Document NQVabzNBd15Nz0nr9aoJNoM8

Envu:o2U2lnt'il Jlcalth Pcrspectipcs Vol. 17,pp. liS^St; 1976 Dose-Dependent Fate of Vinyl Chloride and Its Possible Relationship to Oncogenicity in Rats by P. G. Watanabe* and P. J. Gehring* Studies on the fate of "C-Iabeled 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 cau'e tumors and those that saturate metabolic or detoxifying pathways. Additional studies characterized the depression of liver nonprotein sulfhydryl content (primarily GS1I) 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 Molate the a priori assumption that the dynamics governing the fate of VC in the body are unaltered. Introduction Once the carcinogenicity of a chemical has 1 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, distribution, metabolism and elim:nation 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 dose3 over which an increased incidence of cancer can be measured to predict wh^t per- n'age of individuals may respond at lower * Toxicology Research Laboratory, Health and Envirinnvntal Research, The Dov. Chemical Company, Mid''nd, Michigan 18640. doses. Figure I 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 / r* | o-H O Figure l. 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 tero 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. ,4C-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 ,4C-activity eliminated via various routes following differ ent single oral doses of VC in corn oil to rats (2), The "C-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 "C 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 "C-activity, decreased This demonstrates that the primary route for the elimination of VC from the body is de pendent or. 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 show.a plot of the logarithm of the ,4C-activity elim inated via the urine as a function of time Since the slopes or rates of elimination (t = 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 ? half-life of 1-1 min and was followed by a slower phase with a t v, of 41 min. The rates or t times for elimination at the 100 mg/kg dose correspond well with those reported by YVithey (?) 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 1-16 Environmental Health Perspective- mm r*' SL 101895 Of The Dose A dm inistered 100 doses of VC at 50 and 16.6 mg/kg-day have resulted in 19^4 and IK? induction of hepatic r, angiosarcoma, respectively, while at a dose of 3.33 mg/kg-day no tumors have been observed. These results arc 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 E i. bolic or detoxifying pathways. The dose-re sponse curve generated from the two higher doses (50 and 16.6 mg/kg-day) predicts a 9c/c 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 i of pharmacokinetic data in interpretation of why high doses of a chemical may produce toxi- igure 2. ,4C-activity excreted in the urine expressed as percent of the dose administered (1 and 100 mg/ kg) vs. time. Each point represents the mean , standard error of the mean for five rats. The initial li.iear segments of the curves (1Z-36 hr) were fit by linear regression analysis, tu, =4.6 hr. ites decreases and the chemical is free to find s way to other sites or to be eliminated. Thus, may be concluded that the pulmonary excreon of VC is not a rate-limiting step. Even ore importantly, the data indicate that the ate in which VC exists in the body changes ith dose. Figure 4 summarizes the dose-dependent exetion of VC via urinary excretion (solid line) id pulmonary elimination (broken line). The ea indicated by the rectangle represents the 1 ge of doses where evidence of distributive metabo'ic saturation first occurs. Of parul.u' sigr ificanve is that in a current carcmonesis study in rats by Maltoni (4) daily oral 0 0.5 1 1.5 2 2.5 3 3.5 4 Hours Ficube 3. Expired vinyl chloride expressed as percent of the dose administered (0.05, 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. jr 1976 147 doses of VC at GO and 1G.G nig/kg-day have resulted in 19(F and 110 induction of hepatic angiosarcoma, respectively, while at a dose of 3.33 mg/kg-day no tumors have been observedThese 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.G mg/kg-day) predicts a 9c/c 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- Of The Dose AciinirnsierctJ igure 2. "C-activity excreted in the urine expressed as percent of the dose administered (1 and 100 mg/ kg) time. Each point represents the mean * 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\i =4.6 hr. ites decreases and the chemical is free to find fs way to other sites or to be eliminated. Thus, t may be concluded that the pulmonary excreion of VC is not a rate-limiting step. Even tore importantly, the data indicate that the 'ate in which VC exists in the body changes ith dose. Figure 4 summarizes the dose-dependent exi etion of VC via urinary excretion (solid line) id pulmonary elimination (broken line). The ea indicated by the rectangle represents the uige of doses where evidence of distributive ' metabolic saturation first occurs. Of par- ular significance is that in a current carcinonesis study in rats by Maltoni (4) daily oral 0 0.5 1 1.5 2 2.5 3 3.5 4 Hours Figure 3. Expired vinyl chloride expressed as percent of the dose administered (0.05, 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. Table 1. Percentage of administered 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 HC 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 5.7 4.8 0.7 22.6 1.2 1.0 0.1 11.0 2.7 81.0 2.9 * Mean standard error, five rats/dose except three rats at 20 mg/kg level. 100 mg/kg 66.6 0.7 2.5 0.1 10.8 1.0 0.5 0.1 1.8 0.1 82.3 0.4 Table 2. Percentage of MC activity eliminated during 72 hr following inhalation exposure to 14C-vinyl chloride for 6 hr.* Expired as VC CO, Urine Feces Carcass and tissues Cage wash11 Total VC recovered, _______________% of 14C activity*________________ 10 ppm 1000 ppm 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) 14.43 0.52-(977) 0.23 0.09 (15) (6642) * Expressed as percentage of the total *4C-activity recovered. All values are means standard error from for rats. Values in parenthesis are microgram equivalents of vinyl chloride. b Water and acetone wash of the metabolism cage at termination of the experiment. city and extrapolation of the same toxic effec at lower levels is invalid because the fate o the chemical has changed. Figure: 4. Summary of the dose-dependent excretion of VC: (------) urinary excretion; (--) pulmonary elim ination. Urinary excretion reptesents 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 MC-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 '"C-activity in the expired air, feces, and urine over the subsequent 72 hr. As in the expert ments in which oral doses were given, thf 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 14C-activity in the tissues and carcas? increases slightly as the exposure is increased1 from 10 to 1000 ppm- Although not statistical!) significant, this is remarkable because a much larger fraction was expired as VC. In partic ular, the normalized amount of "C-activity ir 148 Environmental Health Perspective: ^WWPI SL 101898 the liver and skin increased. This may mean t! a larger fraction is being bound to the ma^romolceules of the tissues. This aspect is currently being investigated, since such reac tivity may explain the carcinogenic effect of VC. Figures 5 and G show respectively the elim ination of ''C-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? ici'HE 5. "'C-activity excreted in the urine expressed as percentage of the recovered radioactivity vs. time following a 6-hr exposure to in and 1000 ppm VC. Each point represents the mean rfc standaid error of the mean for four rats. The initial log linear phase of the curves (12-3G hr) were fit by linear regression analysis. Figure 6. Expired vinyl chloride expressed as percent age of the recovered radioactivity vs. time following a C-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 CO*. 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, N-acetyl-S(2hydroxyethyl)cysteine, and metabolite B, is thiodiglycolic acid. Together these metabolites ctober 1976 149 SL 101899 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 5-(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- INH-C-CH, ^0 HO--CHi--CH,--S--CHj--CH--C '''OH Metabolite A: 2V-Acetyl(S-2-hydroxyethyl)cysteine ^f if0 C--CHi-S-CII,--C HO"" ""OH Metabolite B: Thiodijfiycolic acid NHi I HO--CH,--CH,--S--CHj-CH-C ''OH S-(2-Hydioxyethyl)cysteine eter may change with close it need not l reflected by the urinary metabolites which coi stitute ultimate end products of metabolism. Our initial work and subsequently that c others (tf-) indicates that one pathway it volves oxidation of VC by microsomal enzyme to chloroethylene oxide- Other pathways whic involve either nonenzymatic or enzymatic cor jugation with GSH, mediated by soluble ei zymes, and dechlorination reactions, mediate by both soluble and microsomal enzymes, ar all possibly involved in the overall metabolisr of VC. The relative contribution of these er zyme systems in the metabolism of VC ar currently under investigation. Depression of Hepatic Nonprotein Sulfhydrj Content by VC A very important aspect of the metabolisr of VC is the detoxification reaction with hepati nonprotein sulfhydryl groups (composed c primarily GSH). When high doses of som chemicals, for example bromobenzene an acetaminophen, are given, the glutathione i used up at a faster rate than it can be produce' by conjugation with the reactive intermediates As the level of glutathione in the liver is pro gressively depleted, the reactive metabolite react with macromolecules such as protein DMA and RNA leading to toxicity (9, 10) Generally, it is accepted that one mechanisn for chemical carcinogenesis may involve sue! reactions. To assess the effect of VC exposures ot hepatic glutathione levels, rats were expose( to concentrations of 10, 50, 150, 250, 1000, oi 2000 ppm for 1-7 hr (11). The results arc 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 r 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 tc 10 ppm causes no depression. How do these results relate to the carcino genicity of VC? In the studies of JMaltoni and 150 Environmental Health Perspectives I SL 101900 Percent Of Depression sarcomas of the liver were given but also the latency periods for their development were pro vided. The latency peiiods 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. F E 7. Depression of hepatic nonprotein sulfhydryl r-witent vs. duration of exposure to 2000, 1000, 250, 150, 50, and 10 ppm vinyl chloride. Each point repre sents the mean rt 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 ivas 22%. The incidence in rats exposed to 500 and 250 ppm were, respectively, 12 and 7%. Reference *o Figure 7 indicates that the depression of i he hepatic nonprotein sulfhydryl content oberved after 4 hr of exposure coincides avith Hie increased incidence of angiosarcoma. In the same study (12) an incidence of only '7o angiosarcoma of the liver occurred in rats xposed to 50 ppm VC. As indicated previously, xposure to 50 ppm for 7 hr caused a small and ^consistent depression of the hepatic nonpro1 in sulfhydryl content. This exposure appeared > he in the transition zone of the threshold for his biological effect. In a recent publication by ialtoni (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 correla tion exists between doses of VC that cause tu mors 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 (5), protein sulfhydryl groups, RNA (7), and adenosine of DNA (8). Inclusion of glutathione in the system will decrease or preclude these reactions denending 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- K-'*! _ ^-.--^ r' W ^ V --i-vI >' - `fi *3Z^ ' * -VtJ^:^..- .x^,-'-`^` ''"* <- *,`?*,\~f ZiT ^S?V * * " . _' v 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 ,JC-VC. Finally, it must be emphasized strongly that stochastic, ~statistical projections utilizing data collected frotn~rats exposed to hisrh cToses'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. Sci. 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: 3S1 (1976). Maltoni, C,, Gli effetti onogeni del cloruro di vinile 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. Phar macol. 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 rah 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 experimental environmental carcinogenesis. An example: vinyl chloride. Ambio, 4: 18 (1975). f.l i VWWWI Environmental Health Perspectives SL I0l902