Document 72O9y3YL3OOZM8jyydzmkqxV

R&S 041230 PRELIMINARY STUDIES OF THE FATE OF INHALED VINYL CHLORIDE MONOMER IN RATS R. E. Hefner, Jr,, P. G. Watanabe, and P. J. Gehring Reprin ted from ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Volume 246, Pages 135-148 January 31, 19 75 B. Experimental Studies PRELIMINARY STUDIES OF THE FATE OF INHALED VINYL CHLORIDE MONOMER IN RATS* R. E. Hefner, Jr., P. G. Watanabe, and P. J. Gehring Dow Chemical U.S.A. Health and Environmental Research Midland, Michigan 48640 Introduction Vinyl chloride monomer (VCM), extensively used for the production of poly vinyl chloride and other plastics, has been associated with the development of angiosarcoma and portal cirrhosis of the liver as well as other untoward effects in workers exposed to unknown but undoubtedly high concentrations of VCM. Angiosarcomas, zymbal gland carcinomas, and nephroblastomas developed in rats exposed to concentrations of VCM ranging from 50-10,000 ppm, 4 h/day, 5 days/week for 12 months, and subsequently maintained and observed until death.1 No information is available on the fate of VCM in the mammalian organism at the present time. Such information is essential for elucidating the toxicodynamics of VCM and providing a rationale to assess the potential hazard of exposure to low levels of VCM. Results of preliminary studies on the fate of VCM in rats exposed via inhalation are reported herein. Prior to undertaking these studies we conceived the idea that VCM might be metabolized via the alcohol dehydrogenase pathway, and if so, this pathway would very likely be saturable, and that con jugation of VCM or its metabolites with glutathione and cysteine might be ex pected. Therefore, much of the work conducted to date has been directed at evaluating these possibilities. Methods Kinetic Studies of the Uptake (Metabolism) of Inhaled Vinyl Chloride Monomer Male Sprague-Dawley albino rats of Spartan strain weighing from 165-200 g were exposed to initial concentrations of VCM gas ranging from 50.5-1167.0 ppm (0.13-2.99 mg/liter). Exposures ranged from 52.5-356.3 min. FIGURE 1 depicts the closed, recirculating 4.7-liter inhalation apparatus in which a group of 4 rats were concurrently exposed. To minimize contamination of fur and skin, only the nares of the rats protruded through a rubber membrane into the cham ber. Expired carbon dioxide was continuously removed from the chamber by absorption on an Ascarite column in the recirculating system. As carbon dioxide was removed from the system, the pressure drop was detected by a mercury manometer fitted with a photoelectric cell. This activated a solenoid valve and dual syringe pump to inject makeup oxygen into the system. The chamber atmosphere was continuously analyzed for VCM (10.9 n) by an in-line Miran-I infrared analyzer (Wilks). With known concentrations of VCM * These studies were supported in part by a grant from the Manufacturing Chemists Association. 135 136 Annals New York Academy of Sciences R&S 041232 Figure 1. Closed recirculating inhalation apparatus used lor exposing rats to VCM. prepared in 100-liter Saran bags, the absorbance versus concentration adhered to the Beer-Lambert Law, During the exposures, routine checks for carbon dioxide in the chamber atmosphere were made at 4.26 fi. Oxygen consumption for each experiment was determined directly by measuring the oxygen metered into the system using a dry test meter (American Meter Co.). Prior to each exposure, the desired initial concentration of VCM was gen erated in the empty recirculating system and the decline in concentration was followed for a time equal to or exceeding that of the intended experiment. The decline in concentration was in accordance with first-order kinetics as described by dC,/dt - -K, C, (1) In this equation, C, (parts per million) equals the concentration of VCM at time t(minutes), and AT,(minutes'1) is the rate constant for the decline in VCM con centration from the empty recirculating system. Regression analysis of the -V* e e g itg Hefner et alInhaled VCM in Rats 137 logarithm of the concentration of VCM versus time yielded Ky. Initially values for AT, were determined before and after inclusion of rats in the chamber. These values were reproducible; therefore, Kl was not redetermined after removal of the rats in every subsequent experiment. Between experiments, the system was dis assembled, cleaned, and reassembled using a new Ascarite column. The Kx for each reassembled system was unique, within certain limits, which necessitated its redetermination for each experiment. After the rate of decline of VCM concentration in the empty inhalation sys tem was determined, 4 rats were placed in the chamber. The chamber was then charged with the desired concentration of VCM, and the rate of decline of VCM concentration was determined as previously described. The rate of decline, K,, followed apparent first-order kinetics. After an initial equilibration of the tissues of the rats, the decline of VCM concentration from the closed system was as sumed to occur via metabolism of VCM plus the background loss from the sys tem. Since oxygen consumptions were within 10% for all experiments described herein, respiratory parameters were not significantly changed. Therefore, respira tory parameters were not a factor influencing the rates of metabolism. The rate of metabolism, Kc, would therefore be given by the equation K, = K. - K, (2) To assess the effects of potential inhibitors of VCM metabolism, rats were pretreated by intraperitoneal injection with 320 mg/kg of pyrazole (1,2-diazole) 1 h prior to exposure, 5 ml/kg of ethanol 1.5 h prior to exposure, or 75 mg/kg of SKF 525-A (/?-diethylaminoethyldiphenylpropylacetate) 0.5 h prior to exposure. Without cleaning or reassembling the inhalation system, we exposed both un treated control rats and rats treated with the chosen potential inhibitor to the desired concentration of VCM. The percent inhibition was calculated from the equation Kc (control) -- Kc treated = % inhibition Kc control (3) Effects of VCM on Liver Suljhydryl Levels Groups of male Sprague-Dawley albino rats of Spartan strain weighing from 193-250 g at initiation of the experiment were exposed to nominal concentrations of 15,000 ppm of vinyl chloride for 5 days, 5000 or 500 ppm 5 days/week for 1, 3, or 7 weeks, and 50 ppm for 1 h, 7 h, or 5 days. The exposures were carried out in a glass-walled 160-liter chamber under dynamic conditions with the VCM being metered into the chamber airstream. For repeated daily exposures, 7-h ex posures were conducted on the first 4 of 5 consecutive days each week. On the 5th day, if the rats were to be sacrificed, the duration of exposure was reduced to 5-6 h; however, if the rats were being continued on exposure, they received a full 7-h exposure. The body weights and food consumption of rats exposed to 500 and 5000 ppm of VCM were determined before each daily exposure and the rats were obse'ved periodically for signs of toxicity. Between 1 and 2 p.m., immediately fol lowing the fifth exposure of the designated week, the rats were killed by cervical dislocation and the livers removed and prepared for assay of sulfhydryl content. Gross pathological examinations were conducted. The method used for the sulfhydryl assay was a modification of that de scribed by Sedlak and Lindsay.1 Exactly 500 mg of liver from each rat were R&S 041234 138 Annals New York Academy of Sciences homogenized for 1 min in a Dounce tissue homogenizer containing 8 ml of 0.02 M disodium EDTA. For the total sulfhydryl assay, a 0.5-ml aliquot of each homogenate was mixed with 1.5 ml of 0.2 M Tris HC1 buffer (pH 9.2), 0.1 ml of 0.01 M 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and 7.9 ml of methanol. A reagent blank without liver homogenate and a sample blank without DTNB were also prepared. The color generated via the release of nitromercaptobenzoic acid anion was allowed to develop for 15 min, and the samples were centrifuged for 15 min at 4000 x g. Absorbance of each sample was read against the respective sample blank at 412 nm with a Beckman DB spectrophotometer. Subsequently, the molar concentration of total sulfhydryl in the sample was calculated using an extinction coefficient determined from standards of known concentrations of glutathione or cysteine. A plot of absorbance versus the concentration of cysteine or glutathione coincided with that reported by Sedlak and Lindsay.' The nonprotein sulfhydryl content of liver was determined after precipitating out the protein by addition of 1 ml of 50% trichloroacetic acid to a 5-ml sample of liver homogenate. Each sample was diluted with 4 ml of distilled water and after 15 minutes centrifuged at 4000 X g. A 2-ml aliquot of the supernatant was mixed with 4 ml of 0,4 M Tris HC1 buffer (pH 8.9). Immediately before reading the absorbance against a reagent blank, 0.1 ml of 0.01 M D174B was added. Subtraction of the nonprotein sulfhydryl content from the total sulfhydryl con tent yielded a value for protein-bound sulfhydryl. Urine samples collected from rats exposed to 5000 ppm of vinyl chloride for 4, 5, and 7 weeks were analyzed for the presence of S-(2-chloroethyl)cysteine, S(2-hydroxyethyl)cysteine, and S-(2-carboxymethyl)cysteine (5- and 7-week sam ples only). Urine was collected for analysis by applying pressure to the posterior abdomen between the 5th and 6th h of the 5th daily exposure on the designated week. Urine collected on the same day was pooled. For the three aforementioned compounds, 2-15 pi of urine were spotted directly on a 5 by 20 cm Baker-flex silica gel plate. Also spotted were samples of urine collected from control rats and standard aqueous solutions as well as control urine to which approximately 1 pg/pi of each of the compounds had been added. The chromatograms were developed for 5 h in a sealed glass tank containing n-butanol, acetic acid, and water (80:10:10 or 60:20:20). After the plates were air dried, they were sprayed with Ninspray ninhydrin reagent and heated for 2 min at 80"C. The color of the spots and their R( values were used to identify the compounds. A urine sample collected from rats exposed to 5000 ppm of vinyl chloride for 9 weeks was analyzed for the presence of chloroacetic acid. The 10-ml urine sample was acidified with 0.1 ml of 50% v/v HjSO,. Subsequently, the urine sample was extracted 3 times with 2 ml of diethyl ether. The diethyl ether extract was evaporated to 0.1 ml and 2-15 pi of the concentrated extract and an aqueous standard containing 5% w/v chloroacetic acid were spotted on an Eastman fluo rescent silica gel plate. The chromatograms were developed in a sealed glass tank for 5 h using the aforementioned solvent systems. After the plates were air dried, they were examined under ultraviolet light to determine the location of spots. Subsequently, the plates were treated with ninhydrin as described previously. Results Kinetic Studies on the Metabolism of Inhaled VCM Typical declines in the concentration of VCM in the inhalation apparatus containing 4 rats and initial concentrations of approximately 50 or 1000 ppm are Hefner et al.: Inhaled VCM in Rats 139 1 VCM Ettpotvn Concentration Ippmij 30 fio (/) Figure 2. Typical declines in VCM concentration with time at approximately 50 and 1000 ppm of VCM exposure concentrations. Also shown are the respective de clines in VCM concentration for the unoccupied inhalation apparatus, shown in Figure 2. Also shown are the corresponding declines in the concentra tion of VCM from the unoccupied chamber. As indicated under "Methods," the rate of these declines, K. and respectively, were determined by regression analysis. The rate of metabolism, Kt, was assumed equal to K. -- Kx. For seven separate exposures to concentrations of VCM ranging from 50 to 105 ppm, the mean and standard deviation for the apparent first-order rate constant, K,, were --8.04 X 10-3 3.40 X 10'3 min-1. This corresponds to a half-life (tu,) of 86 min. For five separate exposures to concentrations of VCM ranging from 220 to 1167 ppm, Kc was --2.65 X 10'3 1.35 X 10'3 min'1. This corresponds to a txn of 261 min. As indicated by the standard deviations, there was little variation of K, within the indicated range of concentration. Furthermore, there was no con sistent trend for the values of K, within this range. Administration of 320 mg/kg of pyrazole, an inhibitor of alcohol dehydro genase, xanthine oxidase, and other enzymes,3 1 h before exposure to 65 and 1234 R&S 041236 140 Annals New York Academy of Sciences Duration Of Exposure (Min ) Figure 3. Declines in VCM concentration with time at 65 and 1234 ppm of VCM exposure concentrations for both pyrazole-pretreated and untreated control rats. Also shown are the respective declines in VCM concentration for the unoccupied inhalation apparatus. ppm of VCM resulted in 71.2 and 86.9 percent inhibition of metabolism of VCM, respectively (Figure 3). Because of the lack of specificity of pyrazole as an enzyme inhibitor, 5 ml/kg of 95 percent ethanol were administered to rats in an attempt to inhibit specifi cally alcohol dehydrogenase activity. In rats exposed to initial concentrations of 56 and 97 ppm of VCM, ethanol pretreatment caused 96.0 and 82.9 percent in hibition of VCM metabolism, respectively. When rats were pretreated with ethanol and exposed to initial concentrations of 1025 and 1034 ppm of VCM. inhibition of metabolism was 46.5 and 35.7 percent, respectively. Figure 4 illustrates the inhibition for separate experiments conducted at extremes of initial VCM concentrations. Hefner et air Inhaled VCM in Rats 141 Figure 4. Declines in VCM concentration with time at 56 and 1034 ppm of VCM exposure concentrations for both elhanol-pretreated and untreated control rats. Also shown are the respective declines in VCM concentration for the unoccupied inhalation apparatus. Pretreatment of rats with 75 mg/kg of SKF 525-A, an inhibitor of some types of microsomal oxidases* resulted in no inhibition of metabolism of VCM in rats exposed to initial concentrations of 65 ppm and an 18.8 percent inhibition in those exposed to 1038 ppm (Figure 5). Effect oj Inhaled VCM on Liver Suljhydryl Levels No antemortem or postmortem signs of toxicity were noted in rats exposed to any concentration of VCM used in these experiments. Analysis of food con sumption data for rats exposed to nominal concentrations of 500 or 5000 ppm of VCM and unexposed controls revealed no statistically significant differences. In addition, no significant difference was found in the water consumption of rats exposed to 5000 ppm and controls. Throughout the durations of observation, the mean body weights of rats exposed to the various concentrations of VCM were R&S 041238 142 Annals New York Academy of Sciences Figure 5. Declines in VCM concentration with time at 65 and 1038 ppm of VCM exposure concentrations for both SKF 525-A-pretreated and untreated control rats. Also shown are the respective declines in VCM concentration for the unoccupied in halation apparatus. essentially the same as those of the respective controls. No gross pathological le sions related to exposure of VCM were found in any of the rats. Table 1 shows the nonprotein sulfhydryl content of the livers of rats exposed to 50, 500, 5000, or 15,000 ppm of VCM for the indicated durations. Values for concurrent controls are also given. In Figure 6, these data are summarized and expressed as the percent depression of the nonprotein sulfhydryl content of the liver as a function of exposure concentration and duration of exposure. Signifi cant reductions in the levels of nonprotein sulfhydryl occurred in rats exposed to 50 ppm of VCM for 7 h, 500 and 5000 ppm for 1 and 3 weeks, and 15,000 ppm for 1 week. There is no definitive association between exposure concentra tion and the degree of depression. The degree of depression decreases with con tinued exposure which suggests compensatory mechanisms are responding to re lieve this biochemical effect induced by VCM. Hefner et air Inhaled VCM in Rats 143 Table 1 Nonprotein Sulfhydryl Content (] x 10-* mol SH/mg liver S.D.) of Rats Exposed to VCMf Duration of Exposure 15,000 ppm Concur Concur* Concur Concur rent 5000 ppm rent 500 ppm rent *50 ppm rent Controls Controls Controls Controls 7 h/day for 5 0.29* 0.42 days 0.08 0.07 7 h/day 5 days/ week for 3 weeks 7 h/day 5 days/ week for 7 weeks 0.33* 0.46 0.02 0.03 0.41* 0.72 0.03 0.03 0.59 0.70 0.08 0.02 0.35* 0.58 0.09 0.06 0.45* 0.61 0.06 0.08 0.58 0.64 0.06 0.08 0,34 0.01 0.43 0.09 * Significantly different using Student's t test, p < 0.05. f A single 1-h exposure to 50 ppm of VCM resulted in 0.64 0.05 and 0.65 0.06 for the concurrent controls. A single 7-h exposure to 50 ppm of VCM resulted in 0.17 0.07 and 0.44 0.08 for the concurrent controls, t Five rats per group. ,h,d VINYL CHLORIDE CONCENTRATION 7 Hr, Q Week, 50 ppm -1 = 11* ` 11 > 500 ppm r"" ...."i3 L. __,, j'll 5.000 ppm j f j*1 15.000 ppm i iI11................ I.------ 1------- 1 0 10 ?0 30 40 50 60 70 % Depression Of NPSH Mcn Cone. ~ Mean Cant. 1 (tap,TMTM - 01 C.PWI. O' E-w-l Mean Cane. Of Controls t *Sifn<(*Mtlv vying Slkritinil "T" (Ml, Figure 6. Percent depression of nonprotein sulfhydryl in the liver of rats as a function of VCM exposure concentration and duration of exposure. The protein-bound sulfhydryl contents of the liver of rats exposed to 50, 500, 5000, or 15,000 ppm of VCM for the indicated durations were not significantly different from those of the concurrent controls. This was not unexpected be cause the sulfhydryl groups of protein have been shown not to be alkylated readily unless denaturation of the protein renders them available for alkylation.5 Effect of Ethanol Administration on VCM-Indttced Depression of Nonprotein Sulfhydryl in Liver In this experiment, 4 rats pretreated with 5 ml/kg of ethanol and 4 untreated control rats were exposed to 1070 ppm of VCM. After 105 min of exposure, the 144 Annals New York Academy of Sciences concentration of nonprotein sulfhydryl in the liver was determined. For those pretreated with ethanol, the depression of nonprotein sulfhydryl was 77.0 12.8 percent, whereas for controls it was 95.0 3.4 percent. These levels of depres sion were significantly different as determined by Student's t test, p < 0.05. John son* has reported that ethanol alone does not affect the nonprotein sulfhydryl content of the liver. Metabolites of VCM Preliminary results which must be viewed with considerable reservation have been obtained from studies in which rats were exposed to 5000 ppm of unlabeled VCM for 4, 5, or 7 weeks. Chromatograms of urine collected from rats after each of these exposure durations were comparable. S-(2-HydroxyethyI)cysteine R, (0.26-0.28 in 80:10:10 n-butanol-acetic acid-water) appeared to be present. S-(2-Chloroethyl)cysteine and S-(2-carboxymethyl)cysteine were not detected; however, it is conceivable that the latter compound was not adequately resolved from the urine background. In another experiment, chromatograms of urine from rats exposed to 5000 ppm of VCM for 9 weeks revealed the presence of monochloroacetic acid (R, 0.74 using the aforementioned solvent system). The spots for monochloroacetic acid were visualized under ultraviolet light on the fluorescent plates and did not develop with ninhydrin reagent. Studies of the metabolism of VCM were terminated until methods were devel oped to synthesize "C-VCM. "C-VCM in a liquid state polymerized even when an inhibitor was present and a temperature of --70'C was maintained. Recently, a method has been developed to synthesize "C-VCM in a gaseous state from 1,2dichloroethane. In the gaseous state "C-VCM is relatively stable. Currently, the methodology is being refined to allow synthesis of "C-VCM with reproducible specific activity. With "C-VCM, 3 180-g male rats have been exposed in the closed recirculat ing system for 65 min. The initial concentration of "C-VCM with a specific ac tivity of 1.155 mCi/mmol was 49 ppm. If we assume equivalent uptake of VCM by 3 rats, each rat received 0.49 mg/kg of VCM. Immediately following expo sure, the rats were removed from the chamber and placed in Roth type metabo lism cages which allow separate collection of urine, feces, expired carbon dioxide, and any expired VCM. Within 15 h after exposure, a mean of 58.0 percent of the "C activity had been excreted in the urine, 2.7 percent in the feces, and 9.8 percent as expired carbon dioxide. By 75 h after exposure, 67.1 percent had been excreted in the urine, 3.8 percent in the feces, and 14.0 percent as expired carbon dioxide. Only a trace, 0.02 percent of the dose, was expired as VCM and trapped on activated carbon. After 75 h, 1.6 percent of the dose remained in the liver, 3.6 percent in the skin, 0.2 percent in the kidneys, and 7.6 percent in the remaining carcass. The mean percentage of the assumed dose recovered from the 3 rats was 97.9 percent. Various chromatographic techniques are being evaluated for separation and identification of the "C-labeled urinary metabolites. S-(2-Hydroxyethyl)cysteine and S-(2-carboxymethyl)cysteine and their respective N-acetyl derivatives appear to be likely metabolites. Ultimate verification of metabolites will be determined by gas chromatographic-mass spectroscopic analysis. In the experiment just de scribed. monochloroacetic acid has not been detected. In summary, metabolism studies have demonstrated tentatively the following: 1. VCM is quite readily metabolized to polar metabolites which are excreted Hefner et al.: Inhaled VCM in Rats 145 predominantly in the urine of rats exposed via inhalation to an initial concentra tion of 49 ppm. Smaller amounts of VCM are excreted in the expired air as carbon dioxide and in the feces. Very little is excreted in expired air as un changed VCM. 2. A significant but small amount is retained in tissue, particularly liver, as long as 75 h after exposure. 3. Metabolites excreted in the urine appear to be conjugated with glutathione and/or cysteine through covalent linkage to the sulfhydryl group. This is consist ent vvith the reduction of the nonprotein free sulfhydryl levels in the livers of exposed rats. Preliminary in vitro experiments' have shown that direct conjuga tion of vinyl chloride with cysteine or glutathione in aqueous solutions occurs to a small degree but very slowly. 4. Monochloroacetic acid also appears to be a metabolite of VCM, when rats were exposed to 5000 ppm for an extended time. Discussion Because of the preliminary nature of the studies reported herein, the results and subsequent discussion must not be considered conclusive. Justification for premature publication of some of the data contained herein is the magnitude of the impact of recently revealed toxicological effects of VCM and the urgent need for communication of even preliminary results. The studies reported herein have demonstrated reliably in some instances and tentatively in others the following: 1. Rats exposed to concentrations of VCM below 100 ppm metabolize the compound fairly readily and in accordance with first-order rate kinetics, ;1/a = 86 min. 2. In rats exposed to a concentration of VCM exceeding 220 ppm, the rate of VCM metabolism was reduced, r,/a = 261 min. This indicates that the predomi nant pathway for metabolism of VCM by rats exposed to 100 ppm or less is saturable. Residual metabolism at concentrations exceeding 220 ppm may be via this pathway in conjunction with additional pathway(s). 3. Pyrazole inhibits the metabolism of VCM suggesting that metabolism of VCM is via alcohol dehydrogenase. 4. Stronger evidence for the metabolism of VCM by alcohol dehydrogenase was its inhibition by the administration of ethanol. This inhibition was less pro nounced in rats exposed to 1000 ppm than in rats exposed to 100 ppm or less, which suggests that metabolism via pathways other than that of alcohol dehydro genase occurs in rats exposed to 1000 ppm of VCM. 5. SKF 525-A does not affect the metabolism of VCM by rats exposed to 65 ppm of VCM but there is an indication that it slightly depresses metabolism by rats exposed to 1038 ppm of VCM. This suggests that in rats exposed to con centrations of VCM which exceed the capacity of the alcohol dehydrogenase pathway metabolism may occur via oxidases in the microsomes. 6. Monochloroacetic acid was found in urine of rats exposed to 5000 ppm of VCM daily for 9 weeks. 7. Exposure to VCM reduces the nonprotcin sulfhydryl concentration of liver. This reduction is not definitively associated with the exposure concentration of VCM in a range of 50-15.000 ppm. There is a tendency for the reduction to become less pronounced with repeated daily exposures. These results arc con sistent with a saturable mechanism for the metabolism of VCM followed by conjugation of the metabolites of VCM with glutathione and/or cysteine. aj So to 041242 146 Annals New York Academy of Sciences 8. The administration of ethanol significantly reduces the depression of the concentration of nonprotein sulfhydryl in the livers of rats caused by exposure to 1000 ppm of VCM for 105 min. 9. In rats exposed to 49 ppm, VCM is metabolized to polar products which are excreted predominantly in the urine. These products appear to be derived after initial metabolism of VCM and subsequent conjugation of the products with glutathione and/or cysteine through covalent binding with the sulfhydryl. A small but significant fraction of VCM is metabolized to COa and expired. An even smaller but significant amount of "C activity appears to be retained in the liver primarily, but also in other tissues as long as 75 h after exposure. Considering the results in toto, we hypothesize that in rats exposed to con centrations of VCM below 100 ppm, VCM is predominantly metabolized via sequential oxidation to 2-chloroethanol, chloroacetaldehyde, and monochloroacetic acid by the alcohol dehydrogenase pathway: C1HC = CH, -> ClHjC--CHjQH alchohol dehydrogenase ClHjC--CHO - ClHiC--COOH Only small amounts, if any, of monochloroacetic acid are formed at low doses because chloroacetaldehyde reacts rapidly with the sulfhydryl of glutathione and cysteine.* This conjugation accounts for the lack of dose-related reduction in the nonprotein sulfhydryl content of the liver of rats exposed to 50 through 15,000 ppm of VCM. Alternate pathways of VCM metabolism which may be involved at high doses are at this time purely speculative. However, at 220 ppm, metabolism by the more rapid alcohol dehydrogenase pathway appears to be saturated, and metabo lism via oxidation of the accumulating 2-chloroethanol may occur as follows: ClHjC--CHjOH HiOi catalase ClHjC--CHiOOH -* CIHiC--CHO Carter et al.* have demonstrated that microsomal oxidation of ethanol in vitro proceeds via the formation of hydrogen peroxide and catalase which subsequently forms a peroxide of ethanol. Acetaldehyde is the end product of this oxidation. It is conceivable that a similar oxidation of 2-chloroethanol occurs. In addition to this series of reactions, a direct epoxidation of VCM may oc cur as follows: CIHC=CH, onidj'K o /\ H,C--CH - C1HSC--CHO - C1H,C--COOH Cl Zief et al* have shown that chloroethylene oxide spontaneously rearranges to chloroacetaldehyde. Such mechanisms would explain why SKF 525-A causes a slight inhibition of metabolism in rats exposed to 1038 but not 65 ppm of VCM. Also it may explain why monochloroacetic acid may be excreted by rats exposed to 5000 but not 50 ppm of VCM. In the former case, chloroacetaldehyde is pro duced by each of the hypothesized pathways which may result in a greater amount being oxidized to monochloroacetic acid than being conjugated with glutathione and/or cysteine. Inferences from the results of these studies about the toxicodynamics of VCM are premature, but worth mentioning. First, the saturation of a primary meta bolic pathway for VCM degradation and redirection through other pathways Hefner et alInhaled VCM in Rats 147 provide some hope that a threshold concentration for the untoward effects of VCM may exist. Metabolites of VCM formed only via the alternate pathways may constitute the ultimate toxin and carcinogen. It has been reported that the administration of cysteine or glutathione pro vides protection against the untoward effects of various aliphatic and aromatic mustards, triethylenemelamine, x-rays, and ionizing radiation.1,Conceivably, cysteine and glutathione may provide a natural defense against tumor-producing free radicals generated within the body, as well as synthetic or naturally occurring alkylating agents which are absorbed into the body. Therefore, reduction of the nonprotein sulfhydryl content of the liver in animals exposed to VCM may con stitute predisposition to toxicity and carcinogenicity mediated via other materials. Finally, the speculated formation of chloroethylene oxide seems particularly pertinent insofar as postulation of the mechanism of carcinogenesis. This com pound is undoubtedly a very active difunctional alkylating agent. It is most inter esting that inorganic arsenicals have been reported to cause untoward hepatic effects, portal cirrhosis, and angiosarcoma, like those reported for VCM.11 The mechanism of toxicity for arsenic has been shown to occur via its reaction with 6,8-dithiooctanoic acid (a-lipoic acid).11 In this reaction arsenic forms a stable bridge between the two sulfhydryl groups. If chloroethylene oxide were formed, it would readily react with -lipoic acid, bridging the sulfhydryl groups like ar senic. :i SH SH CH--CHj--CH--(CHj),--COOH SS \/ CHS--CH OH Although this postulated mechanism is highly speculative, the rarity of materials known to produce untoward hepatic effects like those of VCM must be given some weight. Acknowledgments The technical advice of Dr. B. K. J. Leong in designing the inhalation ap paratus and the critical review of the manuscript by Dr. V. K. Rowe are grate fully acknowledged. References 1. Maltoni, C. & G. Lefemine. This monograph. 2. SedLaK, J. & R. H. Lindsey. 1968. Anal. Biochem. 25: 192-205. 3. Carter, E. A. & K. J. Isselbacher. 1972. Lab. Invest. 27: 283-286. 4. Soliman, M. R. I,, H. D. Johnson & A. E. Wade. 1974. Drug Nletab. Disp. 2: 87- 96. 5. Stacey, K. A., M. Cobb, S. E. Consens & P. Alexander, 1958. Ann. N.Y. Acad. Sci. 68: 657. 6. Johnson, M. K. 1965. Biochem. Pharmacol. 14: 1383. 7. Dow Chemical Co. Unpublished data. 8. Johnson, M. K. 1967. Biochem. Pharmacol. 16: 185-199. 9. Zief, M. & C. H. Schramm. 1964. Chem. Ind. April IS: 660-661. 10. Ball, C. R. 1966. Biochem. Pharmacol. 15: 809-816. R&S 041244 148 Annals New York Academy of Sciences 11. Calcutt, G., T. A. Connors, L. A. Elson & W. C. J. Ross. 1963, Biochem. Phar macol. 12: 833-837. 12. Goldenthal, E. I., M. U. Nadkarni & P. K. Smith. 1959. Radiation Res. 5: 571583. 13. Patt, H. M. 1953. Physiol. Rev. 33: 35-76. 14. Popper, H. Personal communication. 15. Gonsalus, I. C. 1953. J. Cellular Comp. Physiol. 41 (Suppl. 1): 133. Discussion Dr. E. J. Fairchild (National Institute o/ Occupational Safety and Health, Rockville, Md.): Dr. Gehring noted during the course of his presentation that there is an indication of possible involvement of sulfhydryl mechanisms which may be affected by vinyl chloride. He also mentioned that this could be an effect of vinyl chloride exposure, but that this was not necessarily associated with carcinogenesis. An important aspect of this finding, at least in my own mind, is that it may give some clue in aiding the development of diagnostic tests for exposure to vinyl chloride. Also, it should be kept in mind that some of the work reported by others indicates a thyroid involvement associated with vinyl chloride exposure, and there is some evidence from the literature which would indicate a close relationship between thyroid function and sulfhydryl mechanisms. Further it should be remembered that an action which affects thyroid function generally has an indirect or secondary effect upon pituitary and/or adrenal gland response. Accordingly, these ramifications of indicator response possibly have some potential in diagnostics. Dr. H. Kraybill (National Cancer Institute, Bethesda, Md,): Perhaps this question will be answered later, but I was interested in Dr. Gehring's paper where he talked about alcohol and other stresses. I wonder if in your design you considered such things as protein stress in general, i.e., the effect of dietary constituents? 23056