l/fAi/L CfHjtftpe. TOXICOLOGY AND AYPUED PHARMACOLOGY 44, 391-399 (1978) Comparison of the Fate of Vinyl Chloride following Single And Repeated Exposure in Rats P. G, Watanabe,1 J. A. Zempel, and P. J. Gehring Toxicology Research Laboratory. Health and Environmental Research, Dow Chemical U.S*4„ Midland, Michigan 48640 Received July 11,1977: accepted October 19,1977 Compxriton of the Fate of Vinyl Chloride following Single and Repeated Exposure in Rats. Watanabe, P, G„ Zempel. J. A-, and Gehring. P. J. (1978). Toxicol, Appl. Pharmacol, 44, 391-399. Rats were exposed by inhalation to 3000 ppm of nonlabeled vinyl chloride (VC) 6 hr/d*y. 3 days/week for 7 weeks. On the last day of repeated exposure l4C -labeled VC was used. The fate of the f l4CfVC was compared in a group of rats exposed repeatedly to a group exposed simultaneously for a single 6 hr period to 3000 ppm of l,4C|VC. The routes and rates of excretion of 14C activity were the same for the two experimental groups. The activity of microsomal enzymes, as reflected by aniline hydroxylase and p-nitroanisole O-demethylase derived from 9000 g liver supernatants, was essentially the same in rats exposed once or repeatedly or in nonexposed control rats. Covalent binding to hepatic macromolecules was greater in rats repeatedly exposed as compared to those subjected to a single exposure. These results indicate that repeated exposure to VC does not induce its biotransformation. However, the increase in hepatic macromolecular binding indicates that repeated exposure augments the reaction of electrophilic metabolites with macromolecules, and this may be expected to enhance potential toxicity including carcinogenicity. While many pharmacokinetic and metabolic studies have been conducted on vinyl chloride (VC), most of these investigations have concentrated on the fate of VC during and following single exposure. Since cancer has been induced by long-term repeated exposure of both experimental animals and man to VC, it is important to consider alterations in the disposition of VC in the body which may occur after repeated exposure. In preliminary studies, monochloroacetic acid was tentatively identified as a major urinary metabolite following repeated exposure (6 hr/day, 5 days/week for 9 weeks) to 5000 ppm of nonlabeled VC (Hefner ef al„ 1975). However, this metabolite has not been found in rats given a single exposure to 1,4C|VC (Watanabe et al., 1976). This raised the question of whether the biotransformation of VC may be altered upon repeated exposure. A change in the biotransformation of VC after repeated exposure is particularly important in assessing the hazard of exposure to VC since evidence suggests that VC is metabolized to a reactive metabolite which is ultimately responsible for its toxic manifestations. This concept is supported by studies showing an enhancement of the 1 To whom correspondence sheukj be sent. 39| (XU l -00*X/‘tt/6 liters/min) with a dual syringe pump. The nominal concentration of VC was determined from the ratio of the rate at which the VC gas was dispensed and the total chamber air flow. The analytical concentration of VC was monitored continuously by recirculating a fraction of the chamber atmosphere through an infrared spectrophotometer (Wilks) set at 10,6 pm. The rats repeatedly exposed to VC were exposed 6 hr/day, 5 days/week for 7 weeks (32 exposures in 44 days). The mean analytical concentration of VC over the 7 week exposure period was 4775 + 908 (SD) ppm. On the final day of exposure the animals were subjected to a 6 hr exposure to l4Clabeled VC generated in the same manner as described above. On this final day of 625086 FATE OF VINYL CHLORIDE IN THE RAT 393 exposure, the chamber atmosphere was also analyzed at hourly intervals by gas chromatography, and at the same times the l4C activity was determined by bubbling 1 mi aliquots or the chamber atmosphere into a scintillation solution containing Concifluor (Mallinckrodt Chemical), 2-methoxyethanol, toluene (6:11:83) (Watanabe el al., 1976). The radioactivity was determined by counting in a liquid scintillation spectro­ meter. The mean analytical concentration determined on the final exposure day, when the animals were exposed to ,4C-labeled VC, was 4600 ±311 (SD) ppm. The specific activity was 50 dpm/jrg of VC. The inhalation chamber was operated in a laboratory fume hood to prevent contamination of the working environment. After transit through the inhalation chamber the [,4C|VC was adsorbed on activated charcoal. These traps were disposed as radioactive waste according to standard regulations. Procedure. Eight rats were exposed repeatedly to VC as described previously. On the last day, five additional unexposed rats and the eight exposed repeatedly were exposed to 5000 ppm of l'4C|VC for 6 hr. Following this final exposure to [UC1VC, three of the eight exposed repeatedly and two of the five exposed once were placed in glass Rothtype metabolism cages for the collection of urine, feces, and expired air. Room air was drawn through the cages at 400-500 ml/min. The exiting air was passed through a series of traps to collect the expired [“C]VC and “CO,. The air leaving the chamber was passed first through a glass tube containing about 40 g of Drierite (W. A. Hammond Drierite Co.) to remove moisture. Subsequent transit through a series of two cold finger traps containing 50 ml of toluene, 2-methoxyethanol (80:20) and a single trap containing 120 ml of 5 m ethanolamine in 2-methoxyethanol enabled the collection of luC|VC and “COp respectively. The cold finger traps were immersed in 2-mcthoxyethanol-dry ice baths throughout the collection periods. The trap for CO2 was maintained at room temperature. Samples of excreta were collected for 72 hr after termination of exposure and analyzed for ,4C activity. Expired VC was collected at 0.5-hr intervals for 3 hr; the CO, trap and urine receptacle (immersed in dry ice bath) were changed at 12-hr intervals for 72 hr; and feces were collected every 24 hr. At the termination of the study (72 hr) the animals were killed by a blow to the head, and samples of tissues (fat, kidney, liver) were collected for analysis of ,4C activity. The remaining carcass was skinned and homogenized (50% w/v) in distilled water and analyzed for 14C activity. The samples of excreta and tissue were prepared for scintillation counting as described previously (Watanabe et a/.. 1976). Carbon-14 activity was determined by counting in a Mark II or Mark III liquid scintillation spectrometer (Searlc). External standard-channels ratios were used to deter­ mine the counting efficiency. The counts per minute were converted to disintegrations per minute using a standard quench curve. The remaining rats in the groups exposed repeatedly and singly to VC, five and three respectively, were killed by a blow to the head immediately following exposure. A piece of liver was used to prepare a 9000 g supernatant in 1.15% KC1 in order to determine aniline hydroxylase (LaOu et at., 1971) and p-nitroanisolc O-demethylase (Kinoshita et al., 1966) activity. Macromolecular binding of radioactivity to hepatic tissue was determined by the method of Jollow et al. (1974). This method measures nonextractable radioactivity from a trichloroacetic acid precipitate which includes protein. 625087 394 WATANABE, ZEMPEU AND CEHRING lipids, and nucleic acids. The carcass was analyzed for total radioactivity as described above. A second experiment was conducted to confirm parameters obtained for binding of radioactivity to hepatic macromolecules. The methodology was the same as described above. A group of rats was repeatedly exposed to VC 14821 + 259 (SD) ppml for 8 weeks. On the last day of exposure 1I4C1VC was used and an additional four rats (singly exposed group) were added. The mean analytical concentration of the luClVC on the last day of exposure was 5065 ± 30 (SD) ppm and the specific activity was 34 dpm/^gof VC. RESULTS Excretion of ,4C activity within 72 hr after a single or repeated exposure to 5000 ppm of (14C)VC is shown in Table 1. The percentage of ,4C activity excreted by each route as well as the total milligram equivalents of VC recovered were essentially identical for the singly and repeatedly exposed groups. The majority of 14C activity eliminated was expired as VC per se. TABLE 1 Percentage “C-Acnyirv Eliminated by Rats During 72 hr Following Inhalation Exposure to 5000 ppm of (“CIVinyl Chloride4 Single exposure (2)* Expired: As VC As CO, Urine Feces Carcass and tissues Total VC recovered Repeated exposure (3)* (%) (mg equiv of VC) (%) (mg equiv of VC) 54.5 ± 3.5 8.0 ± 1.4 27.1 * 2-1 3.2 t 2.5 7.3 + 2.5 14.00 2.05 6.93 0.80 1.89 53.7 ±2.1 9.6 ± 1.6 25.7 ± 1.4 1.4 ± 0.4 9.7 ± 1.6 12.94 2.27 6.21 0.32 2.32 25.67 24.07 * Expressed as percentage of trie total "C activity recovered, mean ± SD. * Number of rati. The time course for expiration of 1,4C]VC per se (Fig. 1) and urinary excretion of l4C activity (Fig. 2) were also essentially identical for the singly and repeatedly exposed rats. The curves were fit by linear regression analysis of the logarithmically transformed data. The estimate of the apparent first-order rate constant for expiration of VC was 0.023 t 0.01 (SD) min*1 which corresponds to a half-life of 30 min. The elimination of urinary ,4C activity was biphasic. An estimate of the apparent first-order rate constant for the initial portion of the urinary excretion curve from 12 to 36 hr was 0.155 ± 0.02 (SD) hr'1 which corresponds to a half-life of 4.47 hr. The data for the slow phase of urinary excretion were extremely variable', therefore no attempt was made to estimate the excretion rate. Less than 1% of the radioactivity excreted in the urine occurred during the slow phase. 62S088 FATE OF VINYL CHLORIDE IN THE RAT 395 Urinary MC activity was separated by thin-layer chromatography in n-butanol, acetone, H,0 (50:20:30) on cellulose and in /i-butanol, acetic acid, HjO (80:20:20) on silica gel. The profiles of radioactivity for rats exposed repeatedly or singly were qualitatively similar and no significant radioactivity was associated with the Rvalue of a standard of monochloroacetic acid. The concentration of radioactivity detected in tissue 72 hr after exposure revealed no statistically significant difference between rats exposed once or repeatedly to VC (Table Fms. 1. Expired vinyl chloride lmiUi|rims) versus time following a 6 hr exposure to 5000 ppm of I "ClVC. Repeatedly exposed rats I A) and singly exposed rats (•). 2). It does appear that, in those exposed repeatedly, more radioactivity may have been retained in the liver and skin: however, the number of animals used does not provide for an adequate statistical evaluation. The effect of VC on xenobiotic drug metabolism by liver 9000 g supernatants, as refleaed by aniline hydroxylase and p-nitroanisole O-demethylase activity, is presented in Table 3. Neither single nor repeated exposure to 5000 ppm of VC altered discernibly the enzyme activity in either system when compared to air-exposed controls. The total amount of VC biotransformed and the hepatic macromolecular binding of UC aaivity following single and repealed exposure are shown in Table 4. The total amount of VC biotransformed was not significantly different between the two groups. However the hepatic macromolecular binding in the first experiment appeared to be increased in the rats exposed repeatedly. When the protein binding was corrected for the 625089 396 watanabe, zempel, and CEHRING Fig. 2. ,4C-Activity excreted in the urine expressed as percentage of the recovered radioactivity versus time following a 6 hr exposure to 5000 ppm of l“ClVC. Repeatedly exposed rats (A) and singly exposed rats (•). amount of VC biotransformed (B/A x 100) a statistically significant increase was found. Because the increased binding in repeatedly exposed rats was not definitive in the first experiment, the experiment was repeated and the results are shown in the lower portion of Table 4. The second study confirmed the observation that rats repeatedly TABLE 2 Percentage “C-Acnvrrv 72 hr Following Inhalation Exposure of Rats to 5000 ppm of Vinyl Chloride4 14C Activity/g of tissue (%) Tissue Single exposure (3)* Repeated exposure (2)* Liver Kidney Fat Skin Carcass 0.119 ± 0.022 0.062 ± 0.026 NDr 0.046 ±0.015 0.030 ±0.014 0.157 ± 0.028 0.070 ± 0.006 NDf 0.080 ±0.019 0.039 ± 0.011 * Expressed as percentage of the total “C activity metabolised, mean * SO. * Number of rats per group. ’ Not detectable, detection limit of 3 //g of VC equiv/g of fat or 0.03% “C activity metaboliied/g of tissue. 625090 FATE OF VINYL CHLORIDE IN THE RAT 397 exposed to VC bind about 20-25% more reactive metabolite to hepatic macromolecules than do rats exposed once. It is not clear why the magnitude of the binding was slightly greater in the second experiment. Nonetheless, these results indicate that a larger fraction of the biotransformed VC reacts covalently with hepatic macromolecules in rats exposed repeatedly. TABLE 3 Effect of Vinyl Chloride on Drug Metabolism By a 9000 g Supernatant Fraction of Liver of Rats* Amount (Mg of produci/g of liver/hr) Control (4/ Single VC exposure (3)* Repeated VC exposure (5)* Aniline hydroxylase p-NitroanisoIe O-demethylase 65 ± 16 71 + 7 83 ± 10 226 + 22 254 + 45 217 ± 31 * Animals were killed immediately followinf the last exposure and etuyme activity was assayed. Values are mean ± SD. * Number of rats per {roup. TABLE 4 Total Metabolism and Hepatic Macromolecular Binding Following Single or Repeated Exposure of Rats to 5000 ppm of Vinyl Chloride* A VC equivalents metabolized («) B VC equivalents bound 0/g/gof protein) Binding corrected for metabolism Single exposure Repeated exposure 9265 + 1467 8718 ± 895 114+10 124 + 10 1.12 + 0.13 1.43 +0.16* Experiment was repeated Single exposure Repeated exposure 8746 ± 882 9421 ± 482 148 + 25 195 + 24* 1.69 ±0.28 2.07 ± 0.25* (B/A x 100/ * Values are the mean ± SD (three to five rats per (roup). * The ratio of B/A * 100 was calculated from individual animal data. ' Statistically sifnificamly different from the single exposure. Student t test (p < 0.05). DISCUSSION Repeated exposure of rats to 5000 ppm of luClVC did not alter discemibly the routes or rates of excretion of radioactivity or qualitatively the excretory products formed from VC or VC per se. These results do not substantiate the previous preliminary observation that monochloroacetate may be a major biotransformation product of VC (Hefner et al.. 1975). 625091 398 WATANABE. ZEMPEL. AND CEHRING A most significant finding in the study was a significantly increased amount of radioactivity bound covalently to macromolccules of rats exposed repeatedly to VC versus those exposed once. An associated observation was the retention of an apparently greater level of radioactivity in the liver of repeatedly exposed rats 72 hr after exposure than those exposed once. These results indicate that toxic manifestations, including carcinogenicity, associated with the reaction of reactive metabolites of VC with macro­ molecules may be enhanced by repeated exposure to VC. While the binding of reactive metabolites of VC to hepatic macromolecules was enhanced following repeated exposure, no differences were observed in the activity of hepatic microsomal enzymes to the substrates aniline or p-nitroanisole in any of the treatment groups when compared to nonexposed control rats. Thus, it did not appear that exposure to VC at this concentration influenced microsomal metabolism. However, in contrast to these findings was the observation by Reynolds et al. (1975b) that the cytochrome P-450 content and the oxidative V-demethyladon of aminoantipyrine and ethylmorphine were markedly depressed in rats following exposure to 50,000 ppm of VC for 6 hr. The difference between our study and that of Reynolds et al. (1975b) is that we used substrates which cause a “type II” binding spectra compared to their use of substrates producing a “type I” binding spectra with hepatic microsomes. In addition Reynolds et al. (1975b) used a 10-fold greater concentration of VC. The apparent discrepancy can be explained by the recent observation that VC causes a “type I” binding spectra when incubated with hepatic microsomes (Salmon, 1976; Ivanetich et al., 1977), and furthermore it has been demonstrated (Ivanetich et al., 1977) that high concentrations of VC can degrade cytochrome 7M50. Thus it appears that VC is metabolized by the hepatic microsomal enzymes and is capable of inhibiting metabolism of other substrates by competitive inhibition or by degradation of cyto­ chrome P-450. In conclusion, the results of these studies showed that repeated exposure to high levels of VC did not alter discernibly the routes or rates of excretion of radioactivity when compared to rats subjected to a single 6 hr exposure to 5000 ppm of (14C1VC. Of particular significance was evidence that the binding of reactive metabolites of VC with hepatic macromolccules was enhanced bv repeated exposure to high levels of VC. 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