. R., Rose. M. S.. and Pratt, I. S. (1985). Biochemical, histological, and ultrastructural changes in rat and mouse liver following the administration of trichloroethylene: Possible relevance to species differ ences in hcpatocarcinogenicity. Toxicol. Appl. Phar macol. 79,365-376. Fernandez, J., Guberman. E,, and Caperos, J. (1976). Experimental human exposures to tetrachloro ethylene vapour and elimination in breath after inha lation. Amer. lnd. Hyg. Assoc. J. 37,143-150. Goldsworthy. T. L. and Popp. J. A. (1987). Chlori nated hydrocarbon induced peroxisomeenzyme activ ity in relation to species and organ carcinogenicity Toxicol. Appl. Pharmacol. 88,225-233. Green, T., and Prout, M. S. (1985). Species differ ences in response to trichloroethylene. II. Biotransfor mation in rats and mice. Toxicol. Appl. Pharmacol. 79, 401-411. Greim, M.. Bonse. G.. Radwan, Z., Reichert, D.. and Henschler. D. (1975). Mutagenicity in vitro and potential carcinogenicity ofchlorinated ethylenes as a function of metabolic oxirane formation. Bio chem Pharmacol. 24.2013-2017. Herren-Freund, S. L.. Pereira, M. A., Olsen. G.. and De Angelo, A. B. (1986). The caroinogencity of trichloroethylene and its metabolites, trichloroacetic acid and dichloroacetic acid in mouse liver. Proc. Amer. Assoc. Cancer Res. 27,91. Ikeda, M.. and Ohtbuii, H. (1972). A comparative study of the excretion of Fujiwara reaction-positive substances in urine of humans and rodents given triehloro or tetrachloro-derivatives of ethane and eth ylene. Br. J. lnd. Med. 19,99-104. Ikeda, M.. Oktsujl H,, Imamura, T,, and Komoike, Y. (1972). Urinary excretion of total trichloro-compounds. trichloroethanol and trichloroacetic arid as a measure of exposure to trichloroethylene and tetra chloroethylene. Br. 1. lnd. Med. 29,328-333. Laib, R. J., and bolt, H. M. (1977)-. Alkylation ofRNA by vinyl chloride metabolites in vitro and in vivo: For mation of l-'N-etbcno-adenosine. Toxicology &, (85195. Lowry. O. H.. Rosebrough, N. J., Farr. a. L, and Randall, R. J. (1951). Protein measurement with the Folin phenol reagenL/ Biol. Chem. 193,265-275. Mennear, J. H.. Clarke, W,, Ragan, H., and Miller, R. (1986). Toxicology and carcinogenicity SL 035546 112 ODUM ET AL. studies ofinhaled tetrachloroethylene (PERC) in male and female F344 rats and B6C3F1 mice. Toxicologist 6,133. Monster, a. C, Boersma. C.. and Steenweg, H. (1979). Kinetics of tetrachloroethylene in volunteers: Influence of exposure concentration and work load, /nr. Arch. Occup. Environ. Health 42,303-309. National Cancer Institute (1976). Carcinogenesis Bioas say of Trichloroethylene. CAS No. 79-01 -6. DHEW pub. No. (NIH) 76-802. National Cancer Institute (1977). Bioassay ofTetrachloroethylene for Possible Carcinogenicity. DHEW pub. No. 77-813. National Toxicology Program (1983). National Toxicol ogy Program draft report abstracts on nine chemical carcinogenesis animal bioassays. Chem. Regul. Rep. 6, 767-768. Ohtsukj, T., Sato, K.. Koizumi, a., Kumai. M., and IKEDa, M. (1983). Limited capacity ofhumans to me tabolise tetrachloroethylene. Int. Arch. Environ. Health 51,381-390. Prout, M. S-, Provan, W. M,, and Green. T. (1983). Species differences in response to trichloroethylene. I. Pharmacokinetics in rats and mice. Toxicol. Appl. Pharmacol. 79.389-400. Reddy. J. k., azarnoff, D. L. and Hignite. C. E. (1980). Hypolipidaemic hepatic peroxisome prolifera ters form a novel class ofchemical carcinogens. Nature (London) 283.397-398. Reddy, J. K.. and LalwaNI, N. D. (1983). Carcinogen esis by hepatic peroxisome proWerators: Evaluation of the risk of hypolipidemic drugs and industrial plasti cizers to humans. CRC Crit. Rev. Toxicol. 12,1 -38. Reddy. I. K., Krhhnakantka, T. P.. and Tao. M. S. (1973). Microbody (peroxisome) proliferation in mouse kidney induced by methyl dofenapate. Vir chows Arch. B CellPathol. 17,293-306. Reddy, J. K.. Warren. J. R.. Reddy, m. K., and Lalwani, N. D. (1982). Hepatic and renal effect ofperoxi some prolifeiaion; Biological implications. Ann. .V. Y. Acad. Sci. 386,81-110. Reichert. D. (1983). Biological actions and interactions of tetrachloroethylene. Mutat. Res. 123,411-429. Reitz, R. H.. Watanabe. P. G.. McKenna, M. J., Quast, l. F,, AND Gehring, P. ]. (1980). Effects of vinylidene chlonde on DNA synthesis and DNa re pair in the rat and mouse; A comparative study with dimethylnitrosiminr. Toxicol. Appl. Pharmacol. 52, 357-370. Sakamoto, N. (I976L Metabolism of tetrachloroethyl ene in guinea pip. Japan. J. Health 18,11-16. Schumann a. M,, Quast. J. F,, and Watanabe, P. G. (1980). The pharmacokinetics and macromolecular interactions of perebioroethylene in mice and rats as related to oncogenicity. Toxicol. Appl. Pharmacol. 55, 207-2(9. Weibeu E. R.. Staubu. w . Gnage, H. R., and Hess, F. (1964). Conctaed morphometric and biochemical studies on the livercefl. I. Morphometric modd stereological methodsand normal morphometric data for rat liver. J. Cell BioL 21.68-91. Yllner, S. (196I)l Urinary metabolites of l4C-tetrachloronhykoem mice. Nature (London) 191,820.