Document 93aNR7X7oo3pkVJpyRV41MVVq

if*. (1986). ; pedfidty i aion oxi- T,, Gor'aN Dern Middle |514. ' < u t,, jj NO GOR;Jine *lkaand anaor agents. F., Moot, 18). Semiantitumor 31,1520- 3 .1 TOXICOLOGY AND APPLIED PHARMACOLOGY 101, 285-298 (1989) Species and Strain Sensitivity to the Induction of Peroxisome Proliferation by Chloroacetic Acids1 A. B. DeAngelo,2 F. B. Daniel, L. McMillan, P. Wernsing, and R. E. Savage, Jr.3 Biochemical and Motecular Toxicology Branch. Genetic Toxicology Division, Health Effects Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio45268 Received Febi Species and Strain Sensitivity t( n by Chloroacetic Acids. DeAngelo, A. B,, Daniel tD Savage, R. E., Jr. (1989). Toxicol. Appl. Pharm aguc-Dawley rats were provided drinking water con 39 mM (1-5 g/liter) dichloroaoeti *"**- tic acid (TCA), 8I monochloroacetic acid (MCA) for 14 days. TCA and ____________ (/ relative liver weight in a dose-dependent manner. Rat liver weights were not altered by TCA or DCA treatment, but were depressed by MCA. Hepatic peroxisome proliferation was demonstrated by (1) increased palmitoyl-CoA oxidase and carnitine acetyl transferase activities, (2) appearance of a peroxi some proliferation-associated proton, and (3) morphometric analysis ofelectron micrographs. Mouse peroxisome proliferation was enhanced in a dose-dependent manner by both TCA and DCA, but only the high DCA concentration (39 mM) increased tat liver peroxisome prolifera tion. MCA was ineffective in both species. Three other mouse strains (Swiss-Webster, C3H, and C57BL/6) and two strains of rat (F344 and Osbome-Mendel) were examined for sensitivity to TCA. TCA (12 and 31 mM) effectively enhanced peroxisome proliferation in all mouse strains, especially the C57BL/6. A more modest enhancement in the Osbome-Mendel (288%) and F344 rat (167%) was seen. Dosing F344 tats with 200 mg/kg TCA in water or corn oil for 10 days increased peroxisome proliferation 179 and 278%, respectively, above the vehicle controls. These studies demonstrate that the mouse is more sensitive than the rat with respect to the enhancement ofliver peroxisome proliferation by TCA and DCA and suggest that ifperoxisome proliferation is critical forthe induction ofhepatic cancer by TCA and DCA, then the rat should be less sensitive or refractory to tumor induction, c 1989 Academic Pros, inc. Trichloroacetic acid (TCA) and dichloroacetic add (DCA) are present in finished drink ing water as by-products ofthe chlorine disin fection process at concentrations ranging 1 Presented in part at the meeting oftheSocietyofToxicology. New Orleans, Louisiana, March 3-7,1986. This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommen dation for use. 2 To whom correspondence should be addressed. 1 Present address: National Institute of Occupational Safety and Health, 4676 Columbia Parkway, Cincinnati, OH 45226. from 34 to 160 Mg/liter (Uden and Miller, 1983). They result from the reaction be tween chlorine and humic substances in the surface waters (Miller and Uden, 1983; Coleman et al., 1984). TCA and DCA were also found to be the major chlorinated or ganic compounds formed in the stomach of experimental animals following the inges tion of water containing sodium hypochlo rite (Mink el al,, 1983). The widespread ex posure of large segments ofthe human pop ulation to low levels of TCA and DCA prompted an examination ofthe toxicologi cal effects ofthese chemicals. 0041-008X/89 $3.00 Copyrifbt CI9S9 byAodemic Pit**, Inc. All rifhtt ofreproduction in feayfonn reserved- SL 037275 #86). icity oxi- jORDERRklle . T,, jOR alka- seats. Mooe, i. Semititumor . 1520- toxicology and applied pharmacology 101,285-298 (1989) Species and Strain Sensitivity to the Induction of Peroxisome Proliferation by Chloroacetic Acids1 A. B. DeAngelo,2 F. B. Daniel, L. McMillan, P. Wernsing, and R. E. Savage, Jr.3 Biochemical and Molecular Toxicology Branch. Genetic Toxicology Division. Health Effects Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268 Received February 15, 1989: acceptedAugust 8. 1989 Species and Strain Sensitivity to the Induction of Peroxisome Proliferation by Chloroacetic Acids. DeAngelo, A. B,, Daniel, F. B., McMillan, L., Wernsing, P., and Savage, R. E., Jr. (1989). Toxicol. Appl. Pharmacol. 101, 285-298. B6C3F, mice and Sprague-Dawley rats were provided drinking water containing 6-31 mM (1-5 g/liter) trichloroacetic acid (TCA), 839 mM (1-5 g/liter) dichloroaoetic acid (DCA), or 11-32 mM (1-3 g/liter) monochloroacetic acid (MCA) for 14 days. TCA and DCA, but not MCA, increased the mouse relative liver weight in a dose-dependent manner. Rat liver weights were not altered by TCA or DCA treatment, but were depressed by MCA. Hepatic peroxisome proliferation was demonstrated by (1) increased palmitoyl-CoA oxidase and carnitine acetyl transferase activities, (2) appearance of a peroxi some proliferation-associated protein, and (3) morphometric analysis of electron micrographs. Mouse peroxisome proliferation was enhanced in a dose-dependent manner by both TCA and DCA, but only the high DCA concentration (39 mM) increased rat liver peroxisome prolifera tion. MCA was ineffective in both species. Three other mouse strains (Swiss-Webster, C3H, and C57BL/6) and two strains of rat (F344 and Osbome-Mendel) were examined for sensitivity to TCA. TCA (12 and 31 mM) effectively enhanced peroxisome proliferation in all mouse strains, especially the C57BL/6. A more modest enhancement in the Osbome-Mendel (288%) and F344 rat (167%) was seen. Dosing F344 rats with 200 mg/kg TCA in water or com oil for 10 days increased peroxisome proliferation 179 and 278%, respectively, above the vehicle controls. These studies demonstrate that the mouse is more sensitive than the rat with respect to the enhancement ofliver peroxisome proliferation by TCA and DCA and suggest that ifperoxisome proliferation is critical for the induction ofhepatic cancer by TCA and DCA, then the rat should be less sensitive or refractory to tumor induction, c 19*9 Academic Press, inc. Trichloroacetic acid (TCA) and dichloroacetic acid (DCA) are present in finished drink ing water as by-products ofthe chlorine disin fection process at concentrations ranging ' Presented in part at the meeting ofthe Society ofTox icology, New Orleans, Louisiana, March 3-7,1986. This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommen dation for use. 2 To whom correspondence should be addressed. 3 Present address: National Institute of Occupational Safety and Health, 4676 Columbia Parkway, Cincinnati, OH 45226. from 34 to 160 (tg/liter (Uden and Miller, 1983). They result from the reaction be tween chlorine and humic substances in the surface waters (Miller and Uden, 1983; Coleman et al., 1984), TCA and DCA were also found to be the major chlorinated or ganic compounds formed in the stomach of experimental animals following the inges tion of water containing sodium hypochlo rite (Mink et al,, 1983). The widespread ex posure of large segments of the human pop ulation to low levels of TCA and DCA prompted an examination ofthe toxicologi cal effects of these chemicals. 285 0041-008X/89 $3.00 Copyright C 1989 by Academic Peat lee. All rightsofreproduction in any form reserved. 286 DEANGELO ET AL. Recent studies from our laboratory (Herren-Freund et al., 1987) have demonstrated that the administration of both TCA and DCA in the drinking water increased the inci dence of liver cancer in male B6C3F| mice. Prior initiation with a genotoxic carcinogen was not required for the TCA and DCA to produce tumors. On the other hand, the chloroacetic acids do not appear to act as tumor initiators by directly damaging DNA since neither TCA nor DCA possessed mutagenic activity when tested with various Salmonella strains (Rapson el al., 1980; Herbert et al., 1980). Likewise the two compounds only weakly increased sister chromatid exchanges in cultured Chinese hamster ovary cells at high concentrations and in the absence of metabolic activation (Meier and Blazak, 1985). Neither TCA nor DCA produced mouse bone marrow sister chromatid ex changes or micronucleus formation in vivo (J. Meier, personal communication), and TCA failed to initiate enzyme-altered foci in rat liver (Parnell et al., 1986). One notable excep tion is the study of Nelson and Bull (1988) who, using an alkaline unwinding assay, found that TCA and DCA increased the number of DNA single-strand breaks in the liver of B6C3Fj mouse and the SpragueDawley rat. Our interest in the mechanism by which TCA and DCA enhance liver cancer has fo cused on several parameters including the ability of the chloroacetic acids to stimulate hepatic peroxisome proliferation in rats and mice. Since many peroxisome proliferators have been found to increase the incidence of liver cancer in rodents, these agents have been proposed to constitute a unique class of chemical carcinogens (Reddy et al., 1980a). The mechanism(s) by which peroxisome proliferator chemicals induce liver neoplasia is not well defined. One theory is that the heightened production and subsequent me tabolism of hydrogen peroxide and other ox ygen metabolites resulting from the activities of peroxisomal enzymes initiate DNA strand breakage, crosslinkage, and amelorative re pair processes which may yield mutations and chromosomal aberrations (Cadet and Teoule, 1978). The objective of this study was to measure the ability of TCA and DCA to stimulate he patic peroxisome proliferation in several strains ofrats and mice. In addition, the effect of the vehicle (com oil vs water) on the TCA induction of hepatic peroxisome prolifera tion was measured in order to compare the results from these studies with those already in the literature. MATERIALS AND METHODS Chemicals Trichloroacetic acid (CAS No. 76-03-9), dichloraacetic acid (CAS No. 79-43-6), and monochloroacetic acid (CAS No. 79-11-8) were purchased from Aldrich Chemi cal Co., Inc. (Milwaukee, WI). The chloroacetic acids (purity > 99%) were dissolved in distilled water at con centrations between I and 5 g/liter (6-39 mM), and the pH was adjusted to 6.8-7.2 by the addition of an appro priate volume of 10 N sodium hydroxide. All solutions were prepared at the beginning of each study and stored at S'C. They were administered to the animals in glass water bottles fitted with teflon stoppers and doubleballed sipper tubes. Drinking waters were changed weekly. All reagents used in the preparation of liver ex tracts, analysis of peroxisomal enzymes, and gel electro phoresis, unless otherwise specified, were purchased from Sigma Chemical Co. (St. Louis, MO). Animals The various strains ofyoung adult male rats (SpragueDawley, Osbome-Mendel, and Fisher 344) weighing 225-275 g and male mice (B6C3F,, C57BL/6, C3H, and Swiss-Webster) weighing 20-25 g were purchased from Charles River Laboratories (Portage, MI). The animals were maintained on Purina Laboratory Chow and water ad libitum at 22 2*C and 40-60% humidity under a 12hr light-dark cycle. Following 2 weeks in quarantine, the animals were divided into treatment groups and the ex posure to the various concentrations of chloroacetic acids (6-39 mM) in the drinking water was started. Con trol animals were given drinking watercontaining 34 mM sodium chloride. After 14 days, the animals were killed by C02 asphyxiation and the body and liver weights were recorded. The livers were quickly removed and minced and pieces were preserved in 2.5% glutaraldehyde for ex- SL 037277 TCA, DCA, MCA: SPECIES SENSITIVITY 287 animation by electron microscopy. The remainder of each liver was quick-frozen in liquid nitrogen and stored at --70*C until it could be processed for analysis of per oxisomal enzymes. In order to evaluate the effect of the vehicle upon the induction of peroxisome proliferation by TCA, a group of F344 rats was given by gavage 200 mg/kg TCA dissolved in either com oil or distilled water on each of 10 days. The animals were killed 24 hr follow ing the last dose. Peroxisomal Enzyme Analysis Electron Microscopy For the morphometric measurement of peroxisome proliferation, one liver section from each of two B6C3F, mice or Sprague-Dawley rats from the high-dose treat ment groups was randomly selected. The number of per oxisomes, peroxisomal areas, and the cytoplasmic area in six electron micrograph fields from each section (12 fields/treatment group) were measured using a Bioquant System IV image analysis system. The number of peroxi somes per 100 am3 of liver and volume percentage of cytoplasm occupied by the peroxisomes were calculated according to the methods described by Loud (1968). Portions of the frozen livers were homogenized (1:10 w/v) in a buffer containing 0.25 M sucrose, 0.05 m so dium EDTA, and 0.02 m Tris-HCl, pH 7.4. The homog enates were centrifuged at 800# for 5 min, the fatty layers were removed by aspiration, and the extracts were stored at -70'C until assay. Previous work had shown that the enzyme activities in the frozen extracts did not differ sig nificantly from the activities in liver extracts not frozen prior to assay. Cyanide-insensitive palmitoyl-Coenzyme A (PCO) activity was measured according to the method ofOsumi and Hashimoto (1978). Carnitine acetyl-CoA transferase (CAT) activity was assayed according to the method ofGray etal.( 1982). Gel Electrophoresis The induction of the 80,000 kDa peroxisome prolifer ation-associated (PP-A) protein (Reddy and Kumar, 1977) in the liver extracts from rats and mice exposed to the highest drinking water concentrations of the chloroacetic adds (31 dim TCA, 39 mM DCA, and 32 mM MCA) was demonstrated by polyacrylamide gel electro phoresis (PAGE) using the procedure of Laemmli (1970) as described by Irwin and Dauphinais (1979). A 10% acrylamide separating gel was overlayed with a 3% stack ing gel. The liver extracts (30 Mg protein) were made up to 100 m! with sample buffer (final concentrations: 0.01 M Tris-HCl, pH 6.8, 1% 2-mercaptoethanol, 2% sodium dodecylsuliate, 6% sucrose, 0.001% bromphenyl blue) and heated in a boiling water bath for 5 min. The samples were loaded into the wells, and electrophoresis was car ried out at constant amperage and temperature (30 idA/ gel; 20'C) in a vertical slab gel electrophoresis unit (Hoefer Scientific Instruments, San Frandsco, CA) until the bromphenol blue dye front had moved approxi mately 11 cm into the separating gel. The gels were re moved and stained overnight in a solution containing 0.05% Coomassie brilliant blue R (Serva Fine Biochemioals, Inc., Heidelberg, Federal Republic of Germany), 25% 2-propanol, and 10% acetic acid. The gels were thor oughly destained with a solution containing 25% 2-pro panol and 10% acetic acid. Statistical Analysis Statistical analysis ofthe data was carried out using the Tukey means multiple comparison or the one-way anal ysis of variance. RESULTS Although water consumption measure ments were not made during this study, ear lier analysis in our laboratory had shown that rats and mice will limit their water consump tion with increasing concentrations of the chloroacids. The calculated average daily in takes in mg/kg body wt/day for the B6C3Fi mouse over a 14-day period were: 265, 386, and 482 for a drinking water containing 11, 21, and 32 mM MCA; 90, 166, and 346 for 8, 16, and 39 mM DCA; and 131, 261, and 442 for 6, 12, and 31 g/liter TCA. For the Sprague-Dawley rat the 14-day average daily intakes were: 170, 321, and 501 for 11, 21, and 32 mM MCA; 166, 294, and 666 for 8, 16, and 39 mM DCA; and 212,327, and 719 for 6, 12, and 31 mM TCA. Body and Liver Weight Changes Table 1 shows the effect of a 14-day drink ing water administration to the three chloroacetic acids on the body weights and liver weights (expressed as percentage of the body weight) of male B6C3F! mice and SpragueDawley rats. Rats drinking water containing the three acids experienced a dose-dependent SL 037278 288 DEANGELO ET AL. TABLE 1 Body Weight and Liver Weight Changes for SD RatsandB6C3F1 Mice Given TCa, DCA, or MCA in Their Drinking Water for 14 Days Chloroacetic acid Drinking water concentration Body weight" (g) Liver weight (g/lOOgbody wt) TCA (6)fc (6) (6) DCA (3) (5) (5) MCA (5) (6) (6) NaCl (6) Sprague-Dawley rat 31 mM 21 mM 6 mM 39 mM 16 mM 8 mM 32 mM 21 mM 11 mM 34 mM 285 + 14*+ 319+15 304 + 8* 262 + 16** 270 22" 282 20* 194 10" 220+ 10" 283+ 7" 341 6 4.0+ 0.4 4.4 0.4 3.8 0.4 4.7 0.9 3.8 0.2 4.7 0.4 3.1 0.1* 3.3 0.1* 3.5 0.2* 4.4 0.4 TCA (6) (6) (6) DCA (6) (6) (6) MCA (6) (6) (5) NaCl (6) B6C3F1 mouse 31 mM 12 mM 6 mM 39 mM 16 mM 8 mM 32 mM 21 mM 11 mM 34 mM 21 1 21+0 22 0 21 + 1 21 + 1 20+ 1 20+ 1 20 1 21 + 1 21+3 7.1 0.4" 5.9 0.3 5.5 0.2 7.9 0.3" 6.5 0.2" 6.2 0.1" 4.9 0.1 5.2 0.1 5.2 0.2 5.1 0.2 * Average SEM. * (N) = the number ofanimals surviving and used for analysis. cp< 0.05 (*) or p < 0.01 (**) as determined by the Tukey means multiple comparison test when compared to the NaO control. depression in body weight gain. The severity of the weight gain depression was greatest for MCA (57% of the control value at 32 mM) and least for TCA (84% ofcontrol at 31 mM). The depression ofthe growth rate by the high concentration DCA (39 mM) was intermedi ate (75% of control). TCA and DCA did not affect the relative liver weights. However, MCA treatment depressed liver to body weight ratios and these declines were dose de pendent. In marked contrast, the body weight gains ofthe B6C3F, mice were not altered by drinking water containing any of the three chloroacetic acids. However, DCA and to a lesser extent TCA treatment did increase rela tive liver weights in the mice. MCA in the drinking water had no effect on the relative liver weights of the mice. Alteration ofPeroxisomal Enzyme Activities The effects of the chloroacetic acids on CAT activities in the Sprague-Dawley rat and B6C3F, mouse are shown in Figs. 1A (rat) and 1B (mouse). MCA did not alter CAT activity in either rats or mice at any of its drinking water concentrations (11-32 mM). SL 037279 290 DEANGELO ET AL. DRINKMG WATER CONCENTRATION (Mil Fig. 2. The alteration ofcyanide-insensitive palmitoyl-CoA oxidase activity in the livers of(A) rats and (B) mice by TCA (), DCA (O), and MCA (a), in the drinking water. The enzyme activity for control groups SE were 3.91 0.23 nmoles NAD reduced/min/mg protein for rats and 1.40 0.34 nmoles of NAD reduced/min/mg protein for mice. N = 5 or 6 for all groups except rat on 39 mM DCA where N = 3. *p < 0.05 as determined by the Tukey means multiple comparison test when compared to the NaCl control. tic acids. Only the highest concentration of DCA (39 mM) in the drinking water in creased the activity of this marker enzyme (234% of the control value, Fig. 2A). The mouse, on the other hand, responded with an increased PCO activity to both TCA and DCA (Fig, 2B). TCA was the more potent of the two acids. At 31 mM it increased the PCO activity to 959% of the control value in a dose-dependent manner; this compared to a 430% increase seen for the highest concentra tion of DCA (39 mM). No statistically sig nificant increases in PCO activity were mea sured at the lower DCA concentrations. MCA did not alter PCO specific activity in either species. Gel Electrophoresis SDS-polyacrylamide gel electrophoresis was also used to evaluate liver peroxisome proliferation following chloroacetic acid treatment by demonstrating the presence of a SL 037281 TCA, DCA, MCA: SPECIES SENSITIVITY 291 Fig. 3. Polyacrylamide gel electrophoretic separation of proteins in liver extracts derived from rats and mice treated for 14 days with 31 mMTCA, 39 mM DCA, 32 mM MCA, or 34 mM NaCl (HjO). Ttypsinogen (24 kDa), ovalbumin (45 kDa), and bovine albumin (66 kDa), were corun as molecular weight markers. PP-A protein (Fig. 3). No PP-A protein band was seen for control or MCA (32 mM)-treated mice and rats, or for TCA (31 mM)-treated rats. TCA induced the PP-A protein only in the mouse, while DCA (39 mM) was active in both species. The molecular weight ofthe PPA protein was determined by comparing its migration to those of marker proteins. A mo lecular weight of 74,000 kDa was obtained compared to the value of 80,000 kDa re ported in the literature (Reddy et ai, 1980b). Quantitative Morphometry The enhancement of peroxisome prolifera tion by the highest concentration ofthe chloroacetic acids was scored by determining the number of peroxisomes per unit volume of cytoplasm and calculating the volume per centage of the cytoplasm they occupied. A cellular structure was identified as a peroxi some if it met three of the four following cri teria: (1) it was round to oval in shape; (2) had a single limiting membrane; (3) contained granular material; and (4) had an electron dense nucleoid(s). If any structure possessed a double limiting membrane it was excluded, even if the other three criteria were met. The variable size of the peroxisomes could be re lated to sectioning or to the stage of develop ment of the peroxisome. Good agreement was obtained between the morphometric measurements (Table 2) and the two other in dicators of peroxisome proliferation. In the rat, only DCA increased the peroxisome number per 100 Min3 and the volume per centage ofcytoplasm occupied. In the mouse, both TCA and DCA increased the peroxi some number per moi3 (30.75 2.80 and 30.77 2.85, respectively, vs 6.89 1.23 for the control) and the volume percentage ofthe SL 037282 292 DEANGELO ET AL TABLE 2 Morphometric Analysis of Hepatic Peroxisome Proliferation Induced by Chloroacetic Acids Treatment Number of peroxisomes per 100 iitn1 cytoplasm Volume fraction ofcytoplasm occupied by peroxisomes 31 mMTCA 39 mM DCA 32 mM MCA 34 mM NaCl Sprague-Dawley rat 7.13 1.13" 16.75 2.05* 8.32 0.69 6.60 0.75 0.89 0.17* 2.80 + 0.31* 2.27 0.26 1.94 0.72 31 mMTCA 39 mM DCA 32 mM MCA 34 raM NaCl B6C3F1 mouse 30.75 2.80* 30.77 2.85* 16.67 3.33 6.89 1.23 4.92 0.54* 3.75 0.46* 1.02 0.17 0.61 0.12 " Average SEM for 12 fields examined (6 fields/liver section). * p < 0.05 as determined by the analysis of variance when compared to the Nad control. cytoplasm occupied by peroxisomes (4.92 0.54 and 3.75 0.46, respectively, vs 0.61 0.12 for the control). MCA slightly in creased the peroxisome number, but not to a level of significance (p < 0.05). Taking all the data together, the conclusion may be drawn that the Sprague-Dawley rat is relatively insensitive to the induction of per oxisome proliferation by the chloroacetic acids since only the highest concentration of DCA (39 mM) for 14 days increased the vari ous markers of peroxisomal proliferation. In contrast, both DCA and TCA were active in the B6C3Fi mouse with TCA appearing to be more potent than DCA. MCA was ineffective in increasing liver peroxisome proliferation in both rodent species. TCA Induction of Peroxisome Proliferation in Other Rat and Mouse Strains two other strains of rat, Osborne-Mendel and F344, and four mouse strains, Swiss-Webster, B6C3F,, C57BL/6, and C3H. The latter two are the parental strains of the B6C3F, hy brid strain. Groups of six animals were given 34 mM NaCl, 12 mM TCA, or 31 mM TCA in the drinking water for 14 days. TCA treat ment depressed the body weight of the F344 rat but the effect was not significant (94% of the control value). Only a slight increase (112%) in the relative liver weight for the F344 rat was recorded (Table 3). Dose-dependent increases in the relative liver weights were obtained for all mouse strains. The mag nitude of the hepatotrophic response was the same for the Swiss-Webster, B6C3F,, and C3H strains (126-138% ofcontrol). A greater hepatotrophic response was observed in the C57BL/6 where the relative liver weight in creased to 164% of the control value at both the low and high TCA concentrations. He patic peroxisome proliferation was moni tored by measuring the PCO activity (Table 4). Unlike the results obtained for the Sprague-Dawley rat, a modest increase was measured for the F344 rat (163% of the con trol value) at the high TCA concentration. A greater response of 238% of the control value for exposure to 31 mM TCA was seen in the Osborne-Mendel rat. Relative to the rat, TCA was a good inducer of PCO activity as sociated with peroxisome proliferation in all of the mouse strains examined. TCA in creased PCO activity in a dose-dependent manner and was equally effective in the C3H, B6C3F,, and Swiss-Webster strains showing increases of746,778, and 749%, respectively, at the 31 mM concentration. The C57BL/6 strain was particularly sensitive to the en hancing effects of TCA with a more than 2000% increase over the control value at both TCA concentrations. Corn Oil Effect on Peroxisome Proliferation The ability ofTCA in the drinking water to The vehicle used for TCA induction ofper alter peroxisome proliferation was tested in oxisome proliferation in the liver of the F344 SL 037283 i TCA, DCA, MCA: SPECIES SENSITIVITY 293 TABLE 3 Body Weight and Liver Weight Changes in Rats and Mice Given TCA in the Drinking Water for 14 Days Species/strain Drinking water concentration Body weight" (g) Liver weight (g/IOOgbody wt) Rat Osbome-Mendd F344 Mouse Swiss-Webster B6C3FI C57BL/6 C3H 31 mM 12 mM Nad 31 mM 12 mM NaCl 31 mM 12 mM NaCl 31 mM 12 mM NaCl 31 mM 12 mM NaO 31 mM 12 mM NaCl 323 7 324 8 333 8 252 3 264 4 269 3 28 1 27 2 28 1 25 1 25+ 1 24 1 24 1 25 1 26 0 24 0 23 1 23 0 5.5 0.2 5.6 0.2 5.6 0.1 5.6 0.1* 5.3 0.2 5.0 0.2 8.6 0.2* 8.0 0.3* 6.8 0.3 8.8 0.2* 8.2 0.2* 6.5 0.2 11.1 0.3* 10.9 0.3* 6.7 0.3 9.1 0.2* 8.3 0.2* 6.6 0.1 * Average SEM; N = 6. * p < 0.05 as determined by the Tukey means multiple comparison test when compared to the NaCl control. rat can have an effect (Table 5). The animals were dosed with 200 mg/kg TCA in either 1 ml distilled water or com oil for 10 days. Com oil and TCA independently increased the PCO activity to 140% and 179%, respec tively, of the water control. The TCA dis solved in the com oil increased hepatic PCO activity to 228% of the com oil alone and 314% ofthe value obtained for the water con trol. DISCUSSION TCA and DCA were more effective en hancers of hepatic peroxisome proliferation in the mouse than in the rat. MCA was inac tive in both species. Following a 14-day expo sure to the chloroacetic acids in the drinking water, three indices of the peroxisome prolif eration, PCO activity, the 80,000 kDa PP-A protein, and peroxisome number and vol ume, were increased in the B6C3F, mouse. Only the highest concentration of DCA (39 mM) increased these markers in the SpragueDawley rat. TCA was stronger than DCA as an inducer of peroxisomes in the mouse. The patterns of DCA and TCA enhance ment of hepatic CAT activity were similar to that seen for PCO except that the response in rats to DCA was stronger. CAT is found pri marily in hepatic mitochondrial fractions where it constitutes approximately 60% ofthe total CAT activity (Markwell et al,, 1973). Peroxisomal CAT activity is about 3-fold lower. In view of the study by Leighton et al. (1982) who reported a 21 -fold increase in mi tochondrial C4-fatty acid specific CAT activ ity compared to a 4- to 5-fold enhancement SL 037284 ss;r.,;BUiawj 294 DEANGELO ET AL. TABLE4 TCA Induction of Peroxisome Proliferation Species/strain Drinking water concentration PCO activity"'' % Control Rat Osbome-Mendel F344 Mouse Swiss-Webster B6C3F1 C57BL/6 C3H 31 mM 12 mM NaCl 31 mM 12 mM NaCl 31 mM 12 mM NaCl 31 mM 12 mM NaCl 31 mM 12 mM NaCl 31 mM 12 mM NaCl 9.65 0.76* 6.26 0.50 4.05 0.32 15.40 1.08* 9.21 0.77 9.46 1.22 47.98 4.44* 24.10 3.19* 6.41 0.36 34.21 2.87* 24.44 1.02* 4.40 0.37 69.55 2.65* 81.45 2.35* 3.14 0.73 19.31 1.64* 11.02 1.34* 2.59 0.59 238 154 100 163 97 100 748 376 100 778 556 100 2213 2592 100 744 425 100 * Activity expressed as nanomoles NAD reduced/min/mg protein. b Average SEM; N = 6. * p < 0.05 as determined by the Tukey means multiple comparison test when compared to the NaCl control. of the peroxisomal enzyme in response to ciprofibrate, further investigation into the subcellular distribution of DCA-induced CAT activity appears warranted. TABLE 5 Corn Oil Effect on Hepatic Peroxisome Induction in F344 RATS Treatment PCO activity" Percent control Corn oil TCAincomoir Water TCA in water" 4.68 0.21* 10.50 0.86* 3.34 0.22 5.97 0.59* 100 228 100 179 " Nanomoles NAD reduced/min/mg protein. b Average SEM; N = 4. c Two hundred milligrams per kilogram po for 10 days. * p< 0.05 as determined by the Tukey means multiple comparison test when compared to the vehicle control. In three other mouse strains, Swiss-Webster, C3H, and C57BL/6 examined TCA was a good enhancer of PCO activity. It was par ticularly effective in the C57BL/6 strain. TCA was a relatively weak inducer of peroxi somes in the two other rat strains, the Osbome-Mendel and F344 rats. Only the high est concentration increased the PCO activity to 238% and 163% of the control, respec tively. These results are in contrast to those reported by Elcombe (1985) who found that 200 mg/kg TCA administered in com oil by gavage for 10 days increased hepatic PCO ac tivity to 650% of the control value in male Alderley Park (Wistar-derived) rats. The esti mated average daily intake for the rats in our study was 712 mg/kg/day. Goldsworthy and Popp (1987) found that male F344 rats given 500 mg/kg TCA in com oil for 10 consecu tive days had a liver PCO activity of 284% SL 037285 TCA, DCA, MCA: SPECIES SENSITIVITY 295 above the control value. Parnell and cowork ers (1986) reported that male Sprague-Dawley rats given 31 mM TCA in the water for up to 6 months showed only a slight increase in the PCO activity (113-119% above the con trol value). Finally, Mather et at, (unpub lished observations) in a 90-day study of the effects of TCA and DCA in the drinking wa ter on the male Sprague-Dawley rat found that 31 mM TCA stimulated hepatic PCO 115% above the control value while DCA in creased it nearly fivefold. The differences observed in these studies relative to the extent ofrat hepatic PCO activ ity by TCA may be explained in part by a consideration of the vehicle used to deliver the compound. In this study, the administra tion of 200 mg/kg TCA via a com oil gavage for 10 days stimulated the PCO activity to 224% of com oil alone. By comparison we observed a 179% stimulation of PCO activity when the same dose ofTCA was given in wa ter. Corn oil alone raised PCO activity to 140% of the value of water alone (p < 0.05). ; corn oil appeared to potentiate the TCA Kncement of hepatic PCO activity (175% s water control, p < 0.05). Both TCA and DCA increased the relative liver weights in the mouse. DCA was a partic ularly effective hepatotrophic agent. With one exception (high dose TCA, F344 strain) no increases in the relative liver weights of rats by the chloroacetic acids were measured. The extent to which the liver enlargement in DCA- and TCA-treated mice is due to hyper plasia or hypertrophy is not known. We re cently found that mouse hepatocyte turn over, as assessed by autoradiography of [3H]thymidine incorporation, was not increased after exposure to DCA concentrations up to 39 mM (5 g/liter) for 4 weeks while hepatocy`omegaly and increased liver weights were ob tained (data not shown). This would suggest that hypertrophy is the main mechanism for the DCA-induced increase in relative liver weight in the mouse. The role played by peroxisome prolifera tion in the carcinogenicity of DCA and TCA, if any, in the mouse liver is not known. It has been proposed that chemicals which enhance peroxisome proliferation increase the level of hydrogen peroxide generation by inducing the activity of several oxidases including the multienzyme system for the /3-oxidation of long-chain fatty acids (Osumi and Hashimoto, 1978; Reddy and Lalwani, 1984). While peroxisome catalase activity is also in creased during peroxisome proliferation, its induction is disproportionately smaller than the rise in hydrogen peroxide production (Reddy et at., 1969). The higher cellular levels of hydrogen peroxide, a relatively mild and stable oxidant, could in turn give rise to the production of more reactive oxygen species (e.g., the hydroxyl radical, OH-, Chance et at., 1979). These active oxygen species (AOS) are potent oxidants of many essential cellular components such as lipids, proteins, and nucleic acids (Tappel, 1975; Fridovich, 1978). It seems clear that AOS produce DNA damage. AOS such as OH and OJ have pro duced DNA strand breaks in bacterial (Hagensee and Moses, 1986; Jmlay et at., 1988) and mammalian cells (Mello-Filho et at., 1984; Bimboim and Kanabus-Kaminska, 1985), mutations (Moody and Hassan, 1982; Levin et at., 1982), chromosomal aberrations (Oya et at,, 1986), and sister chromatid ex changes (Larramendy et at., 1987). Thus there is ample reason to speculate that peroxi some proliferation could lead, via the pro duction of AOS, to the formation of cellular DNA damage and hence the initiation of the neoplastic process (Rao and Reddy, 1987). However, attempts to relate peroxisome proliferation to hepatic DNA damage have not been convincing. For example, Kombrust et at. (1984) reported that a sustained feeding of di(2-ethylhexyl) phthalate did not induce any detectable DNA repair activity in hepatocytes. Similarly, treatment with clofibrate, methyl clofenopate, ordi(2-ethylhexyl) phthalate, all effective peroxisome prolifera- SL 037286 296 DEANGELO ET AL. tors, did not lead to increased DNA strand breaks in liver DNA when evaluated by an alkaline elution assay (Elliot and Elcombe, 1987). Using a structure activity comparison, the observation that DCA is the more potent carcinogen and TCA the more effective in ducer of peroxisomes does not directly sup port peroxisome proliferation as the domi nant mechanism for the hepatocarcinogenicity of the chloroacetic acids. Chemicals which have peroxisome proliferator properties also possess mitogenic and hepatomegalic properties which are also characteristic of many tumor promoters (Beckett et al, 1972; Moody et al, 1977). Several peroxisome proliferators (nafenopen, di(2-ethylhexyl) phthalate) have been shown to increase the incidence of liver cancer in rats which were previously initiated with diethylnitrosamine (Reddy and Rao, 1978). However, the fact that some peroxisome pro liferators increase the incidence of liver neo plasia in rodents previously initiated with a carcinogen does not prove that their role in carcinogenesis is confined to that mecha nism, i.e., enhancement of the growth rate of cells initiated by ambient environmental car cinogens. For example, they may act as com plete carcinogens in that they possess both initiating and promoting activities. It will be interesting to determine the carcinogenic po tential of TCA or DCA in the rat since they do not induce high levels of hepatic peroxi some proliferation nor do they stimulate liver growth. These data encourage the prediction that this species might be refractive to the car cinogenicity of the chloroacetic acids. The same argument can be put forward for MCA in both rodent species. The role, if any, of peroxisome prolifera tive processes in the induction of cancer by the chlorinated acetic acids remains un known. 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