Document 2JxQMYrM6p4LYN34onkz3wLGR

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-tabeled 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 metaholic or detoxifying pathways. Additional studies characterized the depression of liver non protein sulfhydryl content (primarily GSII) 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 does 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. ntroduction e the carcinogenicity of a chemical has established a primary consideration is lie hazard of exposure to low levels of the iven chemical. Studies on the pharmacokinetic nd metabolic characteristics of such chemicals re essential in accurately assessing the hazard f low level exposures. Pharmacokinetics is the udy of the dynamic processes involved in the ^sorption, distribution, metabolism and elimiation of chemicals from the body. Pharmaconetic studies alone are insufficient to assess xicity. However, their principal value is in ^relation of toxicity with the time-related disisition of chemicals in the body. An altered sposition of a chemical in the body with dose n explain in certain instances why toxicity, eluding carcinogenicity, is produced at high ses and not at low doses of the same chemi1. In the case of carcinogenicity, stochastic, itistical projections are made from the range doses over which an increased incidence of t'.cer can be measured to predict what per*!age of individuals may respond at lower Toxicology Research Laboratory, Health and Envimental Research, The Dow Chemical Company, Mid- ' 'ichigan 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. tober 1976 S^ 145- 'fife NOTICE: THIS MATERIAL MAY 3E PROTECTED 8/ CQftVftlfiBT LAW fJTLE 17. U. S. CODE) V Figure 1. Plot of hypothetical log dose vs. per cent re sponse curie. 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-dependent 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 14C-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 ,4C 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 14C-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 14C-activity elim inated via the urine as a function of time Since the slopes or rates of elimination (tI/2 - 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 p half-life of 1-1 min and was followed by a slower phase with a 11/ of 41 min. The rates or v. times for elimination at the 100 mg/kg dose correspond well with those reported by Withey (1) 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 SL 041656 "TTTT Environmental Health Perspectives 7 doses of VC at 50 and 1G.G mg/kg-day have resulted in 19 r/i and 14 ff 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 16.6 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- i n 11 11b tC f UCI tilt? L/'Lyj'- :vre 2. MC-activity excreted in the urine expressed s percent of the dose administered (1 and 100 mg/ g) vs. time. Each point represents the mean rt tnndard error of the mean for five rats. The initial :iear segments of the curves (12-36 hr) were fit by near regression analysis. 114 = 4.6 hr. is decreases and the chemical is free to find way to other sites or to be eliminated. Thus, nay be concluded that the pulmonary excrei of VC is not a rate-limiting step. Even re importantly, the data indicate that the te in which VC exists in the body changes h dose. 'igure 4 summarizes the dose-dependent ex:ion of VC via urinary excretion (solid line) pulmonary elimination (broken line). The 1 indicated by the rectangle represents the ge of doses where evidence of distributive metabolic saturation first occurs. Of par lor .significance is that in a current carcino?sis study in rats by Maltoni (4) daily oral 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. 147 doses of VC at GO and 1G.G nig/kg-day have resulted in I9r/c and 14 ft 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 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- Of Tin; Ovjj!. m irin tiis ic rc c i ;URE 2. "C-activity excreted in the urine expressed s percent of the dose administered (1 and 100 mg/ S) -- time. Each point represents the mean tandard error of the mean for five rats. The initial near segments of the curves (12-36 hr) were fit by near regression analysis, fij =4.5 hr. ;s decreases and the chemical is free to find way to other sites or to be eliminated. Thus, nay be concluded that the pulmonary excrei of VC is not a rate-limiting step. Even re importantly, the data indicate that the -e in which VC exists in the body changes h dose. 'igure 4 summarizes the dose-dependent ex ion of VC via urinary excretion (solid line) pulmonary elimination (broken line). The i indicated by the rectangle represents the ge of doses where evidence of distributive metabolic saturation first occurs. Of parlac significance is that in a current carcino sis study in rats by Maltoni (4) daily oral Ficure 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 t4C activity eliminated during 72 hr folio-wing 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 9o of 14C activity* 1.0 mg/kg 20 mg/kg 2.1 0.2 13.3 0.6 69.3 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. lOOmgfkg 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 l4C activity eliminated during 72 hr following inhalation exposure to 14C-vinyI chloride for 6 hr.* Expired as VC CO, Urine Feces Carcass and tissues Cage wash*1 Total VC recovered, pg 10 ppm % of ,4C activity* 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) 14.48 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 fou 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. Figurk 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 14C-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 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 14C~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 SL 041659 Environmental Health Perspectives ll^jver and skin increased. Tins may moan 1 a larger fraction is being bound to the nacromolecules of the tissues. This aspect is urrently being investigated, since such reacivity may explain the carcinogenic effect of TC. Figures 5 and G show respectively the climlation of ''C-nonvolative metabolites in the rine and VC per se in the expired air. Neither limination process is rate-limiting or ovei'helmed by increasing the exposure concentraon. However, it is noteworthy that expij-ed C increased with increasing dose, whereas mretion of urinary metabolites decreased, suggesting a saturation of the metabolism of VC. The results of these studies as those of nrevious 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. Hours 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. Hours vre 5. "C-activity excreted in the urine expressed s percentage of the recovered radioactivity vs. time Mowing a 6-hr exposure to 10 and 1000 ppm VC. ach point represents the mean standard error f the mean for four rats. The initial log linear phase ' the curves (12-36 hr) were fit by linear regression valysis, 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 COs. 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 M ober 1976 SL 041660 149 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- nh-C-CH, I^ HO--CH,~CHi--S--CHj--CH--C "'''OH . Metabolite A: TV-Acetyl(S-2-hydroxyethyl)cysteine C-CII,--S--CH,~C IJO^ ''OH Metabolite B: Thiodiglyeolic acid NH, I HO--CH,--CII,--S--OH,--CH--C ''OH 5- (2-Hydroxyethyl) cysteine eter may change with dose it need not b reflected by the urinary metabolites which con stitute ultimate end products of metabolism. Our initial work and subsequently that o others (C~S) indicates that one pathway in volves oxidation of VC by microsomal enzyme to chloroethylene oxide. Other pathways whicl involve either nonenzymatic or enzymatic con jugation with GSH, mediated by soluble en zymes, and dechlorination reactions, mediate* by both soluble and microsomal enzymes, ar> all possibly involved in the overall metabolisn of VC. The relative contribution of these en zyme systems in the metabolism of VC ar> currently under investigation. Depression of Hepatic Nonprotein Sulfhydry Content by VC A very important aspect of the metabolisn of VC is the detoxification reaction with hepatb nonprotein sulfhydryl groups (composed o primarily GSH). When high doses of som< chemicals, for example bromobenzene am 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 DXA and KXA leading to toxicity (9, 10) Generally, it is accepted that one mechanisn 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, oi 2000 ppm for 1-7 hr (11). The results art 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 p 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 ppin causes no depression. How do these results relate to the carcino genicity of VC? In the studies of Alaltoni and 150 cl 041661 Environmental Health Perspectives nt Of ession 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 vats 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. ' Conclusion i 7. Depression of hepatic nonprotein sulfhydryl 'Crr'itent 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). sfemine (12) f the reported incidence of angio;rcoma of the liver in rats exposed 4 hr/day, days/week to 2500 or 6000 ppm was 22%. re incidence in rats exposed to 500 and 250 >m were, respectively, 12 and 7%. Reference Figure 7 indicates that the depression of e hepatic nonprotein sulfhydryl content obrved after 4 hr of exposure coincides with 2 increased incidence of angiosarcoma. In the same study (12) an incidence of only '> angiosarcoma of the liver occurred in rats posed to 50 ppm VC. As indicated previously, losure to 50 ppm for 7 hr caused a small and onsistent depression of the hepatic nonpron sulfhydryl content. This exposure appeared be in the transition zone of the threshold for s biological effect. In a recent publication by ltoni (13), not only the incidences of angio 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 (6), 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- SL 041662 151 more, this suggests that thresholds exist for 5. Watanabe, P. G., et al. Fate of "C-vinyl chloride 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 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. following exposure to various concentrations of Lancet: 1425 (1975). "C-VC. 8. Barbin, A., et al, Liver-mierosome-mediated forma Finally, it must be emphasized strongly that tion of alkylating agents from vinyl bromide and stochastic, 'statistical protections 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 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 priori assumption that the dynamics governing ing of reactive metabolites with changes in the the fate of the compound are unaltered. incidence and severity of toxicity. Biochem. Phar m macol. 23: 2785 (1974). REFERENCES 10. Gilletter, J. R. A perspective on the role of chem 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). 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). 3. Withey, R. J. Uptake and pharmacodynamic of 12. Maltoni, C., and Lefemine, G. Carcinogenicity bio vinyl chloride administered to rats by different assays of vinyl chloride: Current results. Ann. N.Y. routes. Toxicol. Environ. Health 1: 381 (1976). Acad. Sci. 246: 195 (1975). 4. Maltoni, C., Gli effetti onogeni del cloruro di vinile sommlnistrato per via orale nel ratto. Gli Ospedali della Vita, 2: 102 (1975). 13. Maltoni, C. The value of predictive experimental environmental carcinogenesis. An example: vinyl chloride. Ambio, 4: 18 (1975). St Ofj 663 152 Environmental Health Perspectives