Document mpy4mENRr76X9Kw7Rq0dVaQ7B

Specialty Chemicals Division 340 Elk Street Buffalo. New York 14210 824-5000 March U, 1977 Dr* Walter Troll Institute of Environmental Medicine New York University Medical Center 550 First Avenue New York, New York 10016 Dear Walter: I call your attention to the attached copy of a paper on VCM by Hefner et al (Dow) in Environmental Health Perspectives 11, 85-95 (1975). You may be interested in the proposed parallel between cbloroethylene oxide and arsenic on p. 9^. I wonder if ortho hydroxy-N-hydroxy arylamines also react with ,X-lipoic acid or with glutathione in vivo. Perhaps one or both of the bis-phosphate metabolites of BNA would so react. In any event, the role of difunctional metabolites in carcinogenesis is still a live topic. They could tie up DNA and force error-prone repair. Best regards. K. H. Ferber KHF/bb Enel. cc:w/enc: Dr. Norton Nelson bcc:w/enc: J. B. Charm W. S. Ferguson H. Robinson ASI 000014710 V uscript >[ Fratin-- J - gratcfi. J ical ass. ^ nputeri; j f\ \'v \^s` V \Ca\ V ,-, -..vwj-i;.:Jill. J. N. MA.^ R 13// Environmental Health Perspectives Vol.ll,pp. 85-95,1975 Fate of Inhaled Vinyl ^son, M. ) iiride w.- ' `ality sV. ', > I chior. < /!> (1974 { ' JJjinilf; t it Print - | < of Pi.ju'. i I'opull.tl 1 lire, Wa- ; S. Cue- j uverntv | \ -Adju?'.' i UopiiC. ' V/nshir, { 'notion , Adopt. Gonv-. | h ltd-1 j nscs. I- 2 A.!.,;:- i Go:.<ii : 4i '*h rat' 5 v 5, N'i * R. E. Hefner, Jr.,* P. G. Watanabe,* and P. J. Gehring* Rats were exposed to -vinyl chloride monomer gas (YCM) in a closed recirculating system. The rate at which VCM was removed from the system via metabolism was determined for rats exposed to initial concentrations of VCM ranging from 50 to 1167 ppm. Upon exposure to initial concentrations of 50 to 105 ppm, the rate of metabolism was 8.01 3.40 X 10's min"1. Upon exposure to initial concentrations ranging from 220 to 1167 ppm, the rate constants were less; the mean value being 2.65 . 1.35 X 10'* min'*. Regardless of concentration, the disappearance followed apparent first order kinetics. Pretreatment of rats with pyrazole prior to exposure to initial concentrations of 65 and 1234 ppm VCM caused 71 and 87% reductions in the rate of metabolism. Ethanol caused 06% and 83% reductions in the rate of VCM metabolism by rats exposed to 56 and 97 ppm VCM, respectively. Ethanol was less effective in blocking the rate of metabolism by rats exposed to high concentrations of VCM; 46 and 36% in rats exposed to 1025 and 1034 ppm VCM. In rats exposed to an initial concentration of 65 ppm VCM, SKF-525-A administration caused no inhibition of the rate of VCM metabolism; however, a 19% inhibition was seen in rats exposed to 1033 ppm. The nonprotcin sulfhydryl content of the liver (glutathione and cysteine) of rats exposed to VCM concentrations ranging from 50 to 15,000 ppm VCM is reduced without a relationship to dose. With repeated daily exposure the degree of reduction is reduced. Preliminary results indicate that the primary metabolites of VC.M react with the nonprotein sulfhydryl. Final metabolic products excreted in the urine appear to be $-(2hydroxyethyl)cysteine and iS-(2-carboxymethyl)cysteinc and the respective (V-ncetyl derivatives. Monochloroacetic acid was identified as another potential metabolite. Considering the results in toto, it is hypothesized that VCM is readily and extensively metabolized. Metabolism via the primary pathway, postulated to involve alcohol dehydro genase, is swamped by exposures to concentrations exceeding 220 ppm. In rats exposed to concentrations at and exceeding this level, metabolism occurs via a secondary pathway(s), postulated to be epoxidation and/or peroxidation. These results are considered pertinent in assessing the potential hazard at low level exposures to VCM. AS I 000014711 v.nm - ' inyl chloride monomer (VCM), extensively 1 for the production of poly (vinyl chloride) 1 her plastics, has been associated with the 'lopmcnt of angiosarcoma and portal ciri> of the liver as well as other untoward ',;li in workers exposed to unknown but un' :, dly high concentrations of VCM. Angio1 our., zymbal gland carcinomas, and Research laboratory, Health and En' ` ! Research, Dow Chemical U.S.A., Midland, '> -I8C40. nephroblastomas developed in rats exposed to concentrations of VCM ranging from 50 to 10,000 ppm, 4 hr per day, 5 days per week for 12 months, and subsequently maintained and observed until death (1). No information is available on the fate of VCM in the mammalian organism. Such infor mation is essential for elucidating the toxicodynamies of VCM. This would provide a rationale to assess the potential hazard of expo sure to low levels of VCM. Results of pre liminary studies on the fate of VCM in rats exposed via inhalation are reported herein. Prior to undertaking these studies it was postu- r;-us is a p.iM-rvn ?':: sc;pnc.p'. . in ucu oi- v-. -Y Tr i i-rscviYi-i TUOY OK RLSEARC 85 * 1 '*(' " ** vl - :^A M *4 ^ (V` **__;4>*. v .. V -sSsg:^._yt::J Figure 1. Closed recirculating inhalation apparatus used for exposing rats to VCM, lated that VCM might be metabolized via the alcohol dehydrogenase pathway, and if so, this pathway would very likely be saturable and that conjugation of VCM or its metabolites with glutathione and cysteine might be ex pected. Therefore, much of the work conducted to date has been directed at evaluating these possibilities. Methods Kinetic Studies of the Uptake (Metabolism) of Inhaled Vinyl Chloride Monomer Male Sprague-Dawley albino rats of Spartan strain weighing from 165 to 200 g were exposed to initial concentrations of VCM gas ranging from 51 to 1167 ppm (0.13 to 2.99 mg/1.). Exposures ranged from 52.5 to 356.3 min. Figure 1 depicts the closed, recirculating 4.7-1. inhalation apparatus in which a group of four rats were concurrently exposed. To minimize contamination of fur and skin, only the nares of the rats protruded through' a rubber mem brane into the chamber. Expired carbon dioxide was continuously removed from the chamber by absorption on an Ascarite column in the recirculating system. As carbon dioxide was removed from the system, the pressure drop 4. 1| was detected by a mercury manometer fit*. * L. decline with a photoelectric cell. This activated a sc. -ed syst* noid valve and dual syringe pump to inj<.. i of V ( makeup oxygen into the system. .. system The chamber atmosphere was continuom analyzed for VCM (10.9 /) by an in-!;-. Miran-I infrared analyzer (Wilks). Us;: known concentrations of VCM prepared 100-1. Saran bags, the absorbance versus cccentration adhered to the Beer-Lambert Lav . thin 107 respire, .stly char -. is were vtabolisn rrefore t During the exposures, routine checks for cart dioxide in the chamber atmosphere were na., ; at 4.26 fi. Oxygen consumption for each expe: ment was determined directly by measur.:, the oxygen metered into the system using ; dry test meter (American Meter Co.). Prior to each exposure, the desired ini!;; To asses ; VCM n raperito : !e (1,2-* : ethanol concentration of VCM (Matheson Gas Pm. K&F-525 ucts, Joliet, 111., 99.97c pure) was genern:. ropyl ace in the empty recirculating system and *d- esposur decline in concentration was followed for . azole) 3 time equal to or exceeding that of the intend- : or rea; experiment. The decline in concentration u.-. ..'.treated * in accordance with first-order kinetics i- --."sen pot described by ; sired CO: dC/dt = --K,C -,ii calcui. C where C (ppm) is the concentration of VCT A. (contrc at time t (min) and Kx (min-1) is the ry constant for the decline in VCM concentn tion from the empty recirculating systcr Kfleets of Regression analysis of the logarithm of O concentration of VCM versus time yielded A Initially values for Kx were determined befwand after inclusion of rats in the chamt- These values were reproducible, therefore ' was not redetermined after removal of the r-' in every subsequent experiment. Between >' periments, the system was disassemble cleaned, and reassembled with inclusion of ; new Ascarite column. The Kx for each ." assembled system was unique, within ccrt-> limits, which necessitated its redeterminat. for each experiment. Groups < Spartar. initiatif 1 nominal G-'ride fc r week i ' hr, 7 h rried ou ' der dyn; '-tered ir. ited dai "ducted After determining the rate of decline of ^( >'.* each concentration in the empty inhalation sysV" ' re to be four rats were placed in the chamber. 1 i* reduc\ chamber was then charged with the desf' w being concentration of VCM, and the rate of dt-c " : full 7-hr of VCM concentration was determined a** ] ' I he bod, viously described. The rate of decline A- ' `s expost lowed apparent first-order kinetics. After ''rmined initial equilibration of the tissues of the t* ' ' '-* were 86 Environmental Health Perspcct>'r' *'une 1975 AS I 000014712 Al - ----- .. `-'^ .ihkw.yj/ayjfcVn iii'I.Ja.A.U^^.H ^ --*< ^^tcr fiv t decline of VCM concentration from the .ntcd a j .^1 system was assumed to occur via metabo- P to in-. of VCM phis the background loss from ` ontinuoi *t \ system. Since oxygen consumptions were -in 109b fr all experiments described here- an in*: respiratory parameters were not signifi- ks). IV ;!y changed. Therefore, respiz-atory param- >repared rs were not a factor influencing the rates of versus c mbert U :.ibolism. The rate of metabolism Kc would refore be given by eq. (2): i for car!.. ; were n:.\ 5 K^K' -- K, (2) ach exp- ; To assess the effects of potential inhibitors measur. VCM metabolism, rats were pretreated by m usin,- raperitoneal injection with 320 mg/kg pyr- ':') ; io (1,2-diazole) 1 hr prior to exposure, 5 ml/ ;red ini: vthanol 1.5 hr prior to exposure, 75 mg/kg Gap Pr ; ;.vF-525-A (/? - dicthylaminoethyldiphenyl- gener;.*1 * * 4 pyl acetate) 0.5, 0.6, 1.0, or 2.25 hr prior a and 1 . exposure, or 1000 mg/kg AT (3-amino-l,2,4- wed for ..zole) 3 hr prior to exposure. Without clean- ;e inten. . or reassembling the inhalation system, both ation v rested control rats and rats treated with the inctics . ,'Cn potential inhibitor were exposed to the 4 * -ired concentration of VCM. The inhibition ...s calculated from eq. (3): f vr 1 the r; . concent:.; (control) -- Kc (treated) = % inhibition /fj. (control) (3) r systc ! 1 Teds of VCM on Liver Sulfhydryl Levels in of : \ ; elded i. i 0roups of male Sprague-Dawley albino rats ied bef ' Spartan strain weighing from 193 to 250 g chanii.- initiation of the experiment were exposed refore 1 nominal concentrations of 15,000 ppm vinyl >f the r ride for 5 days, 5000 or 500 ppm 5 days -ween essemb ' week for 1, 3, or 7 weeks, and 50 ppm for nr, 7 hr, or 5 days. The exposures were 'ion each : n cert.' 'Wed out in a glass-walled 160-1. chamber dynamic conditions with the VCM being 'red into the chamber airstream. For re- . minat 'cd daily exposures, 7-hr exposures were -of V i sys'.'" her. 'i dc:-.: f dee 1 as [' K, ' \ft(T I tile r- ti ducted on the first four of five consecutive -,1 cach week. On the fifth day, if the rats ry to be sacrificed, the duration of exposure ; reduced to 5 to 6 hr; however, if the rats /" being continued on exposure, they received ;d 7-hr exposure. 1 he body weights and food consumption of ' exposed to 500 and 5000 ppm VCM were rniined before each daily exposure and the 4 were observed periodically for signs of f>. i fti'f* no 1975 toxicity. Between 1 and 2 PM, immediately following the fifth exposure of the designated week, the rats were killed by cervical disloca tion and the livers removed and assayed for sulfhydryl content. Gross pathological examina tions were conducted. The method used for the sulhydryl assay was a modification of that described by Sedlak and Lindsay (2). Exactly 500 mg of liver from each rat was homogenized for 1 min in a Dounce tissue homogenizer containing 8 ml of 0.02M disodium EDTA. For the total sulfhydryl assay, a 0.5-ml aliquot of each homogenate was mixed with 1.5 ml of 0.2Af Tris HC1 pH 9.2 buffer, 0.1 ml of 0.01M 5,5'-dithiobis(2nitrobenzoic acid) (DTNB), and 7.9 ml of methanol. A reagent blank without liver homo genate, and a sample blank, without DTNB, were also prepared. The color generated via the release of nitromercaptobenzoic acid anion was allowed to develop for 15 min, and the samples were centrifuged for 15 min at lOOOg. Absorbance of each sample was read against the respective sample blank at 412 nm with a Beckman DB spectrophotometer. Subsequently, the molar concentration of total sulfhydryl in the sample was calculated using an extinction coefficient determined from standards of known concentrations of glutathione or cysteine. A plot of absorbance versus the concentration of cysteine or glutathione coincided with that re ported by Sedlak and Lindsay (2). The nonprotein sulfhydryl content of liver was determined after precipitating out the pro tein by addition of 1 ml of a 50% solution of trichloroacetic acid in distilled water to a 5 ml sample of liver homogenate. Each sample was diluted with 4 ml of distilled water and after 15 min centrifuged at 4000f/ for 15 min. A 2-ml aliquot of the supernatant was mixed with 4 ml of 0AM Tris IIC1, pH 8.9 buffer. Immediately before reading the absorbance against a reagent blank, 0.1 ml of 0.01M DTNB was added. Subtraction of the nonprotein sulfhydryl con tent from the total sulfhydryl content yielded a value for protein-bound sulfhydryl. Urine samples collected from rats exposed to 5000 ppm vinyl chloride for .4, 5, and 7 weeks were analyzed for the presence of S- (2-chIoroethyl) cysteine, S- (2-hydroxyethyl) cysteine, and S-(carboxymcthyl)cysteinc (5 and 7 week samples only). Urine was collected for analysis by applying pressure to the pos- 87 AS I 000014713 ,1 ...iNI ` .wh.1. i k 1-Ytr ''f-1' " tir "" iirf* Vii iiiliir1' V> tcrior abdomen between the fifth and sixth hour of the fifth daily exposure on the desig nated week. Urine collected on the same day was pooled. For the three aforementioned com pounds, 2 to 15 /*1 of urine were spotted directly on a 5 by 20 cm Baker-Flex silica gel plate. Also spotted were samples or urine collected from control rats and standard aqueous solu-. tions as well as control urine to which approxi mately 1 fug/fi\ of each of the compounds had been added. The chromatograms were developed for 5 hr in a sealed glass tank containing ?i.-butanoI, acetic acid, and water (80:10:10 or 60:20:20). After being air dried, the plates were sprayed with Ninspray ninhydrin reagent and heated for 2 min at 80C. The color of the spots and their R, values were used to identify the compounds. A urine sample collected from rats exposed to 5000 ppm vinyl chloride for 9 weeks was analyzed for the presence of chloroacetic acid. The 10 ml urine sample was acidified with 0.1 ml of 507c (v/alHiSO^ Subsequently, the urine sample was extracted three times with 2 ml of diethyl ether. The diethyl ether extract was evaporated to 0.1 ml, and 2-15 p1 of the con centrated extract and an aqueous standard con taining 5%(w/v) chloroacetic acid were spotted on an Eastman fluorescent silica gel plate. The chromatograms were developed in a sealed glass tank for 5 hr using the afore mentioned solvent systems. After air drying the plates, they were examined under ultra violet light to determine the location of spots. Subsequently, the plates were treated with nin hydrin as described previously. Urine collected from four rats for 12 hr after exposure to 7855 ppm "C-VCM (specific activity 0.2 mCi/mmole) for 62 min was pooled and analyzed for metabolites. A 3.0-ml portion of urine was applied to 22.2 by 1.5 cm Dowex 50 (4% crosslinked, sodium form, 200-400 mesh) column and eluted with a 0.01M Tris IIC1, pH 7.0 to 3.0 gradient. A 10-12 4 por tion of each fraction containing "C activity was spotted on a 5 by 20 cm silica plate. As standards, 2 /jg of S-(2-hydroxycthyl)cysteine and S-(2-carboxymethyl)cysleinc wore spotted on the plates. The chromatograms were devel oped in a sealed glass tank containing n-pro- panol and ammonia solution (287-), 70:30. After air drying, the plates were analyzed for "C activity by using a Panax thin-layer chro matogram radioscanner and liquid scintillation counting techniques. A single minor frncof "C activity was analyzed as previously , scribed, however 2 /ig of urea was spotted the plates as a standard. Chromatograms developed in scaled glass tanks contair. n-propanol, 28T5 ammonia solution, 70.; ethanol, 287<> ammonia solution, 70:30; n.;. panol, acetic acid, 90:10; or n-butanol, a<v acid, water, 60:20:20. Results Kinetic Studies on the Metabolism of Inhaled VCM Typical declines in the concentration of Vi in the inhalation apparatus containing ( rats and initial concentrations of approxima*. 50 or 1000 ppm are shown in Figure 2. A shown are the corresponding declines in v- Klcnuu; 2. Typical (iodines in VCM concentra'y'* * time at approximately 50 and 1000 ppm posure concentrations. Also shown are the r< ''.J' declines in VCM concentration for the on ' ' inhalation apparatus. 88 Environmental Health Persp^1'1' ASI 000014714 ~^1ll I) RE 3. Declines in VCM concentration with time i- and 1234 ppm VCM exposure concentrations I r both pyrazole pretreated and untreated control r Also shown are the respective declines in VCM j :ccntration for the unoccupied inhalation appa- | !AtUS. j '.rontration of VCM from the unoccupied ! 1 mber. As indicated above, the rate of these ; lines, K,. and Kh respectively, were deter* oil by regression analysis. The rate of \ ' `nbolism, K,., was assumed equal to K,. -- K,. ' seven separate exposures to concentrations M ranging from 50 to 105 ppm, the mean : standard deviation for the apparent firstvr rate constant Iir were --8.04 X 10~* /:| >; 10-1 min'1. This corresponds to a half'J /: of 8G min. r,d' five separate exposures to concentrations ranging from 220 to 1167 ppm, 7CI ; 2.65 x 10-:` 1.35 x 10-1 miir'. This '".'poticia to a t,/_. of 261 min. As indicated j1'1 standard deviations, there was variation h. within the indicated range of concentra- Ficure 4. Declines in VCM concentration with time at 5G and 1034 ppm VCM exposure concentrations for both ethanol pretreated and untreated control rats. Also shown are the respective declines in VCM concentration for the unoccupied inhalation appa ratus. tion, however, there was no consistent trend for the values of 7C,. within this range. Administration of 320 mg/kg pyrazole, an inhibitor of alcohol dehydrogenase, xanthine oxidase, and other enzymes (3) 1 hr before exposure to 65 and 1234 ppm VCM resulted in 71.2 and 86.97 inhibition of metabolism of VCM, respectively (Fig. 3). Because of the lack of specificity of pyrazole as an enzyme inhibitor, 5 ml/kg 95% ethanol was administered to rats in an attempt to spe cifically inhibit alcohol dehydrogenase activity. In rats exposed to initial concentrations of 56 and 97 ppm VCM, ethanol pretreatment caused 96.0 and 82.97 inhibition of VCM metabolism, respectively. When rats were pretreated with ethanol and exposed to initial concentrations of 1025 and 1034 ppm VCM, inhibition of metabolism was 46,5 and 35.7%, respectively. Figure 4 illustrates the inhibition for separate ,n'1 1975 89 mi-* ^ ASI 000014715 | j *" -*----* - t Amt* .f.fc.uV'j - * .*~L-''-- c cc tL Figuhe 5. Declines in VCM concentration with time at 65 and 1058 ppm VCM exposure concentrations for both SK&F-525-A pretreated and untreated con trol rats. Also shown are the respective deadlines in VCM concentration for the unoccupied inhalation apparatus. experiments conducted at extremes of initial VCM concentrations. Pretreatment of rats 0.5 hr previous to expo sure to VCM with 75 mg/kg SK&F-525-A, an inhibitor of some types of microsomal oxidases (-'/), resulted in no inhibition of metabolism of VCM in rats exposed to initial concentrations of G1 or G5 ppm and an 18.8% inhibition in those exposed to 1038 ppm (Fig. 5). Additional exposures of rats pretreated O.G. 1, or 2.25 hr "Oh 75 mg kg SK&F-525-A to approximately 1000 ppm VCM resulted in 8.9, 6.5, and 4.5% inhibition of VCM metabolism, respectively. i FIGURE 6. Declines in VCM concentration with tic. . at 9-10.6 ppm VCM exposure concentrations for h AT pretreated and untreated control rats. Also she - 1 is the respective decline in VCM concentration ' the unoccupied inhalation apparatus. Pretreatment of rats with 1000 mg/kg A' an inhibitor of liver catalase activity (5), r- > suited in a 16.4% inhibition of VCM metal-- : lism in rats exposed to an initial concentrat; of 941 ppm VCM (Fig. 6). ; Effect of Inhaled VCM on Liver Sulfhydryl Levels \ No antemortem or postmortem signs of to*. ity were noted in rats exposed to any concor \ tration of VCM used in these experinur.'' Analysis of food consumption data for r.' exposed to nominal concentrations of 50[> 1 5000 ppm VCM, and unexposed controls vealed no statistically significant diil'entnIn addition, no significant difference was to in the water consumption of rats exposi-i 5000 ppm and controls. Throughout the 4'' tions of observation, the mean body weir' of rats exposed to the various concentrat: of VCM were essentially the same as the respective controls. No gross pathol'Vlesions related to exposure of VCM, were f"-` in any of the rats. Table 1 shows the nonprotein sulfhydryl p tent of the liver of rats exposed to 511. 5000, or 15,000 ppm VCM for the intis- durations. Values for concurrent contp'P ; also given. In Figure 7, these data arc \ OpirtbO" T.Ol'ftE 7. Do; liver of rat$ CL-ntration a: -..irized and - on of the .he liver as >n and du: : ;ctions in ' curred in T hr, 500 a; Tihte 1. Non; exposed a. ''CM concn, ppm :s poo c-rntrol i '.00 Control Control Control ` ''isnidcaru ' \ single i ' * : 0.05 ar. - 7-lir i*Tt : 0.-U0.0.- 90 Environmental Health Perspcfl,,r' June 1975 AS I 000014716 .....'nmx- it-- -------------------------------------------------------------------------------------------------------------- ---- - t .0 VINYL CHLORIOE CONCENTRATION 50 ppm f-- C=J m * 500 ppm r~ p --1. -J- * 5.000 ppm At J zz_______ -- 15,000 ppm J?S j-- ________ i----------- | 11 20 30 40 50 60 70 % Dopreii-on Of NPSH fion vilh itioi:s l\>; its. Also > :centi I jty (>), ; CM mot. ! JllCCIltr.V ; / 4 ns of li : ny nii;,- ; Mv*n Cdnc - Wrin Cone. \ C*prrtvari Of ConuoiVOI Epwd M*4 Cone, Of Controlt 'S ynfi<*ntfy dtpriuid ubnf Studnli "T" Uu, p<l0 0S 7. Depression of nonprotein sulfhydryl in the -r of rats as a function of VCM exposure con ..ration and duration of exposure. rb.ed and expressed as the per cent depresof the nonprotein sulfhydryl content of liver as a function of exposure concentra: and duration of exposure. Significant reinns in the levels of nonprotcin sulfhydryl rred in rats exposed to 50 ppm VCM for *r, 500 and 5000 ppm for 1 and 3 weeks, - porint- >'* 1. Nonprotcin sulfhydryl content in livers of rats i for r fiposcd to VCM and in concurrent controls. of mt 11>1> * i nfj > '.V.-IS J. XJNiv :Jit- ' 'lconcn, Sulfhydryl contentXlO', mole/mg liver after various exposure times --------------------------------------------------7 hr/day, 7 hr/day, 7 hr/day, {or 5 days 5 days/wk, 5 days/wk, for 3 weeks for 7 weeks v. I , :)11 .it tlr fioli n* i i in! i". n 1 IV ' ) Urol ! :Arol "Uol -Urol 0.29 0.08* 0.42*0.07 0.33*0.02* 0.46*0.03 0.35*0,09* 0.58*0.06 0.34*0.01 0.43*0.09 -- 0.41*0.03* 0.72*0.03 0.45*0.OG* 0.61*0.03 ------ -- -- 0.59*0.08 0.70*0.02 0.58*0.06 0.64*0.03 ----- > -- niificmitly different by usini; Student's t test, p <0.05. ';!>ido 1-hr exposure to 50 ppin VCM resulted in 2-05 and 0.05*0.06 for the concurrent controls. A 'In exposure to GO ppm VCM resulted in 0.17*0.07 `**0.03 for the concurrent controls. and 15,000 ppm for 1 week. There is no defini tive association between exposure concentration and the degree of depression. The degree of depression decreases with continued exposure which suggests compensatory mechanisms (s) are responding to relieve this biochemical effect induced by VCM. `The protein-bound sulfhydryl content of the liver of rats exposed to 50, 500, 5000, or 15,000 ppm VCM for the indicated durations were not significantly different from those of the concurrent controls. This was not unexpected because the sulfhydryl groups of protein have been shown not to be readily akylated unless denaturation of the protein renders them avail able for alkylation (<?). Effect of Ethanol Administration on VCM-Induccd Depression of Nonprotcin Sulfhydryl in Liver In this experiment, four rats pretreated with 5 ml/kg ethanol and four untreated control rats were exposed to 1070 ppm VCM. Follow ing 105 min of exposure, the concentration of nonprotein sulfhydryl in the liver was deter mined. For those pretreated with ethanol, the depression of nonprotein sulfhydryl was 77.0 12.8%, while for controls it was 95.0 3.4%. These levels of depression were significantly different as determined by Stu dent's t test, p <0.05. Johnson (7) has reported that ethanol alone does not affect the non protein sulfhydryl content of the liver. Metabolites of VCM Preliminary results which must be viewed with considerable reservation have been ob tained from studies in which rats were exposed to 5000 ppm unlabelcd VCM for 4, 5, or 7 weeks. Thin-layer chromatograms of urine collected from rats after each of these exposure dura tions were comparable. S-(2-Hydroxyethyl) cysteine (Rt = 0.26-0.28 in 80:10:10 7i-butanol, acetic acid, water) appeared to present. S-(2Hydroxycthyl) cysteine was not present in a thin-layer chromatogram of urine collected from control rats. S-(2-Chloroethyl) cysteine and S-(2-carboxymcthyl)cystcine were not de tected ; however, it is conceivable that the latter compound was not adequately resolved from the urine background. 1975 91 AS I 000014 7 } In another experiment, chromatograms of urine from rats exposed to 5000 ppm VCM for 9 weeks revealed the presence of monochloroacetic acid (/?/ = 0.74 in n-butanol, acetic acid, water, 80:10:10). The spots for monochloroacetic acid were visualized under ultraviolet light on the fluorescent plates and' did not develop with ninhydrin reagent. Studies of the metabolism of VCM were terminated until methods were developed to synthesize "C-VCM. 14C-VCM in a liquid state polymerized even when an inhibitor was present and a temperature of --70C was maintained. Recently, a method has been developed to syn thesize "C-VCM in a gaseous state from 1,2dichloroethane (5). In the gaseous state, 14CVCM is stable. Three 180-g male rats were exposed to 14CVCM in the closed recirculating system for 65 min. The initial concentration of HC-VCM with a specific activity of 1.155 mCi/mmole was 49 ppm. Assuming equivalent uptake of VCM by the three rats, each rat received 0.49 mg/kg VCM. Immediately following exposure, the rats were removed from the chamber and placed in Roth type metabolism cages which allow separate collection of urine, feces, expired carbon dioxide, and any expired VCM. Within 15 hr after exposure, a mean of 58.0% of the I4C activity had been excreted in the urine, 2.7% in the feces, and 9.8% as expired carbon dioxide. By 75 hr after ex posure, 67.1% had been excreted in the urine, 3.8% in the feces, and 14.0% as expired carbon dioxide. Only a trace, 0.02% of the dose, was expired as VCM and frapped on activated carbon. After 75 hr, 1.6% of the dose remained in the liver, 3.6% in the skin, 0.2% in the kid neys and 7.6% in the remaining carcass. The mean recovery of the assumed dose from the three rats was 97.9%. Chromatograms of the major fraction (97.9%) of UC activity obtained from ionexchange separation of urine of rats exposed to 7855 ppm "C-VCM for 62 min revealed the presence of three major VCM urinary metabo lites. R, values for these urinary metabolites do not exactly coincide with those for the 5(2-hydroxyethyl)cysteine or S-(2-carboxymethyl)cysteine standards', thus, if these com pounds are present, they are likely present as mcrcapturic acids (A/-acctylatcd) or other de rivatives (sulfoxide, sulfone). Chromatograms of a single minor fraction (1.2%) of "C - : v-idual nr tivity indicated the presence of "C urea as.! - j 220 pp: minor VCM urinary metabolite. `j .action w; In summary, metabolism studies have demy, I'yrazole strated tentatively the following. . ^-costing VCM is quite readily metabolized to poh . ,-..hol den metabolites which are excreted predominate Stronger in the urine of rats exposed via inhalation . alcohol t an initial concentration of 50 ppm. Snin!'. ad mini.- amounts of "C activity are excreted in i.,- less p: expired air as carbon dioxide and in the fee- ;-n than i Very little is excreted in expired air as u: . ..-gesting changed VCM. an that < A significant but small amount of "C active expose is retained in tissue, particularly liver, as lo.\ i ; - Pieth; as 75 hr after exposure. Si<JiF-32- Metabolites excreted in the urine appear . : VCM by be conjugated with glutathione and/or cystcir ,;,:r VCM through covalent linkage to the sulfhydr; duly do; group. This is consistent with the redact:, approxin of the nonprotcin free sulfhydryl levels in A in rat.- livers of exposed rats. Preliminary in vi:- :,;ch excc experiments (unpublished data) have shov .r-genase that direct conjugation of vinyl chloride win olases in cysteine or glutathione in aqueous solutio: O-Amino- occurs to a small degree but very slowly. Monochloroacetic acid also appears to be i metabolite of VCM, when rats were expo.-- ; ri`.'<es the - ,y\1 to 10 rats expo to 5000 ppm for an extended time. u-ood the /`luise pat Discussion j i Because of the preliminary nature of sor- of the studies reported herein, the results .v: ; subsequent discussion must not be considerconclusive. Justification for premature pub'tktion of some of the data contained herein the magnitude of the impact of recently rvealed toxicological effects of VCM and urgent need for communication of even j'" liminary results. The studies reported herein have der.:' strated reliably in some instances and ten1: tivcly in others the following. Rats exposed to concentrations of VCM be! > jtalase. Monochle .;> expos* --.-ks. kxposur k'hydryl a is not 'are cor.15,000 t, . . tint! to : expo. .'.h a satv. VCM to' of VC tile adn 100 ppm metabolize the compound quite road 'rices tin and in accordance with first-order rate kind:- > t1/: = 86 min. When exposed to a concentration of V( ' rprotein -'d by > r .a. exceeding 220 ppm, its rate of metabolism In rats reduced, tuz -- 261 min. This indicates that "' " 'abolizc predominant pathway for metabolism of '1 ''d prci by rats exposed to 100 ppm or less is satura1- ' fs appe; Environmental Health Pcrspecti'^ June 1975 AS I 000014718 a4ii y iiiiil'><k<A Mi*-, lyruw f* 1 of*"C inal metabolism at concentrations excccd- *C urea ; i.'O ppm may be via this pathway in con, "ion with additional pathway(s). i have de.-: .-razole inhibits the metabolism of VCM t-.-ting" that metabolism of VCM is via iaed to p . ~..ii dehydrogenase. rcdomiiur .-linger evidence for the metabolism of VCM inhalatio: ..hohol dehydrogenase was its inhibition by pm. Sm;. .idministration of ethanol. This inhibition reted in , i^ss pronounced in rats exposed to 1000 in the ft than in rats exposed to 100 ppm or less, 1 air as f "C acti-. ;vcr, as 1- .-rsting that metabolism via pathways other ; that of alcohol dehydrogenase occurs in , exposed to 1000 ppm VCM. - lhcthylaminoethyldiphcnylpropyl acetate ytP-525-A) does not affect the metabolism o appear or cysn sulfhyc* reduc: vels in y in r. ave slit oride v, solute. lowly. VCM by rats exposed to approximately 65 VCM but there is an indication that it htly depresses metabolism by rats exposed ;iproximately 1000 ppm VCM. This suggests in rats exposed to concentrations of VCM ih exceed the capacity of the alcohol dehy. cnase pathway metabolism may occur via iases in the microsomes. 'Ami no-1,2,4-triazole (AT) slightly de--cs the metabolism of VCM by rat3 ex- :I to 1021 ppm of VCM. This suggests that rats exposed to concentrations of VCM which >'eil the capacity of the alcohol dehydro- a:se pathway, metabolism may occur via | calase. ; Monochloracetic acid was found in urine of of nor ; ' exposed to 5000 ppm VCM daily for 9 suits a: j '.'Its. msidcr- 1 Exposure to VCM reduces the nonpx-otein pith!:,. t'hydryl concentration of liver. This reduc- 'mn x is not definitively associated with the ex- utly ; ire concentration of VCM in a range of 50 md : !'>.o00 ppm. There is a tendency for the re- en !'' ilcna 'ion to become less pronounced with l'epeated exposures. These results are consistent 'a a saturable mechanism for the metabolism i ter.-.- ` ` \ CM followed by conjugation of the metabo- ' of VCM with glutathione and/or cysteine. !v. J he administration of ethanol significantly :;u'03 the depression of the concentration of ' I'fofciu sulfhydryl in the livers of rats , -wd by exposure to 1000 ppm VCM for 105 \i- .a. n exposed to 49 or 847 ppm, VCM is ''bolized to polar products which are ex'"<1 Predominantly in the urine. These px-od' appear to be derived following initial metabolism of VCM and subsequent conjugation of the products with glutathione and/or cy steine through covalent binding with the sulf hydryl. A small but significant fraction of VCM is metabolized to COj and expired. An even smaller but significant amount of "C activity appears to be retained in the liver primarily, but also in other .tissues as long as 75 hr after exposure. Considering the results in toto, we hypothe size that in rats exposed to concentrations of VCM below 100 ppm, VCM is predominantly metabolized via sequential oxidation to 2chloroethanol, chloroacctaldohydo, and monochloroacetic acid by the alcohol dehydrogenase pathway [eq. (4)]. CIHC = CH, - ClCIIi-CH.-OII dehydro--g-e-n--a-s--e-- * CUI,C-CIIO - CICII^COOU (4) Only small amounts, if any, of monochloroacetic acid are formed at low doses because ehloroacetaldehyde reacts rapidly with the sulfhydryl of glutathione and cysteine (9). This conju gation accounts for the lack of dose related reduction in the nonprotein sulfhydryl content of the liver of rats exposed to 50 through 15,000 ppm VCM. Alternate pathways of VCM metabolism which may be involved at high doses are at this time purely speculative. However, at 220 ppm, metobolism by the more rapid alcohol dehydrogenase pathway appears to be satu rated, and metabolism via oxidation of the ac cumulating 2-chloroethanol may occur as in eq. (5). C1CH,--CHiOH~J--J iGlIICs--CIIiOOII--*CICIT,--CHO cntnlnso (5) Carter et al. (3) have demonsti-ated that mici'osomal oxidation of ethanol in vitro pro ceeds via the formation of hydrogen peroxide and catalase which subsequently forms a pei'oxidc of ethanol. Acetaldehyde is the end product of this oxidation. It is conceivable that a similar oxidation of 2-chloroethanol occurs. In addition to this scries of reactions, a direct epoxidation of VCM may occur [eq. (6)]. C1CH = CHi A , IhC -- CII--------* Cl ClCIIrCHO-------->C1CIIj-COOH (6) liir* bine 1975 AS I 000014719 93 Zicf et al. (10) have shown that chlorocthylcno ,, oxide spontaneously rearranges to chloroace- taldchydc. Such mechanisms would explain why SK&F-525-A causes a slight inhibition of metabolism in rats exposed to approximately 1000 but not G5 ppm VCM. Also it may explain why monochloroacetic acid may be excreted by rats exposed to 0000 ppm but not 50 ppm VCM. In the former case, chloroucetaldehyde is pro-# duced by each of the hypothesized pathways which may result in a greater amount being oxidized to monochloroacetic acid than being conjugated with glutathione and/or cysteine. Inferences from the results of these studies about the toxicodynamics of VCM are pre liminary, but worth mentioning. First, the saturation of a primary metabolic pathway for VCM degradation and redirection through other pathways provides some hope that a threshold concentration for the untoward effects of VCM may exist. Metabolites of VCM formed only via the alternate pathways may constitute the ultimate toxin and carcinogen. It has been reported (6,11-H) that the ad ministration of cysteine or glutathione provides protection against the untoward effects of vari ous aliphatic and az'omatic mustards, triethylenemelamine, x-rays, and ionizing radiation. Conceivably, cysteine and glutathione may pro vide a natural defense against tumor producing free radicals generated within the body, as well as synthetic or naturally occurring alky lating agents which are absorbed into the body. Therefore, reduction of the nonprotein sulfhydryi content of the liver in animals exposed to VCM may constitute predisposition to toxicity and carcinogenicity mediated via other ma terials. Finally, the speculated formation of chloroethylenc oxide seems particularly pertinent in sofar as postulation of the mechanism of car cinogenesis. This compound is undoubtedly a very active difunctional alkylating agent. It is most interesting that inorganic arsenicals have been reported to cause untoward hepatic effects, portal cirrhosis and angiosarcoma, like those reported for VCM (H. Popper, National Institutes of Health, Bethcsda, Md., personal communication). The mechanism of toxicity for arsenic has been shown to occur via its reaction with 6,8-dithiooctanoie acid (p-lipoic acid) (15). In this reaction arsenic forms a stable bridge between the two sulfhydryl groups. If chloro- I | ethylene oxide were formed, it would ror.c ' react with a-lipoic acid, bridging the sulfhyggreups like arsenic [eq. (7)]. OI /\ H.C-CH + CH,--CII--ClI--(CH:),~COO;:. 1 Cl SH SH 1 CHi--CH:--CII--(CH:),--COOH 1' ,* . '5 CH. ` CH | "r.vliomii pretreatr Uiochem. Gohlonth A study < ot tricti Although this postulated mechanism is high'-. ' speculative, the rarity of materials known ; produce untoward hepatic effects like these <: j VCM must be given some weight. ; Acknowledgments These studies were supported in part by ; : grant from the Manufacturing Chemists A- : sociaticr.. The technical advice of Dr. B.KJ ' Leong in designing the inhalation appanUv , and the critical review of the manuscript t: \ Dr. A'. K. Pwowe are gratefully acknowledged. f REFERENCES \ 1. Mahon), C. Ann. N.Y. Acad. .Sci,, in press, 2. Sed'.ak, J,, and Lindsey, R. H. Estimation e.f tot.' protein bound and r.on-protein sulfhydryl groups .- ! tissue with Ellmnn's reagent. Ar.a'l. Bicchcm. 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Rev. 33: 35 (1953). 15. Gonsalus, I. C. The chemistry and function of the pyruvate oxidation factor (lipoic acid). J, Cell Comp. Physiol., 41: 133 (1953). ' 111: OV.',". hose t fc;. f 1.3 il.K | _`I>t ( i'Ml. | i ft. ; I .L .* i "} ' r- ? * i f .I t Jl)nc 1975 ti "'Ml-tP- 95 AS I 000014721 -- *'J'V ,vym,,s