Document bBBdL5X1L0LYj4q0335aX19g

FUNDAMENTAL AND APPLIED TOXICOLOGY 17, 240-253 (1991) Ninety Day Toxicity Study of Chloroacetic Acids in Rats Hari K.. Bhat,* Marv F. KanzT Gerald A. Campbell,! and G. A. S. Ansari*! *Department ofHuman Biological Chemistry and Genetics, and tDepartment ofPathology. University of Texas Medical Branch, Galveston. Texas 77550 Received October !, 1990; accepted March 12, 1991 Ninety Day Toxicity Study ofChloroacetic Acids in Rats. Bhat, H. K., Kanz, M. F,, Campbell, G. A., and AnSari, G. A. S. (1991), Fundam. Appl. Toxicol 17, 240-253. Chloroacetic acids are produced in drinking water as a result of disinfection processes. Chloroacetic acids are also metabolites of widely used and toxic halogenated hydrocarbons. Thus, chronic human exposure to these chemicals is likely to occur. The objective of the present study was to examine the toxic effects of monochloroacetic acid (MCA), dichloroacetic acid (DCA), and trichloroacetic acid (TCA) in a 90-day subchronic study in rats via oral exposure by drinking water. Chloroacetic acid solutions were prepared at concentrations which provided an approximate intake of '/< the LD50 dose per day: MCA, 1.9 idm; DCA, 80,5 mM*, TCA, 45.8 mM. Control rats received distilled water only. After 90 days, major organs were removed, fixed, paraffin embedded, and stained. Light microscopic examination of the major organs revealed variable degrees of alterations in the lung and liver of all three treated groups. In the liver, morphological changes were predominantly localized to the portal triads, which were mildly to moderately enlarged with random bile duct proliferation, extension of portal veins, fibrosis, edema, and occasional foci of inflammation. In the lungs, minimal alterations were observed as foci of perivascular inflammation on small pulmonary veins. Morphological changes in the testes and brain were seen only in the DCA treated group. Testes were atrophic with few spermatocytes and no mature spermatozoa. Focal vacuolation and gliosis were present in the forebrain and brainstem. The results of these studies indicate that, relative to their respective LD50 values, DCA given at 80.5 mM is more toxic than TCA given at 45.8 mM and MCA at 1.9 mM is least toxic. iwi society of Toxicology. Chloroacetic acids are chemically and envi ronmentally important compounds. Mono-, di-, and trichloroacetic acids have been iden tified as by-products of chlorination processes for disinfection of drinking water (Christman et al., 1983; Coleman et al., 1984; Uden and Miller, 1983; Krasner et al., 1989). Chloro acetic acids are also metabolites of several chlorinated hydrocarbons such as 1,1,2-trichloroethane, 1,2-dichloroethane, 1-chloroethene, and 1,1-dichloroethene (Yllner, 1971a,b; Rannug et al,, 1976; Liebler et al., 1985; Liebler and Guengerich, 1983; Reichert et al., 1979). Thus, the human population may be at a constant risk of exposure to these chlo rinated acids. Monochloroacetic acid (MCA), widely used as a herbicide and in the synthesis of various chemicals, is rapidly absorbed through skin and may cause death by systemic exposure (Woodard et al., 1941; Berardi et al. 1987). MCA penetrates the blood-brain barrier since mice surviving an oral LD50 or LD80 dose exhibit a peculiar anomaly after 24 hr in which the front paws are rigidly clasped together and the hind limbs splayed causing difficulty in walking (Berardi et al., 1987). The mechanism by which MCA exerts its toxicity is not well understood. Chaiken and Smith (1969) ascribe the toxicity of MCA to the lability of the halo gen, which allows this compound to react with sulfhydryl compounds. Direct inhibition of 0272-0590/91 $3.00 Copyright 1991 by the Society of Toxicology. All rights of reproduction in any form reserved. 240 TOXICITY OF MCA, DCA, AND TCA 241 thiols in the kidney has been suggested to ac speculation that TCA levels may be important count for anuria in rats receiving toxic doses in TCE carcinogenesis in mice (Prout et ai, of MCA (Hayes et ai, 1973). 1985; Green and Prout, 1985). Dichloroacetic acid (DCA) has been used The objective of this study was to determine as a therapeutic agent for the treatment of sev the subchronic toxicity of MCA, DCA, and eral metabolic disorders; it also causes vaso TCA in rats following oral exposure to these dilation, inhibits nerve impulse transmission chloroacetic acids in water at a standardized in sympathetic ganglia, and protects the liver equitoxic dose regimen. In any subchronic ex against carbon tetrachloride, cyanide, and ar posure, the manifested toxicity of a compound senate toxicity (Stacpoole, 1969). DCA is neu may be in direct relationship to its LD50 value. rotoxic to rats or dogs when administered Although human exposure to these acids is orally at doses higher than 50 mg/kg/day for likely to occur from chlorinated drinking wa several weeks (Stacpoole et ai, 1979; Katz et ter, few comparative toxicity studies using the ai, 1981; Spencer et ai, 1981; Yount et ai, three chloroacetic acids have been done. One 1982; Stacpoole et ai, 1984). Stacpoole et ai previous toxicity study comparing MCA, * (1990) suggest that chronic DCA treatment DCA, and TCA was short in length (14 days) j might induce thiamine deficiency through an and used equimolar dose regimens (DeAngelo increased demand for this vitamin and they et ai, 1989). Other subchronic studies using have found that coadministration of thiamine equimolar dose regimens have examined the '( with DCA to rats significantly reduces the in effects of DCA in dogs and rats (Katz et ai, cidence of hind limb weakness and other be 1981) or DCA and TCA in rats (Mather et ai, havioral changes typical of both DCA toxicity 1990). Therefore, we decided to investigate the and thiamine deficiency. toxicities of the chloroacetic acids based on an Trichloroacetic acid (TCA) is used as a approximate intake of 'A of the LD50 dose per preemergence herbicide, a peeling agent for day. In addition, the equitoxic dose regimens wrinkled, sun-damaged skin and tattoos, and utilized in this study may more nearly ap as a common laboratory agent (Ayres, 1964; proximate human exposure because chlori Collins, 1989; Piggot and Norris, 1988). Be nated drinking water or surface water near in sides being present in drinking water as a result dustrial areas contain much greater concen of chlorine disinfection, TCA is also a major trations of DCA or TCA than MCA (Uden metabolite of trichloroethylene (TCE) and te- and Miller, 1983; Coleman et ai, 1984). trachloroethylene (Coleman et ai, 1976; Uden and Miller, 1983; Daniel, 1963; Dekant et al,, 1985). The metabolism of TCE results, in part, MATERIALS AND METHODS in the formation of TCA as a major metabolite and DCA as a minor metabolite. TCE is an organic solvent with wide industrial applica tion as well as a contaminant of surface and MCA (purity > 99%) and DCA (purity > 99%) were purchased from Aldrich Chemical Co., (Milwaukee, Wl). TCA (purity > 99%) was obtained from Sigma Chemical Co. (St. Louis, MO). ground water (Page, 1981; Love and Elero, 1982). TCA induces liver peroxisome prolif eration (Odum et ai, 1988; Parnell etal., 1986) and an increased incidence of adenomas and hepatocellular carcinomas are observed in Male Sprague-Dawley rats (Harlan, Indianapolis, IN) were acclimatized in our animal facility for one week after arrival with free access to water and food (Purina Rat Chow) and housed in a 12 hr light/dark cycle room. The rats were divided into four groups with five rats in each group. At the start of the study, rat body weights ranged mice exposed to TCA or DCA (Herren-Freund et ai, 1987). An increase in peroxisomal stim ulating activity along with increased metabolic TCA formation, following TCE administra tion in mice compared to rats, has led to the from 220-243 g. Aqueous solutions of MCA (1.9 mM), DCA (80.S mM), and TCA (45.8 mM) were prepared in double distilled wa ter. Our initial studies showed that a rat weighing --240 g consumed ~25 ml of water each day. Solutions were prepared such that each rat would receive approximately SL 036965 242 BHAT ET AL. 'U of LD50 of one of the chloroacetic acids in drinking water per day. LD50 of MCA, DCA, and TCA are 76 mg/ kg, 4.4 g/kg, and 3.3 g/kg, respectively (Stecher, 1976; Woodard et a!.. 1941). These solutions were neutralized to pH 1.2-1A with 1 N sodium hydroxide. Each solution was given in glass bottles and solutions were freshly pre pared and changed on alternate days. Chloroacetic acids are stable for more than I week under these conditions (Herren-Freund et al. 1987; NTP TR 396). Group I re ceived MCA, Group II received DCA, and Group 111 re ceived TCA in water for 90 days. Group [V (controls) received distilled water only. Fixation procedure. Rats were anesthetized with ether and the body cavity opened. The inferior vena cava was severed and major organs were perfused via the heart with 0.9% saline (5 min) and 1% glutaraldehyde (5 min) (Poly Sciences, Warrington, PA) in 0.1 m Pipes buffer (pH 7.35, 365 mOsm) (Sigma Chemical Co., St. Louis, MO). Per fusions were carried out at 37C to maximize blood re moval with saline prior to the initiation of fixation with glutaraldehyde- Different organs were removed, blotted, and immedi ately weighed. Slices of liver, lung, heart, spleen, thymus, kidney, testes, and pancreas were postfixed in 10% neutral buffered formalin, dehydrated, and paraffin embedded. Evaluation oftissues for histopathology was conducted on hematoxylin and eosin stained sections, in addition, liver sections from all animals were stained with Masson's tri chrome for evaluation ofdifferences in collagen deposition. Dilation and extension of portal veins were assessed ac cording to the following criteria: none, minimal (enlarge ment or extension of portal veins in less than one-fourth of portal triads), mild (enlargement or extension of portal veins in one-fourth to one-third of portal triads), or mod erate (enlargement or extension of portal veins in onethird to one-half of portal triads). Differences in collagen deposition were assessed in cross sections of portal triads between 500 and 800 am diameter according to the fol lowing criteria: none, minimal (collagen width external to portal veins, hepatic arteries or major bile ducts approx imately the thickness of one hepatocyte), mild (collagen width greater than 1 but less than the thickness of 2 hepatocytes), or moderate (collagen width greater than the thickness of 2 hepatocytes). Rat brains which had been postfixed in 10% neutral buffered formalin were sliced coronally using standard landmarks (optic chiasm, infundibulum, cerebral pedun cles) as reference points to ensure consistency between animals in the levels examined. The number ofslices pro duced varied between seven and eight per animal and in cluded frontal, frontoparietal, parietal, and occipital levels of the forebrain and midbrain, and pons, medulla, and cerebellum in the hindbrain. Although the plane of section was not stereotaxically determined, the sections closely matched the coronal sections shown in the atlas of Paxinos and Watson (1986). Slices were marked with a punch to identify left and right sides. The slices were then processed through graded alcohols and clearing agent (HistoClear), infiltrated with paraffin, and embedded by standard his tologic techniques. The resulting blocks were sectioned at 7 microns and slides of all blocks were stained with he matoxylin and eosin, (H & E). Luxol fast blue for myelin with H & E counterstain, and cresyl echt violet for nuclei and Nissl substance. Immunohisiochemical staining for glial fibrillary acidic protein (GFAP), an astrocyte inter mediate filament marker, was also performed on selected sections of DCA-treated and control rats to assess the extent of gliosis. GFAP was stained by the avidin-biotin method using a kit from Vector (Burlingame, CA). Statistics. Significant differences were determined by analysis of variance using ABSTAT (Anderson Bell, Boulder, CO), a statistical program for personal computers, A level of p < 0.05 was considered to be significant RESULTS General. Rats treated with DCA and TCA showed a loss in body weight compared to the control group (Fig. 1). On Day 90 the mean body weights of the different groups were: control 448.2 22.8; MCA 426.8 22.1 (95.2% of control); DCA 295.8 9.5 (66% of control, p < 0.0001); and TCA 370.8 17.7 (82.7% of control, p < 0.0001). Organ weight differences among the four groups were found only for liver and testes. The DCA group showed hepatomegaly with increased liver weights but decreased testes weights compared to the other groups and controls. Mean liver weights for the four groups were: control 14.68 -- Control Fig. 1. Body wt profiles of control rats () and rats drinking water containing 1.9 hm MCA ( ). 80.5 mM DCA (A) and 45.8 mM TCA (V) for 90 days. Each point represents the mean body weight of five rats pi us or m i n us the standard deviation. TOXICITY OF MCA, DCA, AND TCA 243 0.78; MCA 13.25 0.64 (90.3% of control, p < 0.03); DCA 16.35 0.41 (111.4% of con trol, p < 0.01); and TCA 12.6 0.72 (85.8% of control, p < 0.002). The percentage liver/ body weight ratios for the different groups were: control 3.3, MCA 3.1, DCA 5.5 (p < 0.0001), and TCA 3.4. Mean testes weights of the different groups were; control 3.73 0.2, MCA 3.70 0.13, DCA 2.47 0.97 (p < 0.01), and TCA 3.56 0.10. The per centage testes/body weight ratios for the groups were: control 0.87, MCA 0.84, DCA 0.84, and TCA 0.96. Light microscopy. At necropsy, no gross le sions were observed. Light microscopic ex amination of the major organs revealed vari able degrees ofalterations in the lung and liver of all three treated groups. In addition, changes in testes and brain were observed only in the DCA group. Liver sections from control rats showed a typical architecture with several central veins and portal triads outlining the lobular sub structure (Fig. 2a). One control rat showed minimal increases in collagen deposition in a minor number of portal triads (Table 1). In DCA-exposed rats, however, portal triads showed alterations ranging from mild to mod erate. In the liver of one moderately affected animal, several portal triads contained greatly enlarged portal veins which were surrounded by increased numbers of bile ducts and duc tules, areas of edema, and variable numbers of inflammatory cells (Fig. 2b). In two other moderately affected livers, portal veins were less dilated but were more tortuous with 6-10 branches per triad (Fig. 3a). Minimal to mod erate increases in collagen deposition were ob served in random portal triads (Fig. 3b) and around larger central veins. Small foci of in flammation were scattered within the peri portal and midzonal parenchyma and were also located peripheral to endothelial cells of central veins; necrotic hepatocytes were rarely observed. Morphological alterations in the livers of MCA- and TCA-exposed rats were similar but generally ranged from minimal to mild (Fig. 4) in the two groups, respectively (Table 1). In the lungs, foci of perivascular inflam mation were observed in all three treatment groups. Such foci were extremely rare in the lungs of control rats which showed normal al veolar architecture (Fig. 5a). These foci were generally found on the periphery of small pul monary veins (Fig. 5b) and were occasionally present at the bifurcations of veins into venules (Fig. 5c). Lymphocytes and macrophages were typically identified in these foci (Fig. 5d). An increase in the thickness ofthe adventitial layer of the adjacent venous walls was also fre quently seen around these foci. The degree of perivascular inflammation in the lungs of treated rats was comparable to the degree of liver injury seen in the three treatment groups: DCA > TCA > MCA. Morphological changes in the testes were seen only in the DCA-treated group. Variable FlC. 2. Light micrographs of the hvers of a control rat (a) and a rat drinking 80.5 dim DCA for 90 days (b). Trichrome stain, original magnification, (a,b) 60x, (a) In the control liver, typical portal triads (P) with normal portal veins (PV) are present at the margins of lobules containing central hepatic veins (CV), (b) The liver of the DCA-treated rat shows an enlarged portal triad with a dilated portal vein (PV) and increased amounts of collagen in the perivascular region (blue staining). Arrowheads indicate increased number of bile ducts and ductules. Gear areas within the portal tn:td represent edema (asterisk). Fig. 3. Light micrographs of the livers of rats drinking 80.5 mM DCA for 90 days (a,b). Trichrome stain, original magnification, (a) 55x, (b) 100X, (a) The portal triad is enlarged and extended with several cross sections (arrows) of the portal vein (PV). (b) Increased amounts of collagen (indicated by the blue staining) are present around the portal triad. Deposition of collagen is beginning to occur within the hepatic cords, shown on the upper left side (arrows) of the portal vein (PV). Fig. 4. Light micrograph of the liver of a rat drinking 45.8 mM TCA for 90 days. The enlarged portal triads (P) contain dilated portal veins (PV) and increased amounts of collagen. Trichrome stain, original magnification, 60x. 244 bhat et al. Figure 2 246 BHAT ET \L TABLE I Hepatic Histopa tholikjv following Subchronic Exposure ro Chloroacetic acids Control MCA DCA TCA Collagen deposition Normal Minimal Mild Moderate Portal vein dilation/ extention Normal Minimal Mild Moderate 4 1 -- -- 5 -- -- -- 1 --1 3 1 > i ?1 --2 1 i ---- 2i1 2 13 --3 1 Note. Cnteria for grading histopathology are described under Matenals and Methods Collagen deposition was assessed on trichrome-stained sections; portal vein dilation/ extension was assessed on H & E-stained sections. Each group contained five animals. degrees of atrophy, from mild to severe, were found within the group of five animals. In control animals, oval to elongated seminifer ous tubules in the testes showed normal sper matogenesis (Fig, 6a). Thin layers of interstitial cells were present between the tubules (Fig. 6b). In the testes of two DCA-treated rats, the seminiferous tubules were severely atrophic and contained enlarged Sertoli cells, very few spermatocytes, and no mature spermatozoa (Fig. 6c). Interstitial hyperplasia, characterized by increased numbers of interstitial cells, was most pronounced in areas of severe atrophy (Fig. 6d). In three animals with mild to mod erate testicular atrophy, individual seminif erous tubules showed disrupted spermatogen esis and the formation of multinucleated giant cells. Testicular alterations were not observed in MCA- and TCA-treatcd animals. The priman alteration seen in the brains of DCA-treated rats included vacuolation and gliosis in major white matter tracts and in structures with mixed gray and white matter. In addition, focal areas with neuronal eosinophilia and shrinkage and nuclear pyknosis were observed in gray matter (cerebral cortex, hippocampus, and cerebellum). Although the neuronal changes seemed subjectively more severe in some of the DCA-exposed animals, they did not differ significantly in distribution from similar changes seen in controls. Focal vacuolation and gliosis, on the other hand, were observed only in the DCA-treated ani mals. The structures most severely affected in the forebrain were the heavily-myelinated superior-lateral portions of the corpus cal losum and contiguous hemispheric white matter, the deep layers of cerebral cortex, and the cerebral peduncles. Brain stem structures that showed selective involvement included the medial lemniscus, inferior colliculus, ventral cochlear nucleus, and medial vestib ular nucleus. Other myelinated white matter structures (cervical spinal tracts, pons, cere bellum, anterior commissure) and basal gan glia were less severely involved. An example of the vacuolation in an area with mixed gray and white matter (inferior colliculus) is shown in Fig. 7a. Such structures (including inferior colliculus, basal cortex, pons, and other brain stem nuclei) also had extensive reactive gliosis, as demonstrated by GFAP immuno- Fig. 5. Light micrographs of the lungs of a control rat (a) and rats drinking 80.5 mM DCA (b). 1.9 mM MCA (c), and 80.5 mM DCA (d) for 90 days. Hematoxylin and eosin stain, original magnification, (a.b) I lOx, (c) 275X. (d) 450X, (a) A typical small pulmonary vein (V) is seen branching into a venule in a control lung. Normal alveoli surround the vein and a bronchus (B) is present in the lower right corner (b) Two perivascular inflammatory foci (arrows) are seen on the margin of a pulmonary vein in a DCA-treated rat. A third focus of inflammation may also be present (arrowhead), (c) An inflammatory focus is seen at the junction of a pulmonary vein into a smaller vein (arrow) in an MCA-treated rat. Another possible focus ofinflammation occurs on the opposite wall of the vein (arrowhead), (d) An enlargement of the inflammatory focus at center right in micrograph b shows the presence of lymphocytes (arrows) and macrophages (arrowheads). SL 036970, TOXICITY OF MCA, DCA, AND TCA 247 FlCiUKt" 5 248 BHAT ET AL. Fig. 6. Light micrographs of the testes from a control rat (a,b) and a rat drinking 80.5 mM DCA for 90 days (c,d). Hematoxylin and eosin slain, original magnification, (a,c) 75X, (b,d) 325X, (a) Normal sper matogenesis is occurring within seminiferous tubules and mature spermatozoa (S) are seen in a majority of the tubules. <b) Higher magnification micrograph shows the thin layer of interstitial cells (arrow) present between normal seminiferous tubules, (c) Seminiferous tubules are atrophic and contain large Sertoli cells (St) in the testes of a DCA treated rat. Spermatogenesis is disrupted and no mature spermatozoa are seen, (d) Higher magnification micrograph indicates an increase in the number of Leydig cells in the abnormal interstitial tissue (arrow) of the testes of a DCA-treated rat. staining (Fig. 7b). Neither vacuolation nor gliosis was present in the brains of control rats (Fig. 7c). DISCUSSION In the United States and other parts of the world, disinfection of water by chlorination is the most common practice. In addition to the treatment of drinking water, chlorine is fre quently used to control biofouling at power plants, disinfect waste water from sewage treatment plants, and bleach paper pulp. Thus, the sources for environmental contamination of both fresh and salt water with chlorinated compounds are ubiquitous. Several epidemi ologic studies have indicated a possible asso ciation between the consumption of chlori nated drinking water and various forms of cancer in humans (Cantor et ai, 1985; Cragle et al,, 1985). Chloroacetic acids are major by-products in chlorine disinfection of drinking water. The presence of these chlorinated compounds, therefore, suggest that the human population is constantly at risk ofexposure. Our objective was to compare the toxic effects of mono-, di-, and trichloroacetic acid in a subchronic study in drinking water. In our earlier studies, we have shown that MCA can react with phos pholipids and neutral lipids, and that it forms a conjugate with cholesterol (Bhat and Ansari, 1989). We have also observed that MCA is SL 036972 TOXICITY OF MCA. DCA, AND TCA 249 distributed into hydrophilic tissues at earlier time points and accumulates into lipophilic tissues at later time periods using whole-body autoradiography (Bhat et al., 1990). In this study using equitoxic doses, altera tions in body weight and organ weights were most severe with oral DCA followed by less severe changes with TCA and minor changes with MCA. DCA (80.5 mM) decreased body weight by 34% and increased liver weight by 11%; testes weight was reduced to 66% of con trol values by 90 days. Katz et al. (1981) noted decreased body and liver weights in rats gavaged with 500 and 2000 mg DCA/kg/day for 13 weeks; however, qualitatively smaller testes were observed only in the 2000 mg/kg group. DeAngelo et al. (1989) found that concentra tions of MCA, TCA, or DCA in drinking water from 1 to 5 g/liter decreased body weight in rats after 14 days; liver weight decreases oc curred only in the MCA group. The MCAexposed rats in this study exhibited the least toxicity, but our results indicate that 1.9 mM MCA is sufficient to cause a 10% reduction in liver weight. Morphological changes observed in brain and testes were limited to DCA-exposed rats only. These results are consistent with the findings of Yount et al. (1982) that DCA is neurotoxic and with the studies of Katz et al. (1981) who indicated that brain and testes are the principal target organs of DCA intoxica tion. Katz et al. (1981) found that brain lesions at all doses occurred primarily in the cerebrum and to a lesser extent in the cerebellum and were characterized by vacuolation of the my elinated white tracts; lesions were not observed in optic or sciatic nerves. In the rats of this study, focal vacuolation was observed not onh in the cerebrum and to a lesser degree in the cerebellum, but also in brain stem, cervical spinal cord, pons, and basal ganglia. In addi tion, reactive gliosis was seen in mixed white and gray matter. The doses of TCA and MCA used in this studs produced no alterations in brain, although MC'A is known to be neurotoxic at higher acute doses (Berardi et al.. 1987; Quick, 1983). The presence of small inflammatory foci in hepatocellular zones 1 through 3 and the vari able alterations in portal veins and portal triads among animals suggests chronic mild loss of injured cells with continual removal of dam aged cellular components and initiation of nonspecific portal fibrosis and bile duct pro liferation (Fuller, 1985). Subchronic admin istration of the model toxin, carbon tetrachlo ride, at 33 mg/kg/day for 12 weeks leads to extensive degenerative changes prior to cir rhosis; these changes include bile duct hyper plasia, portal fibrosis, inflammatory foci, ne crotic hepatocytes, lobular distortion, and hy perplastic nodules (Bruckner et al.. 1986). Many different types of liver injury (chemical, viral, and parasitic) can lead to increased de position ofcollagen and eventually to cirrhosis; the mechanism(s) which allows various forms of injury to progress to the same endpoint is unknown (Diegelmann and Linblad, 1985). The perivascular inflammation observed in the lungs of all three treated groups has cell components characteristic of chronic inflam mation. Since the rats were in our animal fa cility for <100 days, and since the control rats showed very minor signs of inflammation, in fection in the animals at the beginning of the study is unlikely to be the underlying cause of the inflammation. The possibility exists that exposure of these rats to chloroacetic acids for 90 days made them less resistant, and thus more susceptible to infection, but infection in the lung is most commonly present in airways or lung parenchyma. Furthermore, the pro gressive effects of these compounds (MCA < TCA < DCA) on portal triads in the liver were paralleled by the degree of perivascular inflammation of the lung which suggests sys temic toxicity of the chloroacetic acids. Katz et al. (1981) observed no hepatotoxic effects of subchronic administration of DCA at 500 and 2000 mg/kg/day in rats, although alterations in liver Kupft'er cells and gall blad der mucosal hyperplasia were seen in dogs at doses of 50 to 100 mg/kg/day. In addition, secondary effects of DCA exposure in dogs were increases in pulmonary inflammatory le- 50 BHA.T ET AL Fic. 7. Light micrographs of the inferior colliculus region of the brain of a rat drinking SO 5 mM DC A tor 90 days (a,b) and a control rat (c). Hematoxylin and eosin/Luxol fast blue stain (a) and <e>. ai idm-hioun immunohistochemical stain for glial hbrillary acidic protein (GFAP) (b). original magnifications, (a-c) 335 X (a) The lesion shows extensive vacuolation. neuronal loss, and gliosis. Vacuoles are associaied '.Mill myelinated axons (MA) and myelin-staining nms surround other vacuoles (arrows), (b) The GFAP immunohistochemical stain demonstrates cell bodies and processes of reactive astrocvles (A) in the same area, (c) Normal architecture of the control brain shows no vacuolation or gliosis. SL 036974 roxiciiY or mca. dca, and rc\ 251 Fig. 7--Conunued sions and ocular keratitis. Differences between the results of Katz et al. (1981) and the present study may be attributed to the method of ex posure (gavage vs drinking water) and the ini tial weight of the rats (150 vs 240 g). Age and body weight are known to influence toxicity of halogenated hydrocarbons (Bruckner et al., 1986). Recently. Mather et al. (1990) reported de creased body weight gains and increased organ to body weight ratios for liver and kidney in rats exposed to 500 and 5000 ppm DCA in drinking water for 90 days. Increased organ to body weight ratios for liver and kidney were also found in rats exposed to 5000 ppm TCA for 90 days. Histologically, livers of DCA and TCA exposed rats had focal areas of intracel lular swelling or pockets of proteinaceous fluid which occasionally disrupted normal liver ar chitecture in the DCA group. Glycogen ac cumulation was significant in enlarged hepatocytes in both DCA- and TCA-exposed rats. Cells of the tubular epithelium and glomeruli showed degenerative changes in the kidneys of only DCA-exposed rats. Mather et at. (1990) found no alterations in testes and brain of rats exposed to 5000 ppm DCA for 90 days but indicated that testicular atrophy and neurological lesions were manifest by 6 months at 500 and 5000 ppm (unpub lished observations). These authors conclude that liver and kidney are the major targets of subchronic exposure to DCA but only at doses greater than those present in the environment or in chlorinated drinking water. The results of our study are generally consistent with the findings of Mather et al. (1990) with the ex ception of kidney toxicity. Current experi ments in our laboratory investigating the spe cific ultrastructural alterations induced in liver by subchronic exposure to MCA. DCA, and TCA will provide further evidence concerning the differences in toxicity among the chloroacetic acids. ACKNOWLEDGMENTS This research was supported by Grants F.S048I5 awarded by the National Institute of Environmental Health Sciences and OH02149 awarded b> the National Institute 252 BHAT ET AL for Occupational Safely and Health ofthe Centers for Dis ease Control, REFERENCES Ayres, S. (1964). Superficial chemosurgery. Arch Der matol. 89, 395-403. Berardi, M. R., Synder, R., Wariez, R. S,, and Cooper, K. R. (1987). 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