Document 0gryvLdwRpLk1dOgqXNOj8m0k

R&S 111598 BIbrHEDICAl RESEARCH DOCUMENT DESCRIPTION PORH 63 Duplicate in all cards:--^ /iyo 68 69 76 OOOQ.327 year as-1961- Pile number fRight justify [Numeric only] Author(s), as Last Kane PS (No Punctuation) and coden for journal as JAKA preceeded -by. one blank space 77 78 . h- Sub-Xude^ Code Title of Report: end with space-hypnen-hyphen-space. Pollow with lades: Tares, separated froa each other with .coaaa-spsca. Avoid other, punctuation; do 'not abbreviate* 12 . 61 62 r 21 -L. 'i 72 ^ ~ nint / .. , _v r a - i ^ -u K - / 'O i ^ i '/p S' -/ J > I ^ ` -11 -^ x-- O < ^ , r, v 1 ^ 22 JI 23 Ddt*/ , A?t/PA es-s*. ? /Csn 24 Source (Journal, Vol., Number, Pages, Date) 12 '''if " / .........; ' ' -- _ / --7 / ,J, ,A&- 61 61. 31 32 Brief Summary 61 62 61 62 63 64 CP09327 i. Environmental Health Perspectives VoL 17, pp. 145--152,1976 Dose-Dependent Fate of Vinyl Chloride and Its Possible Relationship to Oncogenicity in Rats by P. G. Watanabe* and P. J. Gehring* t1 i * Studies on the fate of "C-labeled vinyl chloride (VC) following oral administration and inhalation exposure in rats demonstrated that the disposition of VC in the body is a function of the dose. More importantly, from the data available, it appears that a corre lation exists between doses of VC which cause tumors and those that saturate metabolic or detoxifying pathways. Additional studies characterized the depression of liver non protein sulfhydryl content (primarily GSH) with the duration and concentration of expo sure to VC. The results of these investigations indicate that statistical projections utilizing data collected from rats exposed to high doses of VC are invalid for predicting the hazard of low level exposure, because such projections violate the a priori assumption that the dynamics governing the fate of VC in the body are unaltered. R&S 111599 Introduction Once the carcinogenicity of a chemical has been established a primary consideration is the hazard of exposure to low levels of the given chemical. Studies on the pharmacokinetic and metabolic characteristics of such chemicals are essential in accurately assessing the hazard of low level exposures. Pharmacokinetics is the study of the dynamic processes involved in the absorption; distributiori, metabolism and elim ination of chemicals from the body. Pharmaco kinetic studies alone are insufficient to assess toxicity. However, their principal value is in correlation of toxicity with the time-related dis position of chemicals in the body. An altered disposition of a chemical in the body with dose can explain in certain instances why toxicity, including carcinogenicity, is produced at high doses and not at low doses of the same chemi cal. In the case of carcinogenicity, stochastic, statistical projections are made from the range of doses over which an increased incidence of cancer can be measured to predict what per centage of individuals may respond at lower * Toxicology Research Laboratory, Health and Envi ronmental Research, The Dow Chemical Company, Mid land, Michigan 48640. doses. Figure 1 shows a hypothetical cumula tive dose-response curve for the percentage of individuals (triangles) in a population respond ing adversely in some manner to selected doses of a chemical. The sigmoid curve represents a response of a population described by a normal or Gaussian distribution. These adverse re sponses (cancer) are discernible only over a range of doses represented by the solid line, because the investigator is limited by the num ber of individuals he can include in such a study. It should be emphasized that an a priori assumption for making such projections is that the chemical is handled in the same manner by the body as the dose decreases. If the dy namics for the fate of the chemical change, such extrapolation is not valid. Conceptually, it is not surprising that toxicity (including carcinogenicity) is expressed only after the capacity to detoxify the chemical in the body has been exceeded. In such cases, the response of the population may be more accurately de scribed by the other two broken lines in the lower left-hand corner of Figure 1. The most important aspect is to determine if an altered disposition of a chemical with dose functions over a range from doses that cause toxicity to those that do not cause toxicity. October 1976 145 009I I I Figure 1. Plot of hypothetical log dose vs. per cent re sponse curve. Measurable responses are represented by triangles. The sigmoid curve (--) represents a population described by a normal distribution; in theory the percentage responding never reaches zero on the low end or 100% on the high end. The other curves (---) and (--) represent simulated responses if there exists a threshold for the response. In initial studies on the fate of vinyl chloride (VC), rats were exposed in a closed recirculat ing inhalation chamber to varying concentra tions of VC (1). By monitoring the concentra tion of VC in the system by infrared spectro photometry, the rate of uptake of VC by the rats was determined. Results on exposure to low and high concentrations of VC and on pre treatment of the animals with metabolic in hibitors indicated that VC was metabolized by at least two pathways. More importantly, it appeared that the primary pathway for the metabolism of VC became saturated as the exposure concentration increased. These initial results suggested a dose-de pendent fate of VC, and this motivated addi tional work to elucidate the fate of VC in rats following both oral administration and inhala tion exposure. 14C-labeled VC was utilized in subsequent investigations which greatly facili tated following the disposition of the admin istered VC. Results and Discussion Fate of VC Following Single Oral Administration Table 1 shows the percentage of 14C-activity eliminated via various routes following differ ent single oral doses of VC in corn oil to rats (). The 14C-activity in urine, feces, and car cass and tissues represents nonvolatile metab olites of VC. If all the processes involved in the disposition of VC in the body could be de scribed by first-order kinetics, implying that * the rates of the processes were proportional to * the amount of chemical available, then the proportions of 14C activity eliminated by each ,, route of excretion would be the same over the range of doses tested. If this were the case, then the fate of VC in the body would be in dependent of the dose administered. However, the results show that as the dose was increased from 0.05 and 1.0 mg/kg to 20 or 100 mg/kg, the percentage expired as VC increased mark edly, while the other parameters, particularly urinary excretion of l4C-activity, decreased. This demonstrates that the primary route for the elimination of VC from the body is^^pendent on the dose administered. Since the urinary and pulmonary excretion of VC were altered dramatically as the dose increased from 1 to 100 mg/kg, the question was raised whether these processes of elimina tion may be a function of dose. Figure 2 shows a plot of the logarithm of the 14C-activity elim inated via the urine as a function of time. Since the slopes or rates of elimination (t 1/3 -- 4.5 hr) are unchanged, it must be concluded that the rate of urinary excretion of nonvola tile metabolites of VC is unaltered by dose. Figure 3 shows similarly the expiration of VC following various doses. Elimination follow ing 0.05 or 1.0 mg/kg occurred in accordance with a first-order rate or monoexponential proc ess with half-life of 53-58 min. When 100 mg/ kg was given, the elimination was biexponen- tial. The initial phase of elimination had a half-life of 14 min and was followed by a slower phase with a 11/2 of 41 min. The rates or 11/2times for elimination at the 100 mg/kg dose correspond well with those reported by Withey (3) for blood. These results are indicative of a material which is bound reversibly to some site in the body having a finite capacity. As the dose increases, the availability of these binding 146 Environmental Health Perspectives doses of VC at 50 and 16.6 mg/kg-day have resulted in 19% and 14% induction of hepatic angiosarcoma, respectively, while at a dose of 3.33 mg/kg-day ne tumors have been observed. These results are after 85 weeks. Figure 4, illustrating the occurrence of saturation as a function of a single oral dose of VC, shows that a correlation exists between doses of VC which cause tumors and those that saturate meta bolic or detoxifying pathways. The dose-re sponse curve generated from the two higher doses (50 and 16.6 mg/kg-day) predicts a 9% tumor incidence at the 3.33 mg/kg-day level. Since no tumors have been observed yet at this low level, this is an example of the use of pharmacokinetic data in interpretation of why high doses of a chemical may produce toxi- SSH FIGURE 2. uC-activity excreted in the urine expressed as percent of the dose administered (1 and 100 mg/ kg) vs. time. Each point represents the mean m standard error of the mean for five rats. The initial linear segments of the curves (12-36 hr) were fit by linear regression analysis, t ^ =4.6 hr. sites decreases and the chemical is free to find its way to other sites or to be eliminated. Thus, it may be concluded that the pulmonary excre tion of VC is not a rate-limiting step. Even more importantly, the data indicate that the state in which VC exists in the body changes with dose. Figure 4 summarizes the dose-dependent ex cretion of VC via urinary excretion (solid line) and pulmonary elimination (broken line). The area indicated by the rectangle represents the range of doses where evidence of distributive or metabolic saturation first occurs. Of par ticular significance is that in a current carcino genesis study in rats by Maltoni (4) daily oral Figure 3. Expired vinyl chloride expressed as percent of the dose administered (0.06,1, and 100 mg/kg) vs. time. Each point represents the mean standard error of the mean of five rats. The linear phases of the curves were fit by linear regression analysis. October 1976 147 Table 1. Percentage of administered 14C activity eliminated during 72 hr following a single oral dose of vinyl chloride. Expired as VC CO, Urine Feces Carcass and tissues Total recovery 0.05 mg/kg 1.4 0.1* 9.0 0.6 68.3 0.5 2.4 0.5 10.1 1.9 91.3 2.5 % of I4C activity* 1.0 mg/kg 20 mg/kg 2.1 0.2 13.3 0.5 59.3 2.8 2.2 0.4 11.1 0.5 88.8 2.0 41.6 6.7 4.8 0.7 22.6 1.2 1.0 0.1 11.0 2.7 81.0 2.9 100 mg/kg 66.6 0.7 2.6 0.1 10.8 1.0 0.6 0.1 1.8 0.1 82.3 0.4 * Mean standard error, five rats/dose except three rats at 20 mg/kg level. Table 2. Percentage of 14C activity eliminated during 72 hr following inhalation exposure to i*C-vinyl chloride for 6 hr.* _______________ % of UC activity*________________ 10 ppm 1000 ppm Expired as VC CO, Urine Feces Carcass and tissues Cage washb Total VC recovered, jig 1.61 0.16 (4) 12.09 0.43 (30) 67.97 1.71 (169) 4.45 0.22 (11) 13.84 1.16 (34) 0.15 0.08 1) (248) 12.26 0.96 (814) 12.30 0.63 (817) 56.29 1.96 (3739) 4.21 1.05 (280) 14.48 0.52 (977) 0.23 0.09 (15) (6642) Expressed as percentage of the total 14C-activity recovered. All values are means standard error from four rats. Values in parenthesis are microgram equivalents of vinyl chloride. 11 Water and acetone wash of the metabolism cage at termination of the experiment. city and extrapolation of the same toxic effect at lower levels is invalid because the fate of the chemical has changed. Figure 4. Summary of the dose-dependent excretion of VC: (------ ) urinary excretion; (--) pulmonary elim ination. Urinary excretion represents polar metabo lites of VC while pulmonary elimination is VC per se. Area demarcated by the rectangle represents range of doses over which distributive and metabolic sat uration occurs. Fate of VC Following Inhalation Exposure Table 2 shows the fate of "C-VC in rats exposed for 6 hr to 10 or 1000 ppm VC (5). Im mediately following the exposure, the rats were placed in cages providing for collection of I4C-activity in the expired air, feces, and urine over the subsequent 72 hr. As in the experi ments in which oral doses were given, the percentage of "C-activity expired as VC in creased as the exposure increased. Also to be noted in Table 2 is that the per centage "C-activity in the tissues and carcass increases slightly as the exposure is increased from 10 to 1000 ppm. Although not statistically significant, this is remarkable because a much larger fraction was expired as VC. In partic ular, the normalized amount of "C-activity in R&S 111602 148 Environmental Health Perspectives R&S 111603 the liver and skin increased. This may mean that a larger fraction is being bound to the macromolecules of the tissues. This aspect is currently being investigated, since such reac tivity may explain the carcinogenic effect of VC. Figures 5 and 6 show respectively the elim ination of 14C-nonvolative metabolites in the urine and VC per se in the expired air. Neither elimination process is rate-limiting or over whelmed by increasing the exposure concentra* tion. However, it is noteworthy that expired VC increased with increasing dose, whereas excretion of urinary metabolites decreased, * suggesting a saturation of the metabolism of VC. The results of these studies as those of previous studies support the conclusions that (1) the fate of VC changes with dose and (2) this occurs because the primary pathway for the metabolism of VC is saturated at high doses or exposures. Hour* Figure 5. "C-activity excreted in the urine expressed as percentage of the recovered radioactivity vs. time following a 6-hr exposure to 10 and 1000 ppm VC. Each point represents the mean m standard error of the mean for four rats. The initial log linear phase of the curves (12-36 hr) were fit by linear regression analysis. Figure 6. Expired vinyl chloride expressed as percent age of the recovered radioactivity vs. time following a 6-hr. exposure to 10 and 1000 ppm VC. Each point represents the mean standard error of the mean of four rats. The curves were fit by linear regression analysis. Since the metabolism of VC appears to occur via at least two pathways, an effort was made to identify the urinary metabolites of VC. Al ready it had been demonstrated that a meas urable amount of VC was metabolized to C02. Using high-pressure liquid chromatography, three major metabolites have been isolated from urine. Two of the three have been iden tified by gas chromatography-mass spectro scopy. These are metabolite A, iV-acetyl-S (2hydroxyethyl) cysteine, and metabolite B, is thiodiglycolic acid. Together these metabolites October 1976 149 R&S 111604 comprise 50 to 60% of the radioactivity found in urine. A third metabolite, comprising' 35% of the metabolites present in the urine has been isolated but remains unidentified. Both metabolites A and B are likely formed from S-(2-hydroxyethyl) cysteine. At one time, it appeared that the third major urinary meta bolite, comprising about 30% of the radio activity, was S- (2-hydroxyethyl) cysteine. Al though some analytical comparisons between the isolated metabolite and 2-hyroxyethylcysteine favored this conclusion, other failed to confirm the identity of this metabolite. Identification of these metabolites of VC in urine indicates that VC is transformed in the body to a reactive intermediate metabolite, which is then detoxified by reaction with gluta thione, (GSH, y-glutamylcysteinylglycine). Subsequently, the glutamic acid and glycine moieties of the tripeptide are cleaved, and the cysteine conjugate of the reactive metabolite of VC is either acetylated or further oxidized and excreted as the aforementioned metabolites. The urinary metabolites of VC were not changed either qualitatively or quantitatively as the dose or exposure level was increased. Since evidence has been presented for several metabolic pathways being involved in the bio transformation of VC, the lack of alteration in the urinary metabolites with dose may seem inconsistent. However, it should be emphasized that toxicity is a result of the balance between the relative rates of intoxicating to detoxificating metabolic pathways, and while this param- o NH-C-CH, ^0 HO--CH,--CH,--S--CH,--CH--C "OH Metabolite A: fV-Acetyl(S-2-hydroxyethyl)cysteine C--CH,--S--CHi--C HO''' '"OH Metabolite B: Thiodiglycolic acid NH, HO--CH,--CHi--S--CH,--CH--C S- (2-Hydroxyethyl) cysteine eter may change with dose it need not be reflected by the urinary metabolites which con stitute ultimate end products of metabolism. Our initial work and subsequently that of others (6-8) indicates that one pathway in volves oxidation of VC by microsomal enzymes to chloroethylene oxide. Other pathways which involve either nonenzymatic or enzymatic con jugation with GSH, mediated by soluble en zymes, and dechlorination reactions, mediated by both soluble and microsomal enzymes, are all possibly involved in the overall metabolism of VC. The relative contribution of these en zyme systems in the metabolism of VC are currently under investigation. Depression of Hepatic Nonprotein Sulfhydryl Content by VC A very important aspect of the metabolism of VC is the detoxification reaction with hepatic nonprotein sulfhydryl groups (composed of primarily GSH). When high doses of some chemicals, for example bromobenzene and acetaminophen, are given, the glutathione is used up at a faster rate than it can be produc^k by conjugation with the reactive intermediator As the level of glutathione in the liver is pro gressively depleted, the reactive metabolites react with macromolecules such as protein, DNA and RNA leading to toxicity (9, 10). Generally, it is accepted that one mechanism for chemical carcinogenesis may involve such reactions. To assess the effect of VC exposures on hepatic glutathione levels, rats were exposed to concentrations of 10, 50, 150, 250, 1000, or 2000 ppm for 1-7 hr (11). The results are shown in Figure 7. Exposure to 150, 250,1000, or 2000 ppm VC caused a progressive depres sion of the hepatic nonprotein sulfhydryl con tent. Exposure to 50 ppm for 7 hr produced a small and inconsistent depression. No depres sion was observed in rats exposed to 10 ppm VC- These results indicate that there is a measurable biological threshold for the depres sion of hepatic glutathione levels induced by exposures to vinyl chloride. Unequivocal de pressions are produced by concentrations ex ceeding 50 ppm, while exposure to 50 ppm seems to be a transition zone and exposure to 10 ppm causes no depression. How do these results relate to the carcino genicity of VC? In the studies of Maltoni 150 Environmental Health Perspectives Percent Of Depression sarcomas of the liver were given but also the latency periods for their development were pro vided. The latency periods were 64, 70, 78, 81, and 79 weeks in rats exposed to 10,000, 6000, 2500, 500, and 250 ppm, respectively. In rats exposed to 50 ppm, the latency period was 135 weeks. Indeed tumors were discovered in these aged rats when they were killed at the end of the study. Since the tumors in the former groups of rats were discovered as they died spontaneously, the discrepancy is even greater than the values indicate. The latency period for the development of other types of tumors showed the same discrepancy. Consideration of these results leads to the conclusion that, in rats, exposure to 50 ppm VC 4 hr/day is in the threshold transition zone for not only hepatic nonprotein free sulfhydryl depression but for tumor induction as well. Figure 7. Depression of hepatic nonprotein sulfhydryl content vs. duration of exposure to 2000, 1000, 250, 150, 50, and 10 ppm vinyl chloride. Each point repre sents the mean standard error of five animals ex cept the point for 50 ppm which represents 25 ani mals. The asterisks (*) represent values statistically different from controls (p < 0.05). Lefemine (12), the reported incidence of angio sarcoma of the liver in rats exposed 4 hr/day, 5 days/week to 2500 or 6000 ppm was 22%. The incidence in rats exposed to 500 and 250 ppm were, respectively, 12 and 7%. Reference to Figure 7 indicates that the depression of the hepatic nonprotein sulfhydryl content ob served after 4 hr of exposure coincides with the increased incidence of angiosarcoma. . In the same study (12) an incidence of only v2% angiosarcoma of the liver occurred in rats exposed to 50 ppm VC. As indicated previously, exposure to 50 ppm for 7 hr caused a small and inconsistent depression of the hepatic nonpro tein sulfhydryl content. This exposure appeared to be in the transition zone of the threshold for this biological effect. In a recent publication by Maltoni (13), not only the incidences of angio Conclusion In summarizing the studies on the pharmaco kinetics and metabolism of VC, the data indi cate that the fate of VC in rats is dose-depen dent following either single oral administration or inhalation exposure. More importantly, it appears from the data available that a correlation exists between doses of VC that cause tumors and those that saturate metabolic or de toxifying pathways. The primary detoxification pathway for VC involves conjugation of its reactive metabolites with nonprotein sulfhydryl groups. Therefore, it seems reasonable to postulate that as the nonprotein sulfhydryl groups are depleted, re active metabolites will be free to react with other macromolecules (DNA, RNA, protein, li pids), resulting in toxicity and carcinogenicity. Recent reports have demonstrated that in the presence of fortified microsomal enzyme prepa rations reactive metabolites of VC are produced which covalently bind to rat liver microsomes (6), protein sulfhydryl groups, RNA (7), and adenosine of DNA (8). Inclusion of glutathione in the system will decrease or preclude these reactions depending on the concentration. It is highly significant that exposure to 10 ppm VC for 7 hr caused no depression of hepatic non protein sulfhydryl content. This indicates that there is a threshold of exposure in rats at which the ability to replace sulfhydryl groups is not overwhelmed and physiologic defense mechanisms remain fully operative. Further- 33 111605 October 1976 151 more, this suggests that thresholds exist for toxic effects which are expressed with greater intensity as this protective mechanism is de pressed. Studies currently in progress are de signed to characterize the in vivo macromolecular binding of VC to protein and nucleic acids following exposure to various concentrations of "C-VC. Finally, it must be emphasized strongly that stochastic, statistical projections utilizing data collected from rats exposed to high doses of VC are invalid for predicting the hazard of low level exposures. Such projections violate the a priori assumption that the dynamics governing the fate of the compound are unaltered. REFERENCES 1. Hefner, R. E., Watanabe, P. G., and Gehring, P. J. Preliminary studies of the fate of inhaled vinyl chloride in rats. Ann. N. Y. Acad. Sd. 246: 135 (1975). 2. Watanabe, P. G., McGowan, G. R., and Gehring, P. J. Fate of "C-vinyl chloride after single-oral ad ministration in rats. Toxicol. Appl. Pharmacol. 36: 339 (1976). 3. Withey, R. J. Uptake and pharmacodynamic of vinyl chloride administered to rats by different routes, Toxicol. Environ. Health 1: 381 (1976). 4. Maltoni, C., Gli effetti onogeni del cloruro di vtnile somministrato per via orale nel ratto. Gli Ospedali della Vita, 2: 102 (1975). 5. Watanabe, P. G., et al. Fate of "C-vinyl chloride following inhalation exposure in rats. Toxicol. Appl. Pharmacol. 37: 49 (1976). 6. Kappus, H., et al. Rat liver microsomes catalyze covalent binding of "C-vinyl chloride to macromole cules. Nature 257: 134 (1975). 7. Bolt, H. M., et al. Metabolism of vinyl chloride. Lancet: 1425 (1975). 8. Barbin, A., et al. Liver-microsome-mediated forma tion of alkylating agents from vinyl bromide and vinyl chloride. Biochem. Biophys. Res. Commun. 67: 596 (1975). 9. Gillette, J. R. A perspective on the role of chem ically reactive metabolites of foreign compounds in toxicity--I. Correlation of changes in covalent bind ing of reactive metabolites with changes in the incidence and severity of toxicity. Biochem. PharmacoL 23 : 2785 (1974). *' . 10. Gilletter, J. R. A perspective on the role of chem ically reactive metabolites of foreign compounds in toxicity--II. Alterations in the kinetics of covalent binding. Biochem. Pharmacol., 23: 2927 (1974). 11. Watanabe, P. G., Hefner, R. E., Jr., and Gehring, P. J. Vinyl chloride induced depression of hepatic nonprotein sulfhydryl content and effects on bromosulphalein (BSP) clearance in rat. Toxicology 6:1 (1976). 12. Maltoni, C., and Lefemine, G. Carcinogenicity bio assays of vinyl chloride: Current results. Ann. N.Y. Acad. Sci. 246: 195 (1975). 13. Maltoni, C. The value of predictive experimenHV environmental carcinogenesis. An example: vinyl" chloride. Ambio, 4: 18 (1975). 3J a* oO) o> 152 j Environmental Health Perspectives