Document O3EpNRkrd24ZKJmQBXaKr65zp

V. L,, Morgan, R. L., is of thiosulfate on cyas. Toxicol. Appl. Phar- n and calcium fluxes in iol. (London) 286, 525- Rink, T. J. (1982). Calnphocytes: Cytoplasmic a new, intracellularly J. Cell Biol. 94, 325- (1984), On the applicaa cells in chemical toxicon. Health 13,511-520. xication and its mecha,'v. Pharmacol. Toxicol. I f i I \ ; 1 I ; TOXICOLOGY AND APPLIED PHARMACOLOGY 88, 225-233 (1987) Chlorinated Hydrocarbon-Induced Peroxisomal Enzyme Activity in Relation to Species and Organ Carcinogenicity T. L. Goldsworthy and J. A. Popp Department ofExperimental Pathology and Toxicology, Chemical Industry Institute ofToxicology, PO. Box 12137, Research Triangle Park, North Carolina 27709 Received August 18,1986; accepted December 8.1986 Chlorinated Hydrocarbon-Induced Peroxisomal Enzyme Activity in Relation to Species and Organ Carcinogenicity. Goldsworthy, T, L., and Popp, J. A. (1987). Toxicol. Appl. Pharma col 88, 225-233. Trichloroethylene (TCE), perchloroethylene (PER), and pentachloroethane (PENT) are widely used industrial chemicals that cause an increased incidence ofhepatocellular carcinoma in mice and a very low incidence of renal tubular adenocarcinoma in rats. A recent study (C. R. Elcombe, M. S. Rose, and I. S. Pratt (1985), Toxicol. Appl. Pharmacol. 79, 365376) suggested that the species difference in the hepatocarcinogenicity ofTCE seen between rats and mice was due to a species difference in peroxisomal proliferation and ceil proliferation. The purpose of the present investigation was to understand better the association of peroxisome proliferation in the species-specific hepatocarcinogenicity, and nephrocarcinogenicity of TCE, PER, and PENT. TCE (1000 mg/kg body wt), PER (1000 mg/kg body wt), PENT (150 mg/kg body/wt), the metabolite trichloroacetic acid (TCA; 500 mg/kg body wt) or the potent peroxi some proliferating agent Wy-14,643 (WY; 50 mg/kg body wt) was administered by gavage to male F-344 rats and B6C3F1 mice for 10 days. Cyanide-insensitive palmitoyl CoA oxidation activity (PCO) was used to measure the peroxisome proliferation response. Of the chlorinated hydrocarbons, TCE and PER elevated PCO activity in mouse liver whereas only TCE elevated rat liver and kidney PCO. All agents increased PCO activity in the kidneys of mice. None ofthe chlorinated hydrocarbons induced a PCO response stronger than WY. These results (1) support an association between peroxisome proliferation and hepatic tumors in mice following TCE and PER, but not PENT, administration and (2) suggest that chlorinated hydrocarbon-induced peroxisome proliferation does not correlate with species-specific renal carcinogenicity. 1987 Academic Press, Inc. Trichloroethylene (TCE), perchloroethylene (PER), and pentachloroethane (PENT) are used extensively as industrial solvents. Ad ministration of TCE (National Cancer Insti tute (NCI), 1976; National Toxicology Pro gram (NTP), 1983a), PER (NCI, 1977; NTP,, 1986), and PENT (NTP, 1983b) caused an increased incidence of hepatocellular carci nomas in B6C3F1 mice and a low (4-8%) but significant increase of renal tubular adeno carcinomas in male Fischer-344 rats. Al though the mechanism(s) by which these chlorinated hydrocarbons cause neoplasia re mains unknown, it is generally believed that these agents require metabolic activation to exert their carcinogenic effects (Henschler et al., 1976; Schumann et al, 1980; Bolt et al, 1982; Stott et al., 1982; Green and Prout, 1985; Prout etal., 1985). Recent work by Elcombe and co-workers (1985) demonstrated that short-term admin istration of TCE-induced peroxisome prolif eration (PP) in the livers of mice but not rats. Trichloroacetic acid (TCA), a metabolite of TCE, induced peroxisome proliferation in the livers of both rats and mice. These studies 0041-008X/87 $3.00 Copyright 1987 by Academic Press. Inc. All rights of reproduction in any form reserved. SI* 03A554 226 GOLDSWORTHY AND POPP suggested that the high rates of metabolism of TCE in mice result in concentrations of TCA (Green and Prout, 1985; Prout et al.. 1985) sufficient to induce peroxisome proliferation whereas low concentrations of TCA found in rats do not induce the peroxisome prolifera tion. These authors suggested that a species difference in metabolism and peroxisome proliferation may explain the TCE-induced species-specific hepatocarcinogenicity ob served. Since TCA is also a metabolite of PER (Costa and Ivanetich, 1980) and PENT (Yllner, 1971), a similar mechanism for mouse liver tumor induction may exist for these two halogenated solvents. Agents that induce liver peroxisome prolif eration also cause hepatocellular carcinomas in both rats and mice (Reddy et al, 1980; Reddy and Lalwani, 1983). On the other hand, a correlation between renal peroxi some proliferation and renal carcinogenesis has not been established. Peroxisomes are lo calized in the tubular epithelium of the kid neys in rats and mice (Beard and Novikoff 1969; Reddy et al., 1975). Proliferation of peroxisomes can be determined morphologi cally or biochemically. Since the assay for cy anide-insensitive palmitoyl CoA oxidation (PCO) is specific for peroxisomes, this assay provides a rapid quantitative indicator for peroxisome proliferation (Reddy and Lal wani, 1983; Elcombe etal, 1985). In the pres ent study we investigated the effects of short term administration of TCE, PER, PENT, and TCA on peroxisome enzyme activity in livers and kidneys of rats and mice. For com parison to the three halogenated solvents, TCA was given in a high dose in these studies to determine if peroxisome proliferation may occur in the kidney as previously described for liver. The animal strain, dose, and route of administraton of TCE, PER, and PENT were chosen to be the same as in the chronic bioassays in order to (1) better understand the role of peroxisome proliferation in the spec ies-specific hepatocarcinogenicity and (2) to determine if chlorinated hydrocarbon-in duced peroxisome proliferation is associated with the development of the renal carcinoge nicity in rats. METHODS Chemicals. Trichloroethylene (99+%, epoxide stabi lizer free), tetrachloroethylene (99+%. epoxide stabilizer free), pentachloroethane (96%, impurities not identi fied), and trichloroacetic acid (99+%, ACS) were ob tained from Aldrich Chemical Co.. Inc. (Milwaukee, Wl). Com oil, NAD, FAD, acetyl coenzyme A, dithiothreitol, bovine serum albumin (BSA) and palmitoyl co enzyme A were supplied by Sigma Chemical Co. (St. Louis. MO). Triton X-100 and KCN were obtained from Fisher Scientific Co. (Pittsburgh, PA). Wy-14,643 (WY) was a generous gift from Wyeth Laboratories (Rad nor, PA). Animals. Male Fischer-344 (CDF(F-344)/Crl BR) rats and male B6C3F1 (B6C3F1/Crl BR) mice from Charles River Breeding Laboratories, Inc. (Kingston, NY), were quarantined on arrival for 2 weeks and found to be free ofpathogenic viruses by a standard virus titer screen (rat/ mouse assessment profile, Microbiological Associates, Bethesda, MD). Animals were housed in polycarbonate shoe-box cages with filter tops at 72 2*F and 50 10% humidity with a 12-hr light/dark cycle. NIH-07 pelleted diet and tap water were available ad libitum throughout the study. At the start of the treatments, rats and mice weighed 170-200 and 20-25 g, respectively. Experiment I. Animals were gavaged (1 ml per rat; 0.05 ml per mouse) with test agents dissolved in com oil for 10 consecutive days. WY was dissolved in a 1:9 di methyl sulfoxide (DMSO)/com oil mixture. Dosages of chemicals follow: TCE (1000 mg/kg body wt), PER (1000 mg/kg body wt), PENT (150 mg/kg body wt), TCA (500 mg/kg body wt), WY (50 mg/kg body wt). Control animals received an appropriate volume ofcom oil vehi cle alone. Experiment 2. In the vehicle comparison studies, the same test agents except PENT were administered to rats at the above concentrations in either com oil or methyl cellulose for 10 days. Naive animals received no treat ment and vehicle controls received the vehicle alone. Experiment 3. In concurrent administration studies, rats and mice were gavaged with TCE (1000 mg/kg body wt), PER (1000 mg/kg body wt), or TCE + PER (separate doses of 1000 TCE mg/kg body wt + 1000 PER mg/kg body wt administered immediately following one an other) dissolved in com oil for 10 consecutive days. All animals were killed 24 hr after the last dose. Tissue collection and enzyme assay. Animals were weighed, anesthesized with methoxyflurane. and killed by exsanguination. After liver and kidney organ weights Et Treatrr Com oil Wy-14,643 (5 Tnchloroethv (lOOOmg/k Perchloroeth\ (1000 mg/k Pentachloroei (150 mg/kg Tnchloroaceti (500 mg/kg " Male Fisc! received com i b x SE ofr, c Significant; d Significant ' Significant! were taken, tis; kidney cortex v from the left lo peroxisome en. lobes to either studies (T. L. G data). Tissues > 0.154 mKC1/5i ume ratio of 1> ney to buffer. strokes ofa rota mzer, and 1-ml Palmitoyl Co somal d-oxidatii fication (Butters (1981), in super 2500^ for 5 mir tion was measui palmitoyl CoA- presence of CN` identical to thos fold increase in t a 1.6-fold incre; that were shown Tissue test sampl f I ney 1:2) and ana f * ion is associated renal carcinoge- * 9+%, epoxide stabi le. epoxide stabilizer ipurities not identie%, ACS) were ob>., Inc. (Milwaukee, ;oenzyme A, dithioA) and palmitoyl co-i Chemical Co. (St. were obtained from V). Wy-14.643 (WY) Laboratories (Rad- (F-344)/Crl BR) rats ) mice from Charles Cingston, NY), were ind found to be free irus titer screen (rat/ 'ological Associates, d in polycarbonate " 2F and 50 10% cle. NIH-07 pelleted / libitum throughout tents, rats and mice ctively. aged (I ml per rat; dissolved in com oil lissolved in a 1:9 di mixture. Dosages of 'kg body wt), PER lg/kgbodywt), TCA ;g body wt). Control ume ofcom oil vehi- iparison studies, the administered to rats r com oil or methyl Is received no treathe vehicle alone, mmstration studies. E (1000 mg/kg body CE + PER (separate + 1000 PER mg/kg following one anmsecutive days. All st dose. oov. Animals were vtlurane. and killed idney organ weights CHLORINATED HYDROCARBON PEROXISOME RESPONSE 227 TABLE 1 Effects of Chlorinated Hydrocarbon Ga vage Treatment on Hepatic Peroxisomal Enzyme Activities of Male F344 Rats11 Liver wt/body wt CN'-insensitive palmitoyl CoA oxidation Treatment %of %of n Ratio control nmol/mg protein control Com oil 5 3.68 + 0.06* Wy-14,643 (50 mg/kg) 5 7,18 0.11c Trichloroethylene (1000 mg/kg) 5 4.52 0.08'*' Perchloroethylene (1000 mg/kg) 5 4.39 0,07"*' Pentachloroethane (150 mg/kg) 5 4.27 0.09' *' Trichloroacetic acid (500 mg/kg) 6 5.18 0.1 r1' 195 122 119 116 141 3.73 0.35 20.94 2.22c 6.72 0.25 5.28 0.31*' 4.73 0.32*' 10.58 + 0.41 c'd 561 180 142 127 284 " Male Fischer 344 rats were administered agents by gavage in com oil for 10 consecutive days. Control animals received com oil vehicle alone. 4 x + SE of n animals. c Significantly different from com oil control, p < 0.05, by Newman-Keuls "multicompare" test. d Significantly different from Wy-14,643, P < 0.05, by Newman-Keuls "multicompare" test. ' Significantly different from trichloroacetic acid, p < 0.05, by Newman-Keuls "multicompare" test. were taken, tissues from the left lobe of the liver and the kidney cortex were processed for enzyme analysis. Tissue from the left lobe of liver was used since no preferential peroxisome enzyme increase was seen in various liver lobes to either WY or nafenopin treatment in previous studies (T. L. Goldsworthy, and J. A. Popp, unpublished data). Tissues were placed in glass tubes with ice-cold 0.154 M KC1/50 mM Tris-HCl buffer, pH 7.4, at a vol ume ratio of 1:4-5 of liver to buffer and of 1:2-3 of kid ney to buffer. Samples were then homogenized by two strokes ofa rotating Teflon pestle using a Braun homogenizer, and 1-ml samples were frozen at -20`C. Palmitoyl CoA oxidase activity, a measure of peroxi somal d-oxidation activity, was measured, using a modi fication (Butterworth et al.. 1984) of Lazarow's method (1981), in supernatants ofthawed samples centrifuged at 2500# for 5 min at -4*C. Briefly, peroxisomal d-oxida tion was measured in liver and kidney homogenates as palmitoyl CoA-dependent reduction of NAD* in the presence of CN-. Assay media for kidney samples were identical to those described for the liver except for a 5fold increase in the concentration of palmitoyl CoA and a 1,6-fold increase in KCN concentration, conditions that were shown to increase the sensitivity of the assay. Tissue test samples were diluted in buffer (liver 1:20, kid ney 1:2) and analyzed using a 1:101 sample to total vol ume ratio in an Abbott VP Bichromatic Analyzer. The protein content of the samples was determined by the biuret method using a commercially available kit (Ab bott Laboratories). Enzyme activity was measured as nmol NAD* reduced min*1 * mg protein-1 (nmol/mg). The Newman-Keuls "multicompare" test was used for comparisons between test groups at a significant p value ofp < 0.05. RESULTS In Experiment 1, the administration of test agents by gavage to rats and mice for 10 days had little or no effect upon the body weight gain of the animals compared to control ani mals with the exception of WY-treated rats which demonstrated no body weight gain (data not shown). All agents significantly in creased the liver to body weight ratio com pared to controls in rats; WY > TCA > TCE - PER = PENT (Table 1). Only WY caused a significantly increased rat kidney to body SL 034556 228 GOLDSWORTHY AND POPP TABLE 2 Effects of Chlorinated Hydrocarbon Gavage Treatment on Renal Peroxisomal Enzyme activities of Male F344 Rats" Kidney wt/body wt CN -insensitive palmitoyl CoA oxidation Treatment % of %of n Ratio control nmol/mg protein control Com oil 5 0.75 +0.014 0.80 + 0.13 Wy-14,643 (50 mg/kg) 5 0.90 0.02' 120 7.66 + 0.25' Trichloroethylene (1000 mg/kg) 5 0.82 0.02 109 2.39 + 0.20'"-' Perchloroethylene (1000 mg/kg) 5 0.79+0.01" 105 1.39 +0.09 Pentachloroethane (150 mg/kg) 5 0.78 + 0.01" 104 0.42 +0.09 Trichloroacetic acid (500 mg/kg) 6 0.80 + 0.01" 107 1.40 + 0.19" 958 300 174 53 175 " Male Fischer-344 rats were administered agents by gavage in com oil for 10 consecutive days. Control animals received com oil vehicle alone. 4 x SE of n animals. c Significantly different from com oil control, p < 0.05, by Newman-Keuls "multicompare" test. " Significantly different from Wy-14,643, p < 0.05, by Newman-Keuls "multicompare" test. ' Significantly different from trichloroacetic acid, p < 0.05, by Newman-Keuls "multicompare" test. f Significantly different from trichloroethylene, p < 0.05, by Newman-Keuls "multicompare" test. g Significantly different from perchloroethylene, p < 0.05, by Newman-Keuls "multicompare" test. ratio (Table 2). All agents except PENT sig nificantly increased the liver to body weight ratio in mice compare to controls (Table 3). In contrast, no increases in the kidney tobody weight ratio were observed in treated mice (Table 4), As anticipated, increases in hepatic cya nide-insensitive palmitoyl CoA oxidation, compared to controls, were seen following administration of WY (561% of control) and TCA (284% of control) to rats (Table 1). He patic PCO activity was not changed in rats following TCE, PER, and PENT treatments. WY (958% of control value) and TCE (300% of control) caused an increase in renal PCO activity in rats (Table 2). The TCE-induced PCO activity was significantly higher than that seen following PER and PENT ad ministration. PCO activity was significantly elevated in the livers of mice in all treatment groups ex cept PENT (Table 3). PCO responses varied between treatment groups: WY > TCE > PER > TCA = PENT. All chemicals increased PCO activity in the kidneys of mice (Table 4). The elevated PCO response was similar in all treatment groups (232 to 360% of controls) with the exception of WY (689% of controls). In Experiment 2, rats were gavaged with TCE, PER, TCA, or WY in methyl cellulose or com oil for 10 days to determine if there was a vehicle effect of the administration of these chemicals on rat liver and kidney PCO activity. PCO activity compared to respective control values was similar in rat liver and kid ney when chlorinated hydrocarbons were ad ministered in either com oil or methyl cellu lose. In general, the data in the vehicle com parison studies confirm the results from the previous experiment (Tables 1 and 2) for the induction of PCO activity compared to con Trea Com oil Wy-14,643 Trichloroe- (1000 mi Perchloroe-. (1000 mi Pentachlon (150 mg/ Trichloroac (500 mg/: 0 Male B( received cor *xSEc 'Significa " Significa ' Significa ^Significa: * Significa trol values contrast tc rat liver weak, wer to control duced PCC lose alone 1 In Expe with TCE PCO respo agent adm effects of ct rinated hyd zyme activi and mice, concurrent was not adi differ from PER alone, or PER, re: OMAL ,ive palmitoyl adation %of n control 958 300 174 g 53 175 s. Control animals est, Rt. test. esponses varied WY > TCE O activity in the te elevated PCO eatment groups th the exception re gavaged with methyl cellulose termine if there iministration of tnd kidney PCO red to respective rat liver and kidjarbons were ador methyl celluthe vehicle cornresults from the s 1 and 2) for the ompared to con CHLORINATED HYDROCARBON PEROXISOME RESPONSE 229 TABLE 3 Effects of Chlorinated Hydrocarbon Gavage Treatment on Hepatic Peroxisomal Enzyme activities of Male B6CF31 Mice" Liver wt/body wt CN`-insensitive palmitoyl CoA oxidation Treatment %of %of n Ratio control ng/mg protein control Com oil 7 4.55 0.13* 1.77 0.34 Wy-14,643 (50 mg/kg) 7 8.81 0.37' 194 21.65+ 1.59' 1223 Trichloroethylene (1000 mg/kg) 7 6.83 0.13' "' 150 11.07 + 0.5 r*' 625 Perchloroethylene (1000 mg/kg) 7 6,48 0.1 V-d 142 7.58+ 1.34t*ft/ 428 Pentachloroethane (150 mg/kg) 7 5.06 0A2d`*fs 111 2.79 + 0.37*^ 158 Trichloroacetic acid (500 mg/kg) 8 6.36 0.10'"* 140 4.96 + 0.38'1' 280 "Male B6C3F1 mice were administered agents by gavage in com oil for 10 consecutive days. Control animals received com oil vehicle alone. 4xSE of n animals. ' Significantly different from com oil control, p < 0.05, by Newman-Keuls "multicompare" test. d Significantly different from Wy-14,643, p < 0.05, by Newman-Keuls "multicompare" test. ' Significantly different from trichloroacetic acid, p < 0.05, by Newman-Keuls "multicompare" test. fSignificantly different from trichloroethylene, p < 0.05, by Newman-Keuls "multicompare" test. * Significantly different from perchloroethylene, p < 0.05, by Newman-Keuls "multicompare" test. trol values in the livers and kidneys of rats. In contrast to Experiment 1, the TCE-induced rat liver PCO values, although relatively weak, were significantly elevated compared to control values and differed from PER-induced PCO activity. Com oil or methyl cellu lose alone had no effect on PCO. In Experiment 3, animals were gavaged with TCE + PER daily for 10 days and the PCO response was compared to that of each agent administered alone to examine the effects of concurrent administration of chlo rinated hydrocarbons on the peroxisome en zyme activity in the livers and kidneys of rats and mice. The PCO response observed by concurrent administration of TCE -I- PER was not additive or synergistic and did not differ from that observed with either TCE or PER alone. The values for TCE + PER, TCE, or PER, respectively, represented as a per centage of PCO activity of control values, are for rat liver 263, 239, 167; for rat kidney 319, 261, 87; for mouse liver 460, 625, 428; and for mouse kidney 232, 360,232. DISCUSSION In an attempt to understand the carcino genicity of chlorinated hydrocarbons, many studies have focused on the metabolism of the chemicals and the potential role of DNA interaction. It has been suggested that the in teraction of reactive metabolites of these agents with critical cellular macromolecules, in particular DNA, may result in somatic mutations and subsequent carcinogenesis (Greim et al., 1975; Simmon et al.. 1977). However, the low level or lack of binding of metabolites to DNA in vivo and the lack of SL 034558 230 GOLDSWORTHY AND POPP TABLE 4 Effect of Chlorinated Hydrocarbon Gavage Treatment on Renal Peroxisomal Enzyme activities of Male B6CF31 Mice" Kidney wt/body wt CN~-insensitive palmitoyl CoA oxidation Treatment %o( %of n Ratio control nmol/mg protein control Com oil 7 1.42 + 0.03 4 Wy-14,643 (50 mg/kg) 7 1.49 + 0.08 Trichloroethylene (1000 mg/kg) 7 1.33+0.05 Perchloroethylene (1000 mg/kg) 7 1.36 0.04 Pentachloroethane (150 mg/kg) 7 1.38 + 0.05 Trichloroacetic acid (500 mg/kg) 8 1.40 + 0.03 104 94 96 97 98 1.11 0.29 7,65 0.56r 4.00 0.35'-tf 2.57 0.21 c'" 3.44 0.40^ 3.38 0.23 ^ 689 360 232 310 305 " Male B6C3F1 mice were administered agents by gavage in com oil for 10 consecutive days. Control animals received com oil vehicle alone. 4 x SE ofn animals. f Significantly different from com oil control, p < 0.05, by Newman-Keuls "multicompare" test. 4 Significantly different from Wy-14,643, p < 0.05, by Newman-Keuls "multicompare" test. overtly detectable genotoxicity of these chemicals (Bolt and Filser, 1977; Schumann et al., 1980; Parchman and Magee, 1982; Stott et al., 1982) does not support the above hypothesis and does not implicate epigenetic mechanisms for the carcinogenicity ob served. In this light it has been suggested that compounds such as TCE and PER enhance the high background incidence of hepatocel lular carcinoma found in B6C3F1 mice through sustained hepatotoxicity and resul tant cell division (Schumann et al., 1980; Stott et al., 1982). On the other hand, the re cent identification in rat urine of small amounts of mercapturic acids following ad ministration of TCE and PER suggests an al ternative activation pathway in the rat the in termediates of which are mutagenic and may thus cause cellular damage (Dekant and Henschler, 1986). Studies that evaluated the role of metabo lism to account for the species difference in the hepatocarcinogenicity of TCE and PER have demonstrated that mice are more active than rats in metabolizing these agents (Schu mann et al., 1980; Stott et al., 1982; Green and Prout, 1985; Prout et al., 1985). One study has suggested that the species difference in the hepatocarcinogenicity of TCE in rats and mice is due to a species difference in per oxisomal proliferation (Elcombe et al., 1985). Although peroxisome proliferation has been linked to neoplasia in rodents, the mechanism(s) involved in the development of tu mors has not been established. One hypothe sis suggests that the carcinogenic response of peroxisome-proliferating agents involves the interaction of increased steady-state concen trations of HjOt, or other reactive oxygen species resulting from carcinogen-stimulated peroxisomal metabolism, with critical cellu lar macromolecules (Reddy and Lalwani, 1983). However, there is no evidence that peroxisome-proliferating agents are reactive with DNA (von Daniken et al., 1983, 1984; Goel et al., 1985; Gupta et al., 1985). SL 034559 XISOMAL nsitive palmitoyl X oxidation itein (C , c,d C,(t , c.d c,d % of control 689 360 232 310 305 iys. Control animals est. : are more active ;se agents (Schual,, 1982; Green ai, 1985). One pecies difference of TCE in rats Jifference in pernbtetal., 1985). ration has been nts, the mediaelopment of tud. One hypotheenic response of nts involves the iy-state concenreactive oxygen ogen-stimulated th critical cellu and Lalwani, o evidence that nts are reactive al.. 1983, 1984; , 1985). CHLORINATED HYDROCARBON PEROXISOME RESPONSE 231 To further test the hypothesis that peroxi some proliferation may be associated with the carcinogenicity of chlorinated hydrocar bons (Elcombe et ai, 1985), we studied the PCO activity following administration of three chlorinated hydrocarbons and com pared this response to their species and organspecific carcinogenicity. Induction of PCO activity correlated to increased organ to body weight ratios. Of the chlorinated hydrocar bons tested, TCE and PER elevated PCO ac tivity in mouse liver whereas only TCE ele vated rat liver PCO activity. PENT resulted in a slight but not significant increase in the liver PCO. Unexpectedly, TCE increased mouse liver PCO activity more than TCA. The peroxisome proliferative response of TCE and PER, coupled with an increased cell proliferation observed in the mouse following administration of TCE and PER (Schumann etal., 1980; Stott et ai, 1982; Elcombe et ai, 1985), may be an additional important factor in the development of mouse hepatic tumor. Since the relationship between the extent of peroxisome proliferation and the carcino genic response observed in rodents has not been established, it is not clear if peroxisome proliferation plays any role in PENT-induced hepatocarcinogenicity. The TCE-induced rat liver PCO response, albeit weak (1.8- to 3.2fold increase over controls), was somewhat contrary to a previous study (Elcombe et al., 1985) which did not demonstrate an in creased rat liver PCO response to TCE expo sure. The use ofdifferent rat strains in the two studies or potential experimental variability may explain the differences observed. If he patic tumor formation in rodents requires both liver cell hyperplasia and increased per oxisome proliferation following TCE expo sure, then the lack ofTCE-induced cell prolif eration in rat liver (Elcombe# al., 1985) may explain the lack of hepatocellular carcinoma development in the rat. In these studies none of the chlorinated hydrocarbons induced a PCO response as strong as that by potent per oxisome proliferator and hepatocarcinogen Wy-14,643 in either species or in either organ in any of the experiments reported herein. PCO data for the concurrent administra tion of TCE + PER did not show additive or synergistic effects. This result was somewhat unexpected in mouse liver since mice had shown a linear relationship up to 2000 mg/ kg between dose of TCE and amount of TCA formed (Prout et al.. 1985). However, prefer ential metabolism of TCE at the dose levels used may in part explain the lack of an addi tive effect following combined TCE and PER treatment. Schumann et al. (1980) reported that only 17% of a 500-mg/kg/day dose of PER was metabolized in mice. Additive dose concentrations of TCE and PER greater than 1000 mg/kg in the rat would not be expected to increase the concentration of TCA in the liver (Prout et al., 1985) and thus would not be expected to increase hepatic PCO activity. There does not appear to be a relationship between chlorinated hydrocarbon-induced PCO activity and the renal-specific carcino genicity. However, the very low incidence of renal tumors induced by chlorinated hydro carbons makes it very difficult to establish a cause and effect relationship between treat ment-related changes in peroxisomes and re nal carcinogenicity. All agents increased mouse kidney PCO but only TCE treatment elevated rat kidney PCO activity. In contrast to these PCO results, an increased incidence of renal tubular adenocarcinomas was ob served only in the male rat following admin istration of these agents. However, it is possi ble that a significant PCO activity in rat kid ney was masked by the heterogeneity of cell types found in the kidney cortex. The PCO activity observed in the kidney following TCE treatment may be the result of strong in duction of peroxisomes in a specific renal tu bular cell population. Factors such as cell rep lication may be necessary along with an in creased peroxisomal response before such changes affect the neoplastic response. In this context, a recent report (Goldsworthy et al., 1986) noted that protein droplet accumula SL 034560 232 GOLDSWORTHY AND POPP tion and increased cell replication were ob served following short-term treatment with PER and PENT but not with TCE. Under conditions similar to the chronic bioassay studies (i.e., animal strains, dose), our results support an association between in creased PCO activity and the development of TCE- and PER-induced hepatocellular carci noma in mice. However, the weak response observed in mouse liver following PENT sug gests that other factors are involved in the de velopment of species-specific hepatocarcinogenicity following chlorinated hydrocarbon administration. The role of the metabolite TCA with peroxisome proliferation and tu mor induction in rats and mice remains to be established. It is clear, with the possible ex ception of TCE, that chlorinated hydrocar bon-induced peroxisome enzyme activity does not correlate with the species-specific re nal carcinogenicity of these compounds. ACKNOWLEDGMENTS The authors thank Douglas A. Neptun and Otis Lyght for excellent technical assistance, and Diane Doach and Linda Smith for typing the manuscript. REFERENCES 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. 42, 501518. Bolt. H. M,, and Filser, J. G. (1977). Irreversible bind ing of chlorinated ethylene to macromolecules. Envi ron. 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