Document ZBxmExe5gQXLOz5Dkx1R81NJ0

TOXICITY OF VINYLIDENE CHLORIDE IN MICE AND RATS AND ITS ALTERATION BY VARIOUS TREATMENTS Robert D. Short, Joseph M. Winston, Jan L. Minor, Chuen-Bin Hong, Joseph Seiftcr, Cheng-Chun Lee Pharmacology and Toxicology, Midwest Research Institute, Kansas City, Missouri The toxicity of vtnvhricne chloride fVOCI win studied posed to ' additionfth^oility of various Compounds to alter parameters i^dilffT^^fB^FUluoted. Mice were more sensitive than rats to the lethal, hepatatoxie. and renal tone effects of VDC. Disulflrant protected mice from these toxic effe IS of inhaled VDC and reduced the levels of cotolent/y bound radioactivity in the hver and kidney after the ip administration of ("Cl VOC. Oiethyldlthiocarbamate and thiram also protected mice Jram the acute lethal effects of VOC. INTRODUCTION Vinylidcne chloride (1,1-dichloroethyleno; VDC) is used as a monomer in the production of plastics and an intermediate in the synthesis of other chemicals. The toxicity of VDC has been reviewed (Haley, 1975; Huffman and Desai'Grccnaway, 1976). A continuous 90 day inhalation of 189 mg/m3 (48 ppm) of VDC produced deaths in monkeys and guinea pigs but not in dogs and rats (Prendergast et al., 1967). Morphological changes occurred in livers from monkeys, dogs, and rats and in kidneys from rats. The hepatic changes included focal necrosis, hemosiderin deposition, and fatty metamorphosis. The primary renal lesion was nuclear hypertrophy of the tubular epithelium. Various parameters influenced the toxicity of VDC. Female rats were The author* (fatefully acknowledge the competent technical assistance of Brett Partition, Tim Unger, and Mary Sawyer. This reiearch wat supported by Environmental Protection Agency contract 6B~01-3242. Preliminary results were presented at the Pint International Congress on Toxicology. March 30 to April 2, 1977, Toronto, Canada, and at the conference on Comparative Metabolism and Toxicity of Vinyl Chloride Related Compounds, May 2-4, 1977, National Institutes of Health, Btihesda, Maryland. ). Scifter't present address It Office of Toxic Substances, Environmental Protection Agency, Washington, D.C. 20460. Requests for reprints should be sent to Robert D. Short, |r., Pharmacology and Toxicology, Midwest Research Institute, 425 Volker Boulevard, Kansas City, Missouri 64110. i j I j j j ' J f I f 1 ' l r j ' Journal of Toxicology and Environmental Health, 3:913-921,1977 Copyright C 1977 by Hemisphere Publishing Corporation M h ! J T (J (j S\ 066321 014 R. D. SHORT ET AL. more sensitive than males to the toxic effects of VDC, which was administered by the oral route (Jaeger ct at., 1973b). Nutritional status affected toxicity, since starved rats were more sensitive to VDC than fed rats (Jaeger ct al.. 1973c). This nutritional effect and a diurnal change in sensitivity to VDC (Jaeger ct al., ,1973a) were correlated with hepatic levels of reduced glutathione (GSH). Additional studies with diethylmaleate (Jaeger ct al., 1974) and cysteine (Jaeger ct al., 1975) pretreat ment also indicated that hepatic levels of glutathione influenced the toxicity of VDC. Conjugation with GSH was a major pathway for the detoxification of VDC and hcpatotoxicity was associated with the covalent binding of reactive metabolites (McKenna ct al., 1977). The purpose of this study was to evaluate the acute toxicity of continuously inhaled VDC in both mice and rats and to evaluate the effect of various treatments on toxicity. The treatments were selected to alter the metabolic activation or promote the detoxification of VDC. In addition, adrenergic blocking agents were used since VDC exposure sensitized rat hearts to catacholamincs (Silctchnik and Carlson, 1974). METHODS CD-I mice and CD rats (Charles River Breeding Laboratories, North Wilmington, Massachusetts) were used in these studies. Animals were given fiee access to feed (Wayne Lab-Blox, Allied Mills, Inc., Chicago, Illinois) and tap water at all times during the study. affl^^^ff^imiiarl^ousc^n^xposccH^oo^air^^^^^^ VDC was obtained from Milwaukee, Wisconsin, ermeon nr cameo me vapors irom in iber atmosphere was sampled from a central point. Preliminary experiments, utilizing at least eight additional sampling points, indicated that there was a uniform distribution of VDC within the chamber. The concentration of VDC was measured with a Varian 2700 gas chromatograph equipped with a flame ionization detector and a stainless steel column packed with 0.4% Carbowax 1500 on Carbopak A. These determinations were made on the average every 2-3 hr during exposure. Mice received one of several compounds in an effort to alter the toxicity of VDC. These compounds were disulfiram (0.10% in feed 2-3 days before and during exposure), dicthyldithiocarhamatc (DUC) (0.12% in feed 3 diys before and during exposure), thiram (0.10% in feed 3 clays before and during exposure), cysteine (0.10 or 0.50% in feed 3 days before and during exposure), methionine (0.10 or 0.50% in feed 3 days before and during exposure), AZ-acctylcystcinc (1,200 mg/kg po every day SL 066322 TOXICITY or VOC AND ITS ALTERATION 91J during exposure), SKF 525-A (50 mg/kg ip every day during exposure), cobaltous chloride (CoClj^HjO) (60 mg/kg ip daily for 2 days before exposure), phcnoxybcnzaminc (10 mg/kg ip daily for first 2 days of exposure), propranolol (10 mg/kg ip daily during exposure), vitamin C (100 mg/kg ip daily during exposure), or DL-cr*tocopherol acetate (vitamin E, 750 mg/kg orally 2 days before exposure and on first day of exposure). Control and treated mice were housed in the same chambers and, consequenjh^exposcd to identical conccntration^^VDC ->rgan tUlllJgP WJA TOGWCU m term^o^erum enzymes anc ustopatnological changes in the liver and kidney. Serum glutamicoxaloacetic transaminase (SGOT) and scrum glutamic-pyruvic transaminase (5GPT) were determined in cardiac blood from mice and aortic blood from rats by the methods described by Amador and Wacker (1962) and Henry et al. (1960), respectively. Covalently bound radioactivity was measured in male mice at 4 and 24 hr after the ip administration of 3 mg/kg (20 pCi/kg) |'*C1VDC (New England Nuclear, Boston, Massachusetts) with a specific activity of 0.652 mCi/mmol. The tissues were homogenized in cold water and an equal volume of 1 N perchloric acid (PCA) was added. The precipitate was washed with successive 5 ml portions of 0.2 N PCA, 0.2 N PCA, 95% ethanol saturated with sodium acetate, absolute ethanol, and ethanol and ether (3:1) and then heated for 1 hr at 37C in 4 ml 0.5 N sodium hydroxide. Afterward, 3 ml of 30% trichloroacetic acid (TCA) was added and the precipitate was washed with 5 ml 5% TCA and heated in 5% TCA for 20 min on a boiling-water bath. The pellet was washed with 5 ml 5% TCA and dissolved in 10 ml 0.3 N sodium hydroxide. Radioactivity and protein (Lowry et al., 1951) were determined on this fraction and the results were expressed as dpm/mg protein. Mortality data were evaluated in terms of the LCS0 (Weil, 1952) and the LTJ0 (Litchfield, 1949) for VDC. Other data were analyzed by the two-sample rank test (Goldstein, 1969) or the Fisher exact probability test (Siegel, 1956). The level of significance selected was p < 0.05. RESULTS Rats and mice had reduced feed consumption and weight loss during exposure to VDC. After 2 days of exposure to 60 ppm VDC, 8 of 10 male mice and 0 of 10 male rats died. In addition, VDC produced more organ damage, as measured by elevated scrum enzymes, in mice than in rats (Tables 1 and 2). A dose-related increase in both SGOT and SGPT occurred in male mice exposed to VDC for 1 day (Table 1). As the duration of exposure increased, these values tended to decline. The scrum enzymes were elevated in male rats exposed to VDC; however, the increase was not as dramatic as that in mice and required a longer exposure period SL 066323 ' 4 *1 91* *. D. SHORT ET AL. TABLE 1. Effect of VOC Exposure on Serum En/ymei in Mile Mice Dayt opened 1 2 3 5 Scrum entyme SCOT sc.rr SCOT SCPT SCOT SCPT SCOT SCPT VOC concentration (ppm) 0 IS 30 56 f S (5)" 32 t 6 (5) 82 t 30 (4) 38 >4 (4) 64 t 14 (5) 2? * 2 (S) 60*5 15) 32 t 2 (S) 249 t 28 (4) 200 t 19 (4) 91 tS (S) 189 t 26 (5) , 138 t 16 (4) 166 4 33 (4) 1181 >8 (6) 47 t 5 (6) 466 t 50 (5) 4 74 t 66 |S) 70S t 120(6) 675 1 81 (6) 448 t 1 79 (4) 514 t 200 (4) 297 * 50 (2) 109 i 8 (2) *Mcan t SE (number of mice). 60 1,946 i 271 |4| 3,043 t 209 (4) TS1 * ISO (2) 1.112 t 226 (2) No lurvivort No lurvivort (" iblc 2). Toxicity, as revealed by these biochemical indicators, was confirmed by histopathological observations. Hepatic and renal lesions were present in male mice exposed to IS, 30, and 60 ppm of VDC for various intervals (Table 3). In addition, hepatic but not renal lesions were observed in male rats similarly exposed to 60 ppm of VDC (Table 4). None of the lesions observed in mice and rats exposed to VDC occurred in control animals exposed to room air. Groups of mice were given one of several compounds in order to investigate their effects on the toxicity of VDC. Among the various compounds tested, disulfiram, DDC, and thiram decreased the acute lethality of VDC (Tables S and 6). Disulfiram, in addition, protected male mice from the hepatotoxic effects of VDC as measured by serum enzymes (Table 7). However, this protective effect was evident after the first, but not the second or third, exposure day. Histopathological confirmation of this protective effect was complicated by the high mortality in nontreated animals exposed to VDC. However, the data indicated that disulfiram TABLE 2. Effect of VOC Expoture on Scrum Entymct in Malt Rill Dayt expoted Scrum enzyme* VDC concentration (ppm) 0 60 1 SGOT 66 t 10 (JJ* 74 * S (S) SCPT 28 1 2 (5) 44 * 7 (5) 2 SCOT 6) t 4 (5) 264 t 33 (5) SCPT 34 t 4 (S) 198 t 29 (5) 3 SCOT 81 t 4 (5) 238 t 47 (5) SCPT 34 t 6 (S) 122 1 29 (S) Mean t SE (number of rati). SL 066324 TOXICITY or VDC AND ITS ALTERATION 917 TABLE 3. Microscopic levens m Organs ol Male Mice Exposed to VDC VDC: 15 ppm 30 ppm 6Q ppm Exposure days: 1 7 3 3 1 7 3 5 1 2 Number mice examined 5 3 5 7 5 S 4 242 Liver leptons Muitonji necrosis Mild 0 0 0 20 40 so 0 0 SO Moderate Marked 0 0 0 0 0 0 0 0 so SO 0 0 0 0 0 0 0 0 so 0 Mitotic figures of hepatoctes Slight increase 0 60 40 0 0 0 0 0 0 0 Moderate increase 0 0 0 0. 0 0 75 0 00 Hepatocellular degeneration 10 40 :o *6** 20 40 0 so 25 0 Kidney teswvn Tubular nephrosis Moderate 0 0 0 57 0 0 0 0 0 0 Marked Severe Tubular regeneration oO 0 0 0 43 0 0 0 0 0 0 100* too* 0 100* too* too* 100* so 100* 0 0 0 6* 0 0 0 0 0 0 "Pficcnutt ol mice examined with the indicatrd lesion. None of these lesions were observed in (he control (roup (five mice a dry) exposed to room sir. ^Significantly different from control group (Fisher execl probability protected mice from the hepatic and renal toxicity of VDC (Tabic 8). Additional evidence supportipg a protective effect of disulfiram was obtained in covalent binding studies (Table 9). Radioactivity, which was covalently bound to proteins, was measured in the liver and kidney at 4 and 24 hr after the ip administration of (14C|VDC. These levels of radioactivity were reduced at both times in mice treated with disulfiram. T ABLE 4. Microscopic Lesions m Organs of Male Rats Exposed to 60 ppm VDC Eiposurt days I2 Number of rats examined S Liver lesions Centrilobular degeneration and/or necrosis Mild 4<f 10* 40 Moderate 0 0 60 Mild hyperplasia of bile duct 60 40 0 ^Percentage of rats examined with the Indicated lesion. None of these lesions were observed in the control troop (five mice a day) exposed to room air. * Significantly different from control troop (Fisher exact probability test). TABLE j. One Diy LC,, (ppm) of VDC in Mice Trcitment Control Disulfirim (0.10%) Cvsteine (0.10%) Methionine (0.10%) CoCt, Mile 91 (82-118) > 320 9S(76-127) 1 1 3 (93-1 38) 113 (81-157) Femite 105 (92-121) > 320 92(74-113) 11 3 (93-1 38) 123 (85-179) LCIt in ppm (95% confidence limiti) or ipproximition of LC,,. TABLE S, Two Diy LC,, (ppm) of VDC in Mice Treilment Mile Control Disulfirim (0.10%) DOC (0.12%) Thirim (0.10%) Af-Acetylcysteine Methionine (0.50%) Cysteine (0.50%) SKf J25-A Phenoxybenrsmine Proprinoiol Vitimin C Vitimin E 35 (25-47) > 160 > 160 > 160 20 (7-25) 38 (28-51) 43 (31-58) 26 (1 7-35) 35 (25-47) 28 (19-37) 35 (25-46) 35 (25-47) LC,, in ppm (95% confidence limits) or ipproximition of LC,,. TABLE 7. Toxicity of 60 ppm VDC in Control end Dilulfirim-Treiled Mele Mice Diet Diys exposed to VDC SGOT (lU/liter) SCPT (lU(luer) Control 1 1,946 1 271 (4| 3,043 1 209 (4) 2 751 t 150(2) 1,112 t 226 (2) Disulfirim 3 1 No igrvivort 140 * 38 (5)* No survivors 66 til (5)* 2 784 1 332 (4) 1,236 t 668 (4) 3 2,292 t 965 (3) 3.182 t 1,488 (3) Mein j SE (number of obscrvilions). ^Sijnificintly different from control (two-simple rink test). 911 I .* SL 066326 TOXICITY F VDC AND ITS ALTERATION 919 TABLE 9, Microxcopn Leuont in Organ* of Mala Mice Expottd lo 60 ppm VOC and Treated with Oisuffiram Treaimenl: None Diiulfirams Expoiure days: 1 2 I2 3 Number of mice examined 4 2S s 3 UvtT Midzorui necrosis Mild 0 SO 0 0 0 Moderate Marked so 50 0 0 0 so 0 0 60 0 Severe 0 0 0 0 67 Hepatocellular degcneniiofl 25 0 0 20 33 Kidney tenon* Tubular nephrons Miltl 0 0 20 40 0 Moderate 0 0 20 20 0 Marked SO 0 0 40 100 Severe SO 100 0 0 0 Dixulfiram (0.I14) in iced 1 day* before and during exposure lo VOL. ^Percentage of mice examined with the indicated lesion. None of these lesiom were observed in group* (five mice a day) fed either control or disulfiram diets and exposed to room air. DISCUSSION Mice arc more sensitive than rats to the lethal, hcpatotoxic, and renal toxic effects of VDC. Disulfiram reduces the severity of these effects in mice. In addition, DDC and thiram protect mice from the lethal effects of VDC. The compounds that protected against the toxicity of VDC are dithiocarbamjtcs. Disulfiram is used clinically in alcohol therapy programs and thiram is used in the agricultural and rubber industries. Disulfiram is metabolized to DDC (Stromme, 1965) and both compounds alter the metabolism of xenobiotics (Zemaitis and Green, 1976). These compounds, TABLES. Covalently Bound Radioactivity alter |'*C|VOC in Control and Diwlfiram-Trcated Male Mice Tiutfc Liver Kidney Time after ['*C| VDC (hr) 4 24 4 24 Activity (dpm/m| protein) Con trot l t 14s $8 x 2 134 t 9 88 t 2 DHulfira' 3t t 7* 23 t S'1 32 t 7 27 t 4 aMcan * SE for four determination!. * Significantly different from control (iwo-iample rank irji). SL 066327 920 ft. 0. SHORT ET AL. in addition, protect against several types of drug-induced toxicitics (Lutz et al., 1973; Wattenberg, 1974). Dithiocarbamatcs also have radio protective properties (Barnes et al., 1975). Speculations concerning the toxicity of vinyl chloride (Watanabc and Gchring, 1976) form a basis for similar speculations concerning the toxicity of VDC. VDC is mctabolicnlly activated to a reactive metaholite that is detoxified by combining with GSH (McKenna et al., 1977), As the levels of GSH arc depleted, the reactive metabolites arc able to combine with macromolccules and produce toxicity or carcinogenicity This proposal is supported, in the present study, by the presence of covalently bound ratioactivity in the liver and kidney after |,4C|VDC. There are a variety of mechanisms by which the dithiocarbamaic compounds tested could reduce the toxicity of VDC. These mechanisms include a decrease in metabolic activation and an increase in detoxifica tion. If protection occurred as a result of reduced activation by the hepatic microsomal mixed-function oxidase system, then SKF 525-A (Conney et al., 1966) and cobaltous chloride (Tephly and Hibbcln, 1971) should have produced similar effects. If protection resulted from an increase in sulfhydryl groups for the detoxification of VDC metabolites, then /V-acctylcystcine (Piperno and Berssenbruegge, 1976) and other sulfhydryl-containing compounds should also have protected. Since these interaction studies arc complicated by dosage and pharmacokinetic con siderations, it is possible that we failed to detect an interaction because of these factors. In addition, since death and organ damage may not be related, it is possible that compounds that failed to protect against death provided protection against organ damage. The results suggested that the mechanism of protection provided by the dithiocarbamatcs involves more than an inhibition of activation or an increase in detoxification of VDC. Possibly both mechanisms are operating at the same time. For example, these compounds may have reduced the activation of VDC and increased the detoxification of reactive metabolites. In this regard, dithiocarbamates may serve as more effective molecules for detoxifying VDC metabolites than some of the sulfhydryl-containing compounds that were tested. REFERENCES Amador, E. and Wacfctr, W. 0. C 1962. Scrum glutamic-oxaloacetic trantammate activity: A new modification and an analytical assessment of current assay technique!. Clin. Chrm. 8:343-341. Barnet, |, H,, Fatome, M,, Etdemont, C. F.. Andriev, L. and Bariy, E. 197S. Synthetet et effett radioprotecteuri d'alkanebitdithiocarbamates dltodiquct, d'acidct w-amlnoalkyldithiocarbamiqom et de leurt derivet N,N'-dimethylet. F.ur. /. tied. Chrm. Chim. Thrr. 10:619-622. Conney, A. H.. Santur, M., Sotoko, F,, Raster, It. and Burnt, |. |. 1966. Entyme induction and inhibition in ttudiet on the pharmacological action! of acatophtnetidin. J. Pharmacol. Etp. Thrr. 141:133-138. Coidttein, A. 1969. Bkistatiitin, on introductory real. New York: Macmilllan. ''C SL 066328 1.. 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Impairment of hepatic microsomal drug metabolism in the rat during daily disulfiram administration. Biochem. Pharmacol, 25:1355-1360. Rci ru ed jut) 21, 7977 Accepted September 9, 7977 w, SL 066329