Document KQL6E97QvbxKMLzB0mBMJjKQ

R&S 107371 B,TO-MEDICAL RESEARCH DOCUMENT DESCRIPTION FORM Duplicate in all cards: 63 68 69 ooQoraj' /fc^l l year as-1961- File number [Right justify [Numeric only] Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded by one blank space 20 21 40 41 1 """ *7 pj WrTPH, ZPF, f 6~ J .3 7778 l'C~ Sub-Index Code 60 61 62 11 12 13 Title of Report; end with space-hyphen-hyphen-space. Follow with Index Terms, separated from each other with comma-space. Avoid other punctuation; do not abbreviate. "7^i' // i'r/i i' Z/-- I) c- JF - c/,6 <3 eT & f,/ ~Ffc. .4--^rs SJyn C' 2.`-,\c /7j ? sF F/Oms s J/ c------ ?, i /(" 'y ' .JT7 ,/)s 21 22 23 24 Source (Journal, Vol., Number, Pages, Date) yO - ,--> 1 2 / (F /j 'F !7 / F . F'? - ?S' . F?? 7 ': 7 j. 61 62 te / f L'f''//"Fr-e77tr\ 31 32 Brief Summary 12 10 SUMMARY: 61 62 61 62 63 64 i t / r- The Relevance of Dose-dependent 0000123 Pharmacokinetics in the Assessment of Carcinogenic Hazard of Chemicals P. J. Gehring, P. G. Watanabe and J. D. Young Toxicology Research laboratory, Health and Environmental Research Dow Chemical, Midland, Michigan 48640 R&S 107372 Typically, the potential of chemicals to cause cancer is evaluated by ad ministering daily a maximum tolerated dose of the agent to animals, usually rats or mice, for their lifetime. The maximum tolerated dose has been selected as the maximum dose not causing death or severe debilitation of the selected species in short-term studies lasting 6 to 12 weeks. Many times the chemical has been administered by routes other than that via which exposure occurs in practice. This methodological concept has resulted in the administration of massive doses of chemicals for which the real-life exposure is much smaller and often via a different route. In essence, this approach reveals whether the potential of the chemical to cause cancer is equivalent to or slightly greater than its potential to cause death or severe debilitation. Frequently the doses supersede those which produce clinical disease. Although this approach may be acceptable for screening chemicals for '-:rther evaluation, translation of the data to predict the hazard of low-level exposure is presumptuous guesswork at best. The presumptuous guesswork has been sophisticated by the statistician who has applied his stochastic, statistical processes, and "stochastic" (which is defined as guesswork) is emphasized. Not considered in making such statistical extrapolations is the fact that the fate of many chemicals changes as the magnitude of the dose. With some chemicals, metabolic changes are induced by large doses, render ing animals receiving such doses different from those receiving low doses. In either case, changes in fate of the chemical or the metabolic status of the animals preclude the use of routine statistical processes to predict the hazard of low doses. Indeed, this violates the a priori assumption for their use. Toxicity, including carcinogenesis, is a dynamic process involving absorp tion of a chemical into the body, distribution to various tissues, reversible or irreversible reactions with cellular components, and ultimately clearance from the tissues and the body via metabolism and/or excretion. Therefore the predictability of animal toxicological data for assessing the hazard of a chemical to man is enhanced if the fate of the chemical per se and/or its 187 ilu t t n-m \ 188 P. J. Gehring, P. G. Watanabe and J. 0. Young degradation products in animals is equated to the fate in man. By fate, we refer to the kinetics for absorption, distribution, reversible or irreversible reactions with cellular components, and elimination of the chemical per se and/or its degradation products. "Pharmacokinetics" is the term used to refer to such studies of the fate of a chemical. Over a selected range of doses, the pharmacokinetics of chemicals are commonly linear, i.e., the kinetics for absorption, distribution, reactions with cellular components, and elimination are proportional to the concentration or amount present. In the simplest form, "linear pharmacokinetics" may be expressed by the linear differential equation ac -5~** where C is the concentration in a tissue at time t, and kt is the rate constant for elimination. Solution of this differential equation gives C = Co exp (--kr) or log C = log C0 2.303 ' where C0 equals the concentration at time zero. A plot of C versus time on semilogarithmic paper will yield a straight line with slope kj2.303. As long as the pharmacokinetics of a chemical remain linear, a tenfold in crease in dose will increase the tissue concentration tenfold at any point in time. However, many metabolic and excretory processes are saturable, and as doses of the chemical begin to saturate or overwhelm these processes, it may be expected that there will be a disproportionate increase in the con centration in tissue and consequently in the toxicity. For these processes, nonlinear pharmacokinetics apply, which can be described by the MichaelisMenten equation: ac _ vmc at Km + c In this equation, dC/dt is the rate of change in the concentration of the chemical at time f; C is the concentration of chemical at time t; Vm is the maximum rate of the process; and Km, the Michaelis constant, is equal to the concentration of the chemical at which the rate of the process is equal to VJ2. There are two important limiting cases to the Michaelis-Menten equation: when the concentration is small compared to Km, SC VmC at ~ Km ' C<< Km', when the concentration is large compared to Km, ac Vmi at C >> Km. wvsQ'Vicpvnucni rate anu wuwuyvniv na&aiu ia Figure 1 Simulated plasma concentration of a chemical (C) or the amount in the body (A) as a function of time for a chemical which displays dose-dependent or nonlinear pharmacokinetics described by the Michaelis-Menten equation. dCldt is the change in concentration with time, Vm is the maximum rate of the process, and Km is the Michaelis constant. Figure 1 depicts a typical tissue concentration or amount in the body--time curve for a chemical whose elimination follows nonlinear or Michaelis-Menten kinetics. As long as the dose incurred via exposure to a chemical results in concentrations or amounts significantly less than Km, the linear portion of the semilogarithmic plot is applicable. Doses that, provide concentrations or amounts approaching or superseding Km lead to the nonlinear portion of the curve. Some criteria that serve as indications that nonlinear pharmacokinetics are applicable for describing the elimination of a chemical from the body were set forth by Levy (1968): 1. The decline of the levels of the chemical in the body is not exponential. 2. The rate constant for elimination decreases with increasing dose. 3. The area under the tissue concentration-time curve increases dispropor tionately with increasing dose. 4. The composition of the excretory products may be changed both quanti tatively and qualitatively by dose. 5. Competitive inhibition by other chemicals metabolized or actively trans ported by the same enzyme system is likely. 6. Dose-response curves may show an unusually large increase in response ;; > i,, I' ; iK < < i i i . R&S 107374 190 P. J. Gehring, P. G. Watanabe and J. D. Young C--------- RM 'RM-M \\ axcretad detoxified TISSUE DAMAGE AND/OR CANCER Figure 2 One-compartment model illustrat ing the chemical process for the fate of a chemical in the body. Each arrow represents a first-order process. (For a more elaborate treatise, see Gillette 1974b.) with increasing dose, starting at the dose level where "saturation" effects become evident. To this list of criteria should be added one more. The reaction of the chemical per se or reactive metabolites produced from it with macromolecules --DNA, RNA, or protein--increases disproportionately with dose. In car cinogenesis, this criterion is very important because it is generally believed that reactions of electrophiles with macromolecules lead to carcinogenesis. Gillette (1974a,b) has given special consideration to the pharmacokinetics of reactive metabolites of foreign compounds that react with macromolecules. The concepts presented in these papers are very important to the toxicologist and oncologist because they elucidate plausible threshold mechanisms for toxicity, including carcinogenesis. Figure 2 shows a simple, one-compartment model illustrating a typical chemical process for the fate of a chemical in the body. Each arrow represents a first-order rate process. The predominant process governing the fate of .the chemical is different for different species, different chemicals, and frequently for different doses of the same chemical. For example, furosamide, a diuretic, is excreted predominantly via the kidney when low doses are administered. However, when high doses are given, the renal clearance of furosamide is overwhelmed, causing a disproportionate increase in the formation of toxic metabolites which react with macromolecules. Bromobenzene is an example of a chemical whose fate in the body occurs almost entirely via biotransformation. Elimination of this compound is via formation of the chemically reactive arene oxide 3,4-bromobenzene epoxide. Detoxification of the epoxide occurs both enzymatically and nonenzymatically, with 70% of a nontoxic dose being converted to the glutathione conjugate by glutathione-S-epoxide transferase in the liver. Administration of toxic doses of bromobenzene depletes available glutathione, thus reducing conjugation of the reactive electrophilic metabolite with glutathione and, consequently, in creasing alkylation of macromolecules and toxicity. Toxicity is not incurred until depletion of glutathione reaches a critical level. Here again is an example of nonlinear pharmacokinetics and a threshold for the toxic response. For a summary of what we have presented thus far, refer to Figure 3. As increasing amounts of fluid flow into the barrel, elimination via the lower slit becomes overwhelmed, leading to disproportionate increases in the amount of fluid in the barrel and/or elimination via the upper slit. In the body, elimination via the upper slit corresponds to excretion via other routes, metabolism via other pathways, or, in some cases, reactions with macro molecules. In the foregoing we have provided a conceptual basis for why metabolic R&S 107376 -I -ij-r ? * J I i I 5 f cfc: Figure 3 Diagrammatic representation of the elimina tion of a chemical via a primary saturable pathway and a secondary pathway whose significance increases upon saturation of the primary pathway. crib s thresholds may lead to a disproportionate increase in toxicity, inclusive of carcinogenesis. For a more complete treatise on the use of pharmacokinetics in assessing the hazard of chemicals, refer to Gehring et al. (1976). To elucidate the concept further, we will utilize the results of 1,4-dioxane and vinyl chloride pharmacokinetic studies conducted in the Toxicology Research Laboratory of The Dow Chemical Company. 1,4-Dioxane 1,4-Dioxane was reported to cause hepatomas and nasal carcinomas in rats maintained for over 13 months on drinking water containing 0.75% to 1.8% dioxane (Argus et al. 1965, 1973; Hoch-Ligeti et al. 1970). Kociba et aL (1974) confirmed these findings in rats maintained for 2 years on drinking water containing 1 % dioxane. These rats also experienced an excessive num ber of deaths and exhibited severe renal and hepatic pathology. In rats main tained on water containing 0.1% dioxane, significant renal and hepatic damage was observed, but no increased incidence of tumors occurred. A further tenfold reduction in the concentration of dioxane in the water, to 0.01%, produced no discernible untoward effects. For the three levels of dioxane in the drinking water of rats, the approximate daily doses of dioxane were 1015 and 1599, 99 and 148, and 10 and 19 mg/kg/day for males and females, respectively. The carcinogenic activity of dioxane, revealed only in rats receiving doses which superseded those necessary to cause death and severe pathology, raised the question of whether dioxane poses a carcinogenic hazard to individuals ex posed to small amounts in their work environment. To provide additional information for making this judgment, a study was conducted to determine whether the fate of dioxane is influenced by dose (Young and Gehring 1976). Figure 4 shows the plasma concentration-time curves for [14C]dioxane in rats given single intravenous doses of [14C]dioxane ranging from 3 to 1000 mg/kg. The clearance of dioxane from plasma is markedly dose-dependent and in accordance with Michaelis-Menten kinetics. The area under the curve increases disproportionately with dose, indicating that the elimination of dioxane is a saturable, dose-dependent or nonlinear pharmacokinetic process. The excretion of 14C-aetivity by rats given various doses of [14C]dioxane also demonstrated dose-dependent kinetics. As the dose was increased, more dioxane per se was eliminated via exhalation; at low doses, essentially all was excreted rapidly as /?-hydroxyethoxyacetic acid in the urine. Thus the bio- a. Vi.: it; i i i Concentration of dioxane per se in plasma of dioxane intravenously. rats given various doses of 192 33 8 CO o 2 N/ Nf t 1 I 1 * t I 1 t1 1 I V*t vy fizczot s?a ' j".'*- ' : it:1:'a. ; it*4 f, ^ Daily body burden of HC-radioactivity of rats given oral doses of 10 or 1000 mg dioxane/kg/day. The body burden was'calculated by subg trading the total cumulative excretion by all routes from the total cumulative dose and expressing the results as a percentage of the total cumulative dose. (Inset) Ratio of the daily body burden of 14C-radioactivity after daily doses of 10 or 1000 mg/kg dioxane. a -r* --- ^ : 7" . .1 rs *T ri:^' i r*?X*rfus.r ?;.\t - >b. tpc - >? -* 1 - \f *? j'- ' _ ; * 'K 1?*TW?lTivPTF*T7j(*( * I rt** ^ 3 1 .JaiiLi* -i. :i4k ih\ f -j.itXu-', Aii-at' J-*`' 4.1 -J- ?. ...lKi MWs0 'MKlS1Si`l. |S* ^UTSfrirS^'^`^: Wif tS 194 P. J. Gehring, P. G. Watanabe and J. D. Young transformation of 1,4-dioxane to the detoxification product /J-hydroxyethoxyacetic acid is a saturable process which is overwhelmed by increasing the magnitude of the dose. The marked retention of dioxane with increasing doseled us to conclude that the metabolism of dioxane must be markedly induced with repeated daily doses. Figure 5 shows the body burden of radioactivity in rats given repeated daily oral doses of 10 or 1000 mg/kg for 17 days. The body burden was calculated by subtracting the total cumulative excretion by all routes from the total cumulative dose and expressing the results as a percentage of the total cumlative dose. The body burden in rats given 10 mg/kg/day dioxane averaged about 5% and ranged between 2% and 9%, with no apparent up ward or downward trend. However, a striking decrease occurred in the body burden of rats given 1000 mg/kg/day dioxane during the first 4 days of administration. This indicates that a dose of 1000 mg/kg/day but not 10 mg/kg/day caused a marked induction of the elimination of dioxane. In essence, the rats receiving 1000 mg/kg/day dioxane had undergone marked biochemical alterations; their responses to dioxane, toxicological or car cinogenic, are no longer extrapolatable to rats receiving low doses. Metabolic induction itself has been shown to increase tumorigenesis. Peraino et al. (1973a) demonstrated an enhancement of tumorigenesis in mice given 0.05% phenobarbital, a well-known inducer of metabolism, in their diet. Furthermore, induction of metabolism in rats by phenobarbital en hanced tumor production by 2-acetylaminofluorene (2AAF), a known hepatic carcinogen (Peraino et al. 1973b), suggesting that induction may enhance the expression of tumors by naturally occurring carcinogens. The marked dose-dependent fate of dioxane and the strong metabolic induc tion by high doses, together with the toxicological results reported by Kociba et al. (1974), negate extrapolation of high-dose carcinogenesis to predict the hazard of low doses of dioxane. Such extrapolations violate common sense as well as the a priori assumptions for such statistical extrapolations. In rats, cancer induction occurs only when the doses are sufficient to cause marked pathology, metabolic alteration, and even death. These effects, including carcinogenesis, are correlative with the dose-dependent fate of dioxane. In light of this correlation, the hazard of low-level exposure to dioxane appears to be nil. Vinyl Chloride In our initial studies, four rats were exposed to varying starting concentrations of vinyl chloride (VC) in a closed, recirculating, 4.7-liter system (Hefner et al. 1975). The concentration of VC in the system was monitored by infrared analysis, and the rate at which the rats took up and chemically altered VC was determined as indicated in Figure 6. It is obvious that the rate of uptake, indicated by the slope, is markedly dependent on the initial exposure concentration. At an initial concentration of approximately 75 ppm VC, the rate of loss was much faster than the rate of removal from the chamber when the initial concentration was approximately 1000 ppm VC. The steeper the slope, the faster the rate of removal. In similar experiments, we demonstrated that the rates of removal between initial concentrations of 50 ppm and 105 ppm were the same, having a half-life (rw) of 86 minutes. Between con- Dose-dependent Fate and Oncogenic Hazard 195 Figure 6 Semilogarithmic plot of the decline in the vinyl chloride concentration in a 4,7-liter, osed, recirculating chamber containing iour rats (controls). Upper portion shows decline when initial con centration was approximately 1000 ppm; lower portion shows decline when initial concentration was ap o -Nj proximately 75 ppm. CO 00 o centrations of 220 ppm and 1167 ppm VC, the rates of removal were the same, with t^ =261 minutes. From the results of these studies, it was concluded that VC is metabolized or degraded readily at low concentrations, and that the primary pathway for degradation is swamped at concentrations exceeding 220 ppm. Another objective means of demonstrating that the pathway for the metab olism or degradation of chemicals may be overwhelmed is to administer potential inhibitors of its metabolism. Figure 7 shows that the administration of 5 ml/kg of 95% ethanol inhibits profoundly the metabolism of VC by rats exposed to an initial concentration of 50 ppm ethanol but not that by rats exposed to approximately 1000 ppm. The percentages of inhibition were 96% and 40%, respectively. In other studies, we found that SKF-525A inhibited by approximately 20% the metabolism of VC by rats exposed to initial con centrations of approximately 1000 ppm but caused no inhibition in rats ex posed to initial concentrations of less than 100 ppm. SKF-525A inhibits the mixed-function oxidase system of the cellular microsomes; this pathway is VC ixpoture cone, ppm Figure 7 Semilogarithmic plot of the decline in the vinyl chloride con centration in a 4.7-liter, closed, recirculating chamber containing four control rats and four rats treated with 5 ml/kg ethanol. 196 P. J. Gehring, P. G. Watanabe and J. 0. Young Table 1 Percent of Administered MC-activity Eliminated following a Single Oral Dose of Vinyl Chloride Percent eliminated after doees (mg/kg) of Route Expired as: VC C02 Urine Feces Carcass and tissues Total recovery 0.05 1.0 20 100 1.4 2.1 41.6 66.6 9.0 13.3 4.8 2.5 68.3 59.3 22.6 10.8 2.4 2.2 1.0 0.5 10.1 11.1 11.0 1.8 91.3 88.8 81.0 82.3 responsible for the metabolic degradation of many foreign compounds enter ing the body. The results of these studies led to the conclusion that VC must be metab olized by at least two different pathways. The primary pathway for metab olism is overwhelmed as the exposure concentration is increased, until, at concentrations exceeding 220 ppm, the secondary pathway predominates. Using the barrel analogy, more escapes through the second slit as the flow into the barrel is increased. In subsequent studies, we were fortunate to have radioactively tagged car bon-14 to follow the fate of VC. This greatly facilitated our ability to follow the disposition of the administered VC. Table 1 shows the percentage of "C-activity eliminated via various routes following different single oral doses of VC in corn oil to rats (Watanabe et al. 1976b). The ^C-activity found in urine, feces, and carcass and tissues represents nonvolatile metabolites of VC. The key item to note in this table is that as the dose is increased from 0.05 and 1.0 mg/kg to 20 and 100 mg/kg, the percent expired as VC increases markedly, whereas the other parameters, particularly urinary excretion of uC-activity, decrease. Once again this demonstrates that the primary route for the elimination of VC from the body is dose-dependent, i.e., the primary route is overwhelmed and more begins to spill out via other routes (the upper slit in the barrel). Figure 8 summarizes the dose-dependent excretion of VC via urinary excretion and pulmonary elimination. The area demarcated by the rectangle represents that range of doses over which distributive or metabolic saturation occurs. It is noteworthy that in ongoing carcinogenicity studies, Maltoni et al. (1975) have reported 19% and 14% incidences of tumors in rats given oral doses of 50 and 16.6 mg/kg/day, respectively. In rats given 3.33 mg/kg/day, no tumors have occurred, although extrapolation of the incidences of the two higher doses predicts an incidence of approximately 9%. Although the data collected from studies in rats given oral doses of VC elucidate the dose-dependent fate of VC, the pharmacokinetics of inhaled VC are more pertinent to assessing the hazard of exposure in the industrial en- R&S 107381 I ` Dose-dependent Fete and Oncogenic Hazard 197 Percent of dose excreted in urine Percent of dose expired ms VC i- )at s. w ir- 1W as ies )le g, TS, ns to 33 ary So CO gle on Q al M CO in CD W !a *V vc VC en- i Figure 8 Summary of the dose-dependent excretion of VC via urinary excretion (--) and pulmonary elimination (------ ). Urinary excretion represents polar metabolites of VC, and pulmonary elimination is VC per se. The area demarcated by the rectangle represents the range of doses over which distrib utive and metabolic saturation occurs. vironment (Watanabe et al. 1976c). Table 2 depicts the fate of [14C]VC in rats exposed for 6 hours to 10 ppm or 1000 ppm VC. Immediately following the exposure, the rats were placed in cages equipped for collection of 14Cactivity in the expired air, feces, and urine during the subsequent 72 hours. As in the experiments in which oral doses were given, the percentage of re activity expired as VC increased as the exposure increased. Also note in this table that the percent l4C-activity found in the tissues and Table 2 Percent 14c-activity Eliminated during 72 Hours following Inhalation Exposure to 14C-labeled Vinyl Chloride for 6 Hours Percent eliminated after exposure to Route 10 ppm 1000 ppm Expired as: vc co2 Urine Feces Carcass and tissues Cage wash' Total fig equivalents VC recovered 1.61' 0.16* (4) 12.09 0.43 (30) 67.97 et 1.71 (169) 4.45 0.22 (ID 13.84 et 1.16 (34) 0.15 0.08 1) (248) 12.26 0.96* (814)h 12.30 0.63 (817) 56.29 1.96 (3139) 4.21 1.05 (280) 14.48 0.52 (977) 0.23 0.09 (15) (6642) Values are percent of the total 14C-activity recovered. " Mean = standard error from four rats. b Microgram equivalents of vinyl chloride. c Water, acetone wash of the metabolism cage at termination of the experiment. 198 P. J. Gehring, P. G. Watanabe and J. D. Young NH- HO--CH^-tHj--S--CH2--CH--C : 'OH (A) N-acetyl($-2-hydroxyethyl)cysteine C---^H2~"S--. HO OH (B) Thiodiglycolic Acid NH, HO^--CH2--CH2--S--CH2--CH--C S-(2-hydroxyethyl)cysteine OH Figure 9 Metabolites of vinyl chloride found in the urine of rats. Metabolites A and B comprise approximately 30% and 25%, respectively, of the metabolites of vinyl chloride found in urine. A third metabolite, com prising 35% of the metabolites present in the urine, has been iso lated but remains unidentified. These metabolites were isolated us ing high-pressure liquid chroma tography. Authentic standards of each have been synthesized and cochromatographed. The unidenti fied metabolite was thought to be S-(2-hydroxyethyl) cysteine but it did not cochromatograph with an authentic standard. carcass, though not statistically significant, increases slightly as the exposure is increased from 10 ppm to 1000 ppm. This is remarkable since a much larger fraction was expired as VC. In particular, the normalized amount of 14C-activity in the liver and skin increased. This may mean that a larger fraction is being bound to the macromolecules of these tissues. This aspect is currently being investigated as such reactivity may explain carcinogenesis. The results of these studies support the conclusions of previous studies that (1) the fate of VC changes with dose, and (2) this occurs because the pri mary pathway for the metabolism of VC is saturated at high doses or exposures. Since the metabolism of VC appears to occur via at least two pathways, a considerable effort has been made to identify the metabolites of VC. It had been demonstrated previously that a measurable amount of VC was metab olized to the end product of mammalian metabolism CO>. By means of highpressure liquid chromatography, three major metabolites have been isolated from urine. Two of the three have been identified by gas chromatography-mass spectroscopy. These are shown in Figure 9. Metabolite A is N-acetyl(S-2hydroxyethyl) cysteine. Metabolite B is thiodiglycolic acid. Together these metabolites comprise 50% to 60% of the radioactivity found in urine. Both of these metabolites are likely formed from the compound shown at the bottom, S-(2-hydroxyethyl)cysteine. At one point it appeared that the third major metabolite in urine, comprising about 30% of the radioactivity, was this compound. Even though some analytical comparisons between the isolated metabolite and S-(2-hydroxyethyl) cysteine favored this conclusion, others failed to establish identity. The finding of these metabolites of VC in urine indicates that VC is trans- R&S 107384 Figure 10 Percent depression of hepatic nonprotein sulfhydryl content vs. duration of exposure to 2000, 1000, 250, 150, 50, and 10 ppm vinyl chloride. Bars represent the mean standard error for five animals, except the point for 50 ppm which represents 25 animals. Depressions were significantly different for exposure concentrations of 150 ppm and greater but not for 50 ppm. formed in the body to a reactive intermediate metabolite. This reactive metabolite is detoxified by reaction with glutathione. Subsequently the glutamic acid and glycine moieties of glutathione are cleaved, and the cysteine conjugate of the reactive metabolite of VC is either acetylated or further oxidized and excreted as the aforementioned metabolites. Before leaving the subject of metabolism, it is important to state that the metabolites of VC were not changed either qualitatively or quantitatively as the dose or exposure level was increased. Since evidence exists that there are at least two pathways for the metabolism of VC, this indicates that both pathways produce reactive metabolites leading to the same end products. Our initial work and subsequently that of others (Kappus et al. 1975; Bolt et al. 1975; Barbin et al. 1975) indicate that one pathway involves oxidation of VC by microsomal enzymes to chloroethylene oxide. As indicated previously, the reactive metabolites of VC are detoxified by reaction with glutathione. This is very important because it has been demon strated that when high doses of some chemicals (e.g., bromobenzene and acetaminophen) are given, the glutathione is used up at a rate faster than it can be produced by conjugation with the reactive intermediates. As the level of glutathione in the liver is progressively .depleted, the reactive metabolites react with macromolecules such as protein, DNA, and RNA, which leads to toxicity (Gillette 1974a,b). It is generally accepted that at least one mechanism for chemical carcinogenesis occurs through such reactions. To assess the effect that exposures to VC may have on hepatic glutathione levels, rats were exposed to concentrations of 10, 50, 150, 250, 1000, or 2000 ppm for durations of from 1 to 7 hours, (Watanabe et al. 1976a). The results are shown in Figure 10. Exposure to 150, 250, 1000, or 2000 ppm VC caused a progressive depression of the hepatic nonprotein sulfhydryl content; the hepatic nonprotein sulfhydryl content is primarily glutathione. Exposure to 50 ppm for 7 hours produced a small and inconsistent depression. No de- J , :i i'llll H!|i II i' I ft f '! *i R&S 107385 200 P. J. Gehring, P. G. Watanabe and J. 0. Young Table 3 Correlation of the Incidence of Angiosarcoma and the Depression of Hepatic Nonprotein Sulfhydryl in Rats Exposed to Vinyl Chloride Exposure concentration (ppm) 10,000 6000 2500 2000 1000 500 250 150 50 10 Percent angio sarcoma* 19 22 22- -- -- 12 7 -- 2 --- Mean latencyb (weeks) 64 70 78 -- -- 81 79 -- 135 -- Nonprotein sulfhydryl depression 4 hr 7 hr __ -- -- -- -- 45 65 30 45 -- 25 35 20 20 0 15 (?) 00 * Maltoni and Lefemine (1975). *> Maltoni (1975). pression was observed in rats exposed to 10 ppm VC. These results indicate that there is a measurable biological threshold for the depression of hepatic glutathione levels induced by exposures to vinyl chloride. Unequivocal de pressions are produced by concentrations exceeding 50 ppm, whereas 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 carcinogenicity of VC? Maltoni and Lefemine (1975) reported the incidence of liver angiosarcoma in rats exposed to various concentrations of VC 4 hours/day, 5 days/week for 1 year and subsequently maintained until death. A summary of these data together with our data on the depression of the nonprotein sulfhydryl content of the liver is given in Table 3. At exposure concentrations of 250 ppm VC and greater, the incidence of angiosarcoma correlates well with the depression of hepatic non protein sulfhydryl content. In the study of Maltoni and Lefemine (1975), a liver angiosarcoma in cidence of 2% occurred in rats exposed to 50 ppm VC. As indicated pre viously, exposure to 50 ppm for 7 hours caused a small and inconsistent depression of the hepatic nonprotein sulfhydryl content. This exposure ap peared to be in the transition zone of the threshold for this biological effect. In a recent publication by Maltoni (1975), not only are the incidences of liver angiosarcomas given but also the latency periods for their development. 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 latencies for the R&S 107386 . 0ose-dependent Fate end Oncogenic Hazard 201 development of other types of tumors showed the same discrepancy. Con sideration of these results leads to the conclusion that, in rats, exposure to 50 ppm VC 4 hours/day is in the threshold transition zone not only for hepatic | nonprotein free sulfhydryl depression but for tumor induction as well, j In summarizing the studies on the pharmacokinetics and metabolism of VC, j the data indicate that the fate of VC in rats is dose-dependent following either j 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 detoxifying pathways. The primary detoxification pathway for VC, which appears saturable at high dose levels, involves conjugation of its reactive metabolites with non protein sulfhydryl groups. -Therefore it seems reasonable to postulate that as the nonprotein sulfhydryl groups are depleted, reactive metabolites will be free to react with other macromolecules (DNA, RNA, protein, lipids), re sulting in toxicity and carcinogenicity. Recent reports have demonstrated that in the presence of fortified microsomal enzyme preparations, reactive metab olites of VC are produced which covalently bind to rat liver microsomes (Kappus et al. 1975), protein sulfhydryl groups, RNA (Bolt et al. 1975), and adenosine of DNA (Barbin et al. 1975). Inclusion of glutathione in the system will decrease or preclude these reactions, depending on the concentra tion. It is highly significant that exposure to 10 ppm VC for 7 hours caused . no depression of hepatic nonprotein sulfhydryl content. This indicates that there is a threshold of exposure in rats where the ability to replace sulfhydryl ! groups is not overwhelmed and physiologic defense mechanisms remain fully { operative. Furthermore, this suggests that thresholds exist for toxic effects * which are expressed with greater intensity as this protective mechanism is I depressed. Studies currently in progress are designed to characterize the macromolecular binding of VC to protein and nucleic acids following exposure to various concentrations of [14C]VC. j Finally, it must be emphasized strongly that stochastic, statistical projec!- tions utilizing data collected from rats exposed to concentrations exceeding j 100 ppm VC are invalid for predicting the hazard of lower level exposures. * Such projections violate the a priori assumption that the dynamics governing J the fate of the compound are unaltered. j, As an overall conclusion, the dose-dependent alterations in the fate of chemicals must be considered when using toxicological data, including car{ cinogenesis, obtained at high doses to assess the hazard of low doses. For , many chemicals, large doses exceed metabolic and physiological thresholds, ; leading to prolonged retention in the body, formation of different metabolites, and in some cases disproportionate increases in reactions between reactive electrophilic metabolites and macromolecules, and, consequently, there is a disproportionate increase in toxicity, including carcinogenesis. Other com! pounds foT which there is evidence indicating that their fate may be dosedependent include styrene, ethylene glycol, aniline, carbon disulfide, 2-naphthylamine, benzopyrene, bis-hydroxycoumarin, salicylamide, amphetamine, and sulfobromophthalein (Gehring et al. 1976). It is likely that as more com pounds are evaluated, dose-dependent fate will be found to be the rule rather ' than the exception. ! i < i i { t 202 P. J. Gehring, P. G. Watanabe and J. 0. Young REFERENCES Argus, M.F., J.C. Arcos and C. Hoch-Ligeti. 1965. 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