Document Ozw34NggJn6yMMYmZp7m8NmMj

FUNDAMENTAL AND APPLIED TOXICOLOGY 8, 432-442 (1987) ^ ~f ``t* Application of Microencapsulation for Toxicology Studies II. Toxicity of Microencapsulated Trichloroethylene in Fischer 344 Rats Ronald L. Melnick, C. W. Jameson, Thomas J. Goehl, Robert R. Maronpot, Brad J. Collins, Arnold Greenwell, Frank W. Harrington, Ralph E. Wilson, Konrad E. Tomaszewski, and Deepak K. Agarwal National Toxicology Program, National Institute ofEnvironmental Health Services, P. 0. Box 12233, Research Triangle Park, North Carolina 27709 Application of Microencapsulation for Toxicology Studies. II. Toxicity ofMicroencapsulated Trichloroethylene in Fischer 344 Rats. Melnick, R. L., Jameson, C. W., Goehl, T. J., Mar onpot, R. R., Collins, B. J., Greenwell, A., Harrington, F. W., Wilson, R. E., TomasZEWSKi, K, E., AND Agarwal, D. K. (1987). Fundam, Appl. Toxicol. 8, 432-442. Gelatinsorbitol microcapsules containing 44.1% trichloroethylene (TCH) were prepared and mixed in NIH-07 rodent meal diet and provided at microcapsule concentrations of 0 (untreated control group), 1.25,2.5, 5.0, or 10% (equivalent to 0,0.55, 1.10, 2.21, or 4.41% TCE, respectively) to groups of 10 male F344 rats for 14 days. An additional control group received diets containing 5% empty capsules. For comparisons, TCE dissolved in com oil was administered by gavage to different groups of 10 male rats for 14 consecutive days at dose levels adjusted to correspond to those in the feed study. Treatment-related deaths occurred only in the highest dose group ofthe gavage study. Body weight gain and feed consumption were reduced in high-dose groups ofboth the feed and gavage studies. There was no measurable loss of TCE in feed sampled from the cages during the study. Dose-related increases in organ (liver and kidney) weight/body weight ratios, individual cell necrosis in the liver, and hepatic microsomal NADPH cytochrome c re ductase and peroxisomal palmitoyl-CoA oxidase and catalase activities were found in both the dosed-fed and gavage groups. Induction of cytochrome P-450 occurred only in the dosed-feed study. There were no significant compound-related pathologic lesions observed in the kidneys, the only other organ examined microscopically. Differences in lethality, cytochrome .P-450 lev els, and induction ofmicrosomal or peroxisomal enzyme activities were attributed to differences in the method ofdosing (gavage versus dosed-feed). The demonstration of no significant loss of TCE from the feed and ofsimilar toxic effects produced by microencapsulated TCE via feed and TCE in com oil via gavage indicate that microencapsulation can provide an excellent alternative exposure route for studying the oral toxicological properties ofvolatile chemicals, such as TCE, in rats. 1987 Society ofToxicology. In the preceding paper we showed that tri chloroethylene (TCE) was stabilized in gela tin-sorbitol microcapsules (Melnick et al,, 1987). In addition, microencapsulation did not appear to interfere with the extent of ab sorption of TCE in male F344 rats (unpub lished results). The present studies were de signed to compare the toxicity of TCE ad ministered in microcapsules mixed in rodent feed to that of TCE administered by gavage in com oil in Fischer 344 rats. TCE is a volatile, colorless liquid (bp = 87"C; vapor pressure at 25'C = 77 mm Hg) which has been used as a vapor degreasing agent of fabricated metal parts, an industrial solvent, a chemical intermediate, a dry-clean ing agent, and an anesthetic (Torkelson and Rowe, 1981; Merck, 1983; U.S. Environ mental Protection Agency, 1985). Produc tion of TCE in the United States in 1981 was about 235 million pounds (USITC, 1982). TCE has been found in foodstuffs and in 0272-0590/8713.00 Copyright 1987 by the Society ofToxicology. All rights of reproduction in any form reserved. SL 035066 drinking w. States (Ka' mental Pro Ingestion tential rout because of volatility, i' water or dc Microencar to provide i oral toxicit\ TCE has icity; the o (Smyth et i LD50 of 24 and 2443 n 1982). Cent the predom; tion exposu 1981). Livei as target orj mice (Tork 1976; Kjell; 1982; Tuckt appear to be ations indue 1982). Som. physiologies sure to TCE body weight strand et al. crease in ki (Kjellstrand toxic nephrt 1987; NTP, lular necrosi patic peroxi not in rats ( in proteins s (Tucker eta> microsomal morphine de droxylase ac somal protei tochrome c and p-nitrop tivities (Pess studies were < TOXICITY OF MICROENCAPSULATED TRICHLOROETHYLENE 433 drinking water in various cities in the United of microencapsulated TCE given in feed with States (Kavlock et al, 1979; U.S. Environ that of TCE administered by gavage in mental Protection Agency, 1985). com oil. Ingestion of TCE in drinking water is a po- .ential route of human exposure. However, lecause of its low water solubility and high METHODS - olatility, it is difficult to perform drinking ater or dosed-feed toxicity studies of TCE. Chemical. High-purity grade TCE (99*%, obtained \licroencapsulation of TCE was investigated o provide an alternative vehicle to study the iral toxicity of this chemical. TCE has a moderate to low acute oral tox icity; the oral LD50 in rats is 7207 mg/kg from Missouri Solvents, Kansas City, MO) ws encapsu lated at Southwest Research Institute (San Antonio, TX) by the centrifugal extrusion process (Goodwin and Som erville, 1974; Melnick et al., 1987). The concentration ofTCE in the gelatin-sorbitol microcapsule preparation (Batch 07; 29.8% sorbitol/70.2% gelatin) was 44.1% (w/ (Smyth et al, 1969) while in mice an oral LD50 of 2402 mg/kg was reported for males md 2443 mg/kg for females (Tucker et al., 1982). Central nervous system depression is the predominant response from acute inhala w). Placebo microspheres lacking TCE were prepared by the same procedure and were ofthe same composition as the microcapsule shells containing TCE. The prepara tion of test diets ofground NIH-47 rodent feed contain ing microencapsulated TCE and the methods for deter mining TCE concentrations in feed samples were de tion exposure to TCE (Torkelson and Rowe, 1981) . Liver and kidney have been identified as target organs to TCE toxicity in rats and mice (Torkelson and Rowe, 1981; NCI, 1976; Kjellstrand et al., 1981; Stott et al., scribed previously (Collins etal., 1986). Animals and treatment. Six-week-old male Fischer 344 rats (obtained from Charles River Breeding Labora tories, Wilmington, MA) were used in this study. The animals, quarantined for 1 week prior to treatment, were weighed, ear tagged, and randomized by weight into 10 1982; Tucker et al, 1982; NTP, 1987). Rats appear to be less susceptible to hepatic alter ations induced by TCE than mice (Stott et al, 1982) . Some of the toxicological effects and physiological responses resulting from expo groups of 10 rats each. The average initial group body weights ranged from 89 to 92 g. Test diets, control diets, and tap water were available ad libitum. Temperature in the animal room was maintained at 22 2*C and relative humidity was 50 10%. Fluorescent lighting was pro vided 12 hr per day. These conditions are consistent with sure to TCE include increase in liver weight/ body weight ratio (Pessayre et al., 1979; Kjell strand et al, 1981; Tucker et al, 1982), in crease in kidney weight/body weight ratio (Kjellstrand et al. 1981; Tucker etal., 1982), the requirements of the National Toxicology Program (NTP, 1984). Groups of 10 male F344 rats were fed diets containing 0 (untreated control), 1.25, 2.5, 5.0, or 10% of TCEloaded microcapsules for 14 days. Since the content of TCE in the microcapsules was 44.1% (w/w), the concen toxic nephropathy in rats and mice (NTP, 1987; NTP, unpublished results), hepatocel lular necrosis in mice (Stott et al, 1982), he patic peroxisome proliferation in mice but not in rats (Elcombe et al, 1985), increases trations of TCE in the diets were 0,0.55,1.10, 2.21, and 4.41 %, respectively. A vehicle control diet containing 5% placebo microspheres was provided to an additional group of 10 animals. Freshly filled feeders were provided after 7 days ofthe study. A parallel corn oil gavage study was also conducted where groups of 10 male F344 rats in proteins and ketones in the urine of mice (Tucker et al, 1982), decreases in rat hepatic microsomal cytochrome .P-450 and ethylmorphine demethylase and hexobarbital hy droxylase activities, and increases in micro were administered TCE dissolved in corn oil by gavage (5 ml/kg body weight) for 14 consecutive days. The dosage levels ofTCE in the gavage study were adjusted five times during the 14-day treatment period to be similar to the dosage levels ofTCE in the feed study. Clinical chemistries and urinalyses. Five animals from somal protein and microsomal NADPH cy tochrome c reductase, aniline hydroxylase, and p-nitrophenol glucuronyltransferase ac tivities (Pessayre et al, 1979). The present studies were designed to compare the toxicity the two highest dosed-feed groups, the two highest dose gavage groups, and the placebo, corn oil, and untreated control groups were housed individually in metabolism cages. Blood samples (2 ml) were collected from the or bital sinus ofC02 anesthetized rats after 1,3, and 14 days ofthe study, and serum samples were analyzed for sorbi- SL 035067 434 MELNICK ET AL. tol dehydrogenase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, urea nitrogen, creatinine, glucose and total protein with a CentriAchem 400 chemistry analyzer (Baker Chemical Co., Allentown, PA). Twenty-four-hour urine samples were also collected at those time periods and analyzed for glucose, total pro tein, creatinine, and blood urea nitrogen. In addition, microscopic appearance of urine sediments, speciAc gravity, and pH ofthe urine samples were recorded. Biochemical measurements. Livers were removed and weighed, and portions not taken for histopathologic ex amination were homogenized in 3 vol of ice-cold 0.25 M sucrose. Catalase and peroxisomal palmitoyl-CoA oxi dase activities in the whole liver homogenates were deter mined by spectrophotometric and fluorometric assay procedures (Aebi, 1974; Walusimbi-Kisitu and Harri son, 1983). The remainder ofthe homogenates were cen trifuged at 10,000? for 10 min and the subsequent super natant fractions were recentrifuged at 140,000? for 45 min. Measurements were made of NADPH cytochrome c reductase activity (Sottocasa el al., 1967) and cyto chrome P-450 (Omura and Sato, 1964) in the micro somal fractions. Protein concentrations were determined by the method ofLowry el a/. (1951) using bovine serum albumin as a standard. Histopathologic examination. Liver samples and the right kidney from each animal were Axed in neutral buffered Formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined mi croscopically in a blind (coded) fashion for evidence of chemically induced morphological changes. Statistical evaluation. The statistical signiAcance ofthe parametric data were evaluated by analysis of variance, and group means were compared by a Student t test (Ryan, 1981). Histopathological evaluations were ana lyzed by the Mann-Whitney V test for nonparametric data (Sokal and Rohlf, 1969). The minimum level of probability accepted for signiAcance was p < 0.05. RESULTS Stability of TCE in Rodent Feed during the 14-Day Study The feed blends containing microencapsu lated TCE were analyzed for TCE content af ter 3 and 7 days of the study to see if the rats promoted deterioration ofthe microcapsules. Deterioration would be expected to cause a decrease in the TCE concentration. How ever, as shown in Table 1, there was essen tially no loss of TCE in samples taken from the surface of the feeders at any ofthe micro capsule concentrations. The slight elevations in TCE concentrations at the 2.5, 5.0, and 10% microcapsule concentrations may be in dicative of selective feeding by the animals. The changes in TCE concentrations were generally much less between Days 3 and 7 than between Days 1 and 3 ofthe study. Feed Consumption and Dosage Levels of TCE The time-weighted average dosage levels of TCE in the feed study were 0.6, 1.3,2.2, and 4.8 g/kg/day (Table 2). The average daily feed consumption for untreated control and pla cebo control animals was between 11 and 12 g per animal during the first week of the study. Similar amounts of feed were con sumed by animals in the 1.25 and 2.5% mi croencapsulated TCE dose groups. The aver age daily feed consumption per animal in the 5.0 and 10% dose groups was between 8 and 9 g. The lower level of feed consumption in these groups may have been due to either a lower palatability of feed containing high concentrations of microencapsulated TCE or to a toxic effect of the TCE. Feed consump tion was also reduced in the high-dose gavage group; however, this decrease may have re sulted from CNS depression caused by TCE, as postgavage lethargy was noted in this group ofanimals. During the second week of the study, feed consumption values were ele vated in all groups; however, feed consump tion for the groups receiving 5 and 10% mi croencapsulated TCE in their diets was still 15-20% lower than those of the placebo con trol or untreated control groups. The two lower dose levels in the gavage study matched closely with the two lower dose levels in the feed study, while the high-dose level in the ga vage study was intermediate between the two higher dose levels ofthe feed study. It was not practical to include a higher dose level in the gavage study since such a dose would be ap proaching the single-dose oral LD50 of TCE in rats. % Targets cone * The concer b Results are | c Different frr Survival ana There wer treated with feed, while t ring in the fi of the gavagi the other g. weight gain. untreated cc as that in the 67.8 g, respe of the two 1 study and of vage study v mean body Tn Mic Group Untreated conti Placebo control 1.25% microcar 2.5% microcaps 5.0% microcaps 10.0% microcar age dosage levels of re 0.6, 1.3, 2.2, and e average daily feed :d control and plabetween 11 and 12 j first week of the of feed were con1.25 and 2.5% mi.e groups. The averm per animal in the k was between 8 and Fed consumption in een due to either a ;d containing high ncapsulated TCE or CE. Feed consumphe high-dose gavage rease may have re ion caused by TCE, was noted in this ' the second week of cion values were ele;ver, feed consumpving 5 and 10% mitheir diets was still ; of the placebo conil groups. The two wage study matched er dose levels in the -dose level in the gaiate between the two eed study. It was not her dose level in the a dose would be ape oral LD50 of TCE -j | j ; TOXICITY OF MICROENCAPSULATED TRICHLOROETHYLENE 435 TABLE 1 Stability of TCE in Rodent Feed during the 1 4-Day Feed Study % Targeted microcapsule concentration" DayO TCE concentration, mg/g feed* Day 3 Day 7 1.25 2.5 5.0 10.0 5.7 0.1 10.9 0.6 21.7+0.5 43.5 2.1 5.4 0.2 (95) 11.00.3(101) 23.7 0.9(109)' 48.0 2.2(110)' 5.6 0.2 (98) 11.8 0.2 (108)' 24.4 0.9 (112)' 47.9 0.9 (110)' The concentration ofTCE in the microcapsules was 44,1 % (w/w). Results are given as means SD; n = 4-8. Numbers in parentheses are percentage of Day 0 concentration, .different from Day 0 concentrations, p < 0.05. rvival and Body Weight Changes There were no deaths in any of the groups ated with microencapsulated TCE in the jd, while there were four deaths, all occurng in the first week, in the high-dose group \ the gavage study. There were no deaths in le other gavage groups. The mean body eight gain after 14 days of treatment in the mtreated control group was nearly the same is that in the placebo control group, 65.4 and 67.8 g, respectively. Mean body weight gains of the two highest dose groups of the feed study and of the highest dose group of the ga vage study were significantly lower than the mean body weight gains of the respective control groups (Fig. 1). Most of the body weight gain in these groups occurred during the second week; this probably reflects in creases in feed consumption and accommo dation to TCE. Postgavage lethargy was ob served only in the 2.8 g/kg dose group of the gavage study. Liver and Kidney Changes At necropsy, liver and right kidney weights were recorded since they have been identified as target organs of TCE toxicity. There were significant and similar dose-related increases in liver weight/body weight ratios at each dos- TABLE 2 Time-Weighted Averages of FEed Consumption and Dosage of TCE in Male F344 Rats during the 14-Day Study Microencapsulated TCE (feed) TCE in com oil (gavage) Group Feed/day (g) TCE dose (g/kg/day) Group Feed/day (g) TCE dose (g/kg/day) Untreated control Placebo control 1.25% microcaps. 2.5% microcaps. 5.0% microcaps. 10.0% microcaps. 12.7 13.2 13.0 13.6 10.5 10.1 0 0 Com oil control 10.6 0.6 Low-dose gavage 11.5 1.3 Mid-dose gavage 11.0 2.2 High-dose gavage 6.5 4.8 0 0.6 1.2 2.8 SL 035069 436 MELNICK ET AL. FEED STUDY GAVAGE STUDY DAYS DAYS Fig. 1. Growth curves for male F344 rats treated with microencapsulated TCE (dosed-feed study) or TCE in com oil (gavage study) for 14 days. Dosed-feed groups: , placebo control; , 0.6 g/kg; , 1.3 g/ kg; O, 2.2 g/kg; A, 4.8 g/kg. Gavage groups: , com oil control; , 0.6 g/kg; , 1.2 g/kg; O, 2.8 g/kg. Standard deviations were generally 7% ofthe mean and never exceeded 15%. `Different from controls, P < 0.05. age level in the dosed-feed and gavage groups (Table 3). Relative to controls, absolute liver weights were increased only at the two lower dose levels (0.6 and 1.2 g/kg groups) of the dosed-feed and gavage studies. Neither the placebo capsules nor the com oil treatment appeared to have an effect on liver weight. Kidney weight/body weight ratios were ele vated at all TCE dose levels compared to con trols in both the dosed-feed and gavage groups; however, there were no treatment-re lated effects on absolute kidney weight (re sults not shown). The only treatment-related lesion observed microscopically in rats from either the dosedfeed or gavage groups was individual cell ne crosis in the liver. The frequency and severity ofthis lesion were similar at each dosage level of TCE administered microencapsulated in the feed or in com oil by gavage (Table 4). Individual cell necrosis of the liver is a subtle lesion which was subjectively graded into one of five categories. A minimal grade was as signed for cases in which 1 to 3 necrotic hepatocytes were found in approximately 10 mi croscopic fields at a magnification of 200X. Similarly, 4 to 7 necrotic hepatocytes per 10 fields were graded as mild, while 8 to 12 were graded as moderate. There were no instances where more extensive lesions (i.e., marked or severe) were found. The cytological features of the individual cell necrosis were nuclear condensation and pyknosis with attendant increased cytoplasmic eosinophilia. There was individualization of affected hepatocytes manifested by shrinkage away from the sur rounding parenchyma. This individual cell necrosis was randomly distributed through out the liver with no apparent lobular distri bution and the change was not accompanied by an inflammatory response. There was no histologic evidence ofcellular hypertrophy or edema in hepatic parenchymal cells. There were no significant compound-related lesions M Dose group (g/kg) Untreated control Placebo control 0.6 1.3 2.2 4.8 " Results art only six surviv 4 Increased c f Different ft observed ir group. Th< changes in z try paramet The activ chrome c r chrome P-4 iNDIVIDl Mic Dose group (g/kg) Untreated control Placebo control 0.6 1.3 2.2 4.8 "Number of; 4 Severity of1< ` Different fro rieo ioo -|---------- (-80 14 16 osed-feed study) or 0.6 g/kg; , 1.3g/ ! g/kg; O, 2.8 g/kg. rent from controls. limal grade was as1 to 3 necrotic hepaproximately 10 minification of 200X. hepatocytes per 10 1, while 8 to 12 were re were no instances ions (i.e., marked or cytological features crosis were nuclear osis with attendant .osinophilia. There iffected hepatocytes away from the surfhis individual cell iistributed througharent lobular distrias not accompanied onse. There was no iular hypertrophy or ,'hymal cells. There ound-related lesions TOXICITY OF MICROENCAPSULATED TRICHLOROETHYLENE 437 TABLE 3 Liver Weights of Male F344 Rats Treated with TCE for 14 Days0 Microencapsulated TCE (feed) TCE in corn oil (gavage) 'C ::roup Liver weight (g) Liver weight/ body weight ratio x 100 Dose group (B/kg) Liver weight (g) Liver weight/ body weight ratio X 100 cuted ,'irol urol 'O 3 -8 8.1 0.8 8.4 0.8 9.50.54 10.1 1.2* 8.9 1.3 7.4 0.5 5.2 + 0.3 5.3 0.2 6.0 0.3 4 6.5+0.54 7.0 0.9* 7.1 0.54 Corn oil control 0.6 1.2 2.8 7.1 1.3 9.3 1,2* 9.1 0.9` 7.7 0.4 5.0 0.4" 6.0 0.4 ` 6.1 0.34,c 7.3 0.5 4 .'. .-suits are given as means SD. N - 10 for all groups except the 2.8 g/kg dose group (gavage study) which had si\ surviving rats at the time ofthe terminal sacrifice, i ncreased compared to control, p < 0.05. Different from corresponding feed group, p < 0.05. ^served in the kidneys in any treatment roup. There were also no remarkable ranges in any of the serum or urine chemist> parameters which were measured. The activity of microsomal NADPH cyto chrome c reductase and the level of cyto chrome P-450 were measured in the present studies (Table 5) since the rat microsomal mixed function oxidase system had been re ported to undergo various changes after re peated administration of TCE (Pessayre et ai, 1979). Microsomal NADPH cytochrome c reductase activity was elevated in the 2.2 and 4.8 g/kg dose groups of rats fed diets con- TABLE 4 Individual Cell Necrosis of Hepatocytes in Male F344 Rats Treated with TCE for 14 Days Microencapsulated TCE (feed) TCE in com oil (gavage) Dose group (g/kg) Frequency oflesion" Mean severity4 Dose group (g/kg) Frequency oflesion" Mean severity4 Untreated control Placebo control 0.6 1.3 2.2 4.8 1/10 0/10 2/10 2/10 9/10 9/10 0.1 0.0 Com oil control 0.4 0.6 0.3 1.2 2.0" 2.8 2.5" 0/10 0/10 1/10 5/6 0.0 0.0 0.1 1.8" " Number of animals with lesion/number of animals examined. 4 Severity oflesion: I = minimal; 2 = mild; 3 = moderate; 4 = marked; 5 = severe. " Different from controls, p < 0.05. SL 035071 438 MELNICK ET AL. TABLE 5 Effect of TCE on Hepatic Microsomal NADPH Cytochrome c Reductase Activity and Cytochrome P-ASO Content in Male F344 Rats'1 Microencapsulated TCE (feed) Dose group (g/kg) NADPH cytochrome c reductase* Cytochrome A450' Untreated control Placebo control 0.6 1.3 2.2 4.8 402 48 347 48 409 83 410 89 732 118'' 775 130" 472 98 421 95 475 83 451 95 719 92l/ 718 84 TCE in com oil (gavage) Dose group (g/kg) NADPH cytochrome c reductase* Cytochrome P-450' Com oil control 0.6 1.2 2.8 461100 439+ 95 790 73`t' 680 78'' 461 83 433 23 585136 426 25' " Results are means SD; n = 5. * Values are expressed as nmol cytochrome c reduced X min'1 X mg protein'1. c Values are expressed as pmol/mg protein. " Different from controls, p < 0.05. ' Different from corresponding feed group, p < 0.05. taining microencapsulated TCE and in the 1.2 and 2.8 g/kg dose groups of rats treated with TCE in corn oil by gavage. Cytochrome P-450 levels were elevated only in the two highest dose groups of the feed study. There were no significant decreases in cytochrome P-450 levels. Neither the placebo capsules nor the com oil treatment affected the levels of NADPH cytochrome c reductase activity or cytochrome P-450. Elcombe et al. (1985) suggested that TCE-induced hepatocarcinogenicity may be unique to mice and possibly may be related to a species-specific proliferation of hepatic peroxisomes resulting from treatment with TCE. At doses of 0.5 to 1.5 g/kg, TCE in duced peroxisome proliferation in mice but not in rats (Elcombe et al., 1985). In the pres ent studies, rat hepatic peroxisomal palmitoyl-CoA oxidase and catalase activities were measured. Palmitoyl-CoA oxidase activity is a sensitive marker of peroxisome prolifera tion (Tomaszewski et al., 1986). We observed dose-related increases in peroxisomal palmitoyl-CoA oxidase and catalase activities in liver homogenates prepared from rats treated with either microencapsulated TCE in the feed or with TCE in com oil by gavage (Table 6). Increases in palmitoyl-CoA oxidase and catalase activities were observed at similar dose levels in the dosed-feed and gavage stud ies. Treatment with com oil, but not with the placebo capsules, caused a slight increase in palmitoyl-CoA oxidase activity. DISCUSSION Microencapsulated TCE was incorporated into rodent feed to compare the toxicity of TCE administered in microcapsules mixed in rodent feed to that of TCE administered by gavage in com oil. Previously, we had shown that microencapsulated TCE was stable when mixed in rodent feed and stored for 7 days in an open vessel at room temperature (Melnick et al., 1987). In the present studies, there were no significant losses of TCE after 7 days of treatment of male F344 rats with feed mix tures containing up to 10% microencapsu M Dose group (E/kg) Untreated control Placebo control 0.6 1.3 2.2 4.8 "Results are * Values are i "Values are i d Different fr ' Different fr lated TCE. did not cam sules. Since weekly in tc for the Nati does not app ration ofthe would occur centration ir may be indi rats. This in ent between 1 and 3 and of the study, tion at the 5 capsulated 4.41% TCE i ished palatal concentratio doses of TC present stud} tween these t tion was also high-dose ga pression was might have c of feed consu CnVTTY /age) Cytochrome P-450' 461 83 433 23 585136 426 25' Jfrom rats treated 'ted TCE in the by gavage (Table "oA oxidase and erved at similar and gavage stud. but not with the slight increase in dty. >N was incorporated re the toxicity of capsules mixed in administered by ly, we had shown E was stable when ored for 7 days in terature (Melnick tudies, there were E after 7 days of ts with feed mix% microencapsu- TOXICITY OF MICROENCAPSULATED TRICHLOROETHYLENE 439 TABLE 6 Effect of TCE on Hepatic Peroxisomal Palmitoyl-CoA Oxidase and Catalase Activities in Male F344 Rats' Microencapsulated TCE (feed) TCE in com oil (gavage) toup died :oI :ol 'v Palmitoyl-CoA oxidase* 270 12 242 17 298 64 424+ 55* 651 + 148* 999 266* Catalase* 8.49 + 0.81 7.98 1.62 8.49+1.92 8.59 1.31 13.03 + 2.02* 15.76+1.11* Dose group (g/kg) Com oil control 0.6 1.2 2.8 Palmitoyl-CoA oxidase* 318 27' 369 26 413 40* 1002 273*' Catalase* 8.59 0.91 10.10 1.82 12.83 3.43*-' 13.54 2.32* 'suits are given as means SD; n = 5. uues are expressed as nmol H202 produced X min'1 X g liver*1. jlues are expressed as mmol H2d2 consumed X min'1 x g liver*1. - Iferem from controls, p < 0.05, `iflerent from corresponding feed group,p < 0.05. :d TCE. Thus, rats feeding on these diets i not cause deterioration of the microcapdes. Since feeders are normally changed .ekly in toxicity studies conducted by and 'r the National Toxicology Program, there ioes not appear to be a concern that deterio ration ofthe capsules and loss ofthe chemical would occur. The slight increase in TCE con centration in the feed blends during the study may be indicative of selective feeding by the rats. This increase, however, was less appar ent between Days 3 and 7 than between Days 1 and 3 and had no impact on the outcome of the study. The decrease in feed consump tion at the 5 and 10% dose levels of microen capsulated TCE (equivalent to 2.21 and 4.41% TCE in the feed) may be due to dimin ished palatability of the dosed-feed at these concentrations or to a toxic effect at these doseS of TCE (2.2 and 4,8 g/kg/day). The present study does not clearly distinguish be tween these two possibilities. Feed consump tion was also decreased in the corresponding high-dose gavage group; however, CNS de pression was evident in these animals and might have contributed to their reduced level of feed consumption. Administration of microencapsulated TCE in the diet of F344 rats for 14 days at levels up to 10% microcapsules (the timeweighted average dosage level of TCE at this concentration of microcapsules was nearly 5 g/kg/day) caused no deaths, while the highest dose level of TCE in the gavage study caused four deaths during the first week of the study; the time-weighted average dose of TCE dur ing that time was nearly 3 g/kg/day. The dose level of TCE in the highest dose group of the feed study is nearly equal to the single dose oral LD50 of 7.2 g/kg for TCE in rats (Smyth et al,, 1969). Therefore, microencapsulation can provide a means of administering higher daily dose levels of unstable chemicals than is possible by single daily gavage treatments. Higher peak blood levels ofTCE would be ex pected in animals administered single daily bolus doses of TCE by gavage than in those fed diets containing TCE. Thus, the method of dosing was probably the cause for the differences in lethality and clinical signs of toxicity (lethargy in only the high-dose ga vage group) in TCE-treated rats. The use of microencapsulation in a feed study also elim inates potential problems of gavage trauma SL 0350733 440 MELNICK ET AL. and of bolus dosing effects, and in the case of TCE, more closely resembles the pattern of human exposure to this chemical. The de crease in body weight gain at the higher dose levels in the feed and gavage studies may be due to a toxic manifestation ofthe TCE treat ment, CNS depression in the high-dose ga vage group, decreased feed consumption, or a combination of these factors. The liver and kidney were evaluated for ev idence of TCE toxicity since they have been identified as target organs of this chemical (Torkelson and Rowe, 1981; NCI, 1976; Pessayre et al., 1979; Kjellstrand et al., 1981; Stott et al., 1982; Tucker et al., 1982; Elcombe et al., 1985; NTP, 1987). In the pres ent studies, microencapsulated TCE and TCE in com oil produced dose-related in creases in liver weight/body weight ratios and in kidney weight/body weight ratios. The effects of TCE on the liver, but not the kid ney, are considered to be toxicologically sig nificant since there were also increases in ab solute liver weight and dose-related histo pathologic alterations in this organ. The effects of TCE on liver weight and the fre quency and severity of hepatic lesions were similar at comparable dosage levels in the feed and gavage studies. A similar increase in liver weight/body weight ratio was reported in male Osbome-Mendel rats treated with TCE by gavage for 3 weeks at a daily dose level of 1.1 g/kg (Stott et al., 1982) or for 10 days at daily dose levels of0.5,1.0, and 1.5 g/ kg (Elcombe et al,, 1985). Stott et al. (1982) did not observe any increase in the kidney weight/body weight ratio. Neither Stott et al. (1982) nor Elcombe et al. (1985) observed any histopathological changes in the livers of rats treated with TCE, whereas in the present studies individual cell necrosis was discerni ble in animals in the 2.2 or 4.8 g/kg dose groups of the feed study and in the 2.8 g/kg dose group of the gavage study. At dose levels similar to those used by Stott et al. (1982) and Elcombe et al. (1985), the frequency and se verity of this lesion were not significantly different from those of controls. Individual cell degeneration and necrosis in this study were characterized by nuclear pyknosis and karyorrhexis and cytoplasmic condensation of scattered single cells. These changes are morphologically compatible with what has been described as "piecemeal" ne crosis as well as apoptosis (Zimmerman, 1980; Wyllie et al., 1980). Apoptosis is the death of single isolated cells occurring with normal cell turnover in healthy tissues, dur ing embryonic development, in endocrinedependent atrophy and hyperplasia, or after exposure to cytoxic agents (Wyllie et al,, 1980). The observation of an increased num ber of necrotic cells in TCE-treated rats may reflect a perturbation in the kinetics of nor mal cell survival such that proportionally more cells are dying than in the normal steady state. The induction of microsomal NADPH cy tochrome c reductase activity in livers of rats treated with TCE in com oil by gavage was similar to that reported by Pessayre et al, (1979). The minimal effective dose for induc tion of NADPH cytochrome c reductase ac tivity was higher in animals treated with mi croencapsulated TCE in feed (2.2 g/kg) than in those that were administered TCE by ga vage (1.2 g/kg). This difference may be due to the method of administration of TCE, since higher peak blood levels and perhaps higher tissue levels of TCE would be expected after the gavage treatment. Cytochrome P-450 lev els were elevated in rats treated with microen capsulated TCE in the feed, but not in rats treated with TCE in com oil by gavage. Pessayre et al. (1979) reported a decrease in cyto chrome P-450 levels in rats treated with TCE by gavage. The extent of destruction of cytochrome P-450 following TCE treatment may be related to the tissue concentration ofactive metabolites of TCE. Low concentrations of TCE may lead to induction of cytochrome P450, while high concentrations may also cause induction of microsomal enzymes, but in addition, result in levels of reactive metab olites ofTCE which inactivate cytochrome P450. Higher peak liver concentrations ofTCE ( ; may occur al in a gavage f TCE in feed The hepat and palmito since Elcoml duction of p occurred in bome-Menc in response differences ii In the prese related incre zymes in li\ F344 rats, pe idase. Indue CoA oxidase nounced in animals whit difference m dosing rathe ing animals patic peroxi; tivity in F34 ported by E mice. Furthe the present greater than combe et al. that TCE-in is not specifi nouncedini In concluis not alterec ing microer part, the tox sulated TCf to that of T gavage. Diffi chrome P-4 or peroxisoi the method feed) rather capsulation TCE so tha volatile che feed and use /., 1987). eration and necrosis acterized by nuclear jxis and cytoplasmic ed single cells. These eally compatible with i as "piecemeal" neDtosis. (Zimmerman, SO). Apoptosis is the cells occurring with healthy tissues, durment, in endocrinehyperplasia, or after gents (Wyllie et al., of an increased numTCE-treated rats may 1 the kinetics of northat proportionally than in the normal rosomal NADPH cy:tivity in livers of rats m oil by gavage was d by Pessayre et al. fective dose for induc^rome c reductase ae rials treated with mi1 feed (2.2 g/kg) than inistered TCE by gaerence may be due to ration of TCE, since s and perhaps higher uld be expected after ytochrome P-450 levtreated with microenfeed, but not in rats -n oil by gavage. Pesed a decrease in cytoats treated with TCE f destruction of cyto; TCE treatment may mcentration ofactive >w concentrations of ion ofcytochrome Pntrations may also osomal enzymes, but ils of reactive metabtivate cytochrome Pmcentrations ofTCE TOXICITY OF MICROENCAPSULATED TRICHLOROETHYLENE 441 cur after single bolus doses of TCE as wige study, compared to ingestion of :ced over an extended period oftime, jpatic peroxisomal enzymes catalase nitovl-CoA oxidase were measured combe et al. (1985) reported that in)t peroxisome proliferation by TCE : in B6C3F! mice but not in Oslendel rats, and that this difference nse may be related to the species ::s in hepatocarcinogenicity of TCE. esent studies, TCE produced doseicreases in the activities of these en- liver homogenates prepared from , s. particularly for palmitoyl-CoA oxduction of catalase and palmitoylidase activity was slightly more proo in gavage-treated animals than in ^ which received TCE in the feed. This .ce may be related to the method of rather than to the vehicle used for dosumals with TCE. The increase in heeroxisomal palmitoyl-CoA oxidase acn F344 rats was not as great as that reJ by Elcombe et al. (1985) in B6C3Fi . furthermore, the doses of TCE used in ">resent study included levels 2-3 times .iter than the highest level used by Elrabe et al. (1985). The present study shows .it TCE-induced peroxisomal proliferation r ot specific for mice, but may be more prounced in this species than in rats. !n conclusion, the stability of TCE in feed is not altered by rats feeding on diets contain ing microencapsulated TCE. For the most part, the toxicological profile of microencap sulated TCE administered in feed is similar to that of TCE administered in com oil by gavage. Differences in lethality, levels ofcyto chrome P-450, and induction of microsomal or peroxisomal enzymes were attributed to the method of dosing (gavage versus dosedfeed) rather than the dosing vehicle. Microen capsulation affords a high level of stability to TCE so that homogeneous mixtures of this volatile chemical can be prepared in rodent feed and used for toxicology studies (Melnick et al., 1987). Microencapsulation provides an excellent alternative for studying the oral tox icological properties of volatile chemicals, such as TCE, in laboratory animals. ACKNOWLEDGMENT The authors thank Ms. Nancy Mitchell for excellent secretarial help in preparation ofthis manuscript. REFERENCES Aebi, H. (1974). Catalase. In Methods in Enzymatic Analysis (H. H. Bergmeyer, Ed.), pp. 673-674. Verlag Chemie, Weinheim. Collins, B. J., Goehl, T. J., Jameson, C. W,, Kuhn, G., and Dux, T. (1986). Analytical methods for the determination of neat and microencapsulated trichlo roethylene in dosing vehicles and whole blood. J. Anal. Toxicol. 10,236-240. Elcombe, C. R., Rose, M. S., and Pratt, I. S. (1985). 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