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Int.Arch.Occup.Environ.lllth 38,69-75 (1976)
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identification of Two Urine Metabolites of Vinyl Chloride by GC-MS-Investigations
G. MULLER1, K. NORPOTH'', and R. ECKARD'
'institut fOr Staublungenforschung ur.d Arheitscedizin der Universitit, Westring 10, D-4400 KOnster. Federal Republic of Germany ^Institut fur Pharmakologie und Toxikologie der UniversitAt, D-4400 Munster, Federal Republic of Germany
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Summary. Thiodiacetic acid and S-(carboxymeir.yi)cysteine are found in the urine of rats after a 43 h exposure to lOOO ppa vinyl chloride. The structure of both compounds could be clarified by GC-KS investigations. Chloroethylene oxide, chi.~>roacetaldehyde and chloroacetic acid are assumed to be intermedi ates in. vinyl chloride metabolism. Compounds which can be transformed to one of these alkylating agents in vivo should also lead to renal excretion of thiodiacetic acid and S-(carboxymethyl)cystei.-.e.
key words: Vinyl chloride - Thiodiacetic acid - S-(carboxymethyl)cysteine Urinary metabolites.
Recently we described the detorminaticr. of thiodiacetic acid and S-(carboxymethyl)cysteine in urine specimens after vinyl chlor
ide (vc) exposure [l2j . The structure of these VC metabolites
was clarified by GC-KS investigations or, the dimethyl ester of thiodiacetic acid and on the N-trifluoroacetyl-n-butyl ester of S-(carboxymethyl)cysteine. The results of our investigations and the methods used are reported here.
MATERIAI5 AND METHODS
Animals Male SPF Wistar rats of the AF/Han strain of the Zentralinstitut fUr Versuchstierzucht, D-3000 Hannover-Linden, were used. Their weight was 130-150 g.
To whom offprint requests should be sent
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Cheni-cals
Vinyl chloride of 99.99% purity was purchased from Messer-Griesheim GmbH, 0-4600 Dortmund 1, thiodiacetic acid (98% purity) and S-(carboxymethyl)cysteine from Janssen Pharmaceutica, Dpt. Aldrich Europe, B-2340 Beerse, Belgium. Diazomethane was gen erated from N-methyl-K'-nitroso-p-toluene sulfonamide according to de Boer and Backer L1J . All other substances including trifluoroacetic acid anhydride were purchased at analytical grade from E. Merck AG, D-6100 Darmstadt.
Procedures
Inhalation Experiments. Several rats were exposed over 48 h to 1000 ppm vinyl chloride in the air in an inhalation chamber of 60 SO * 40 cm. The concentration of vinyl chloride was -ad justed by GC determinations. During the inhalation experiments the urine was collected and then prepared for analytical pro cedures.
Urine Preparations. Five ml samples of the collected urines were subjected to ion exchange column chromatography using 20 ml of Dowex 50 WX8, 50-100 mesh, H, of Serva Feinbiochemica GmbH I Co, D-6900 Heidelberg. To obtain a fraction containing thio diacetic acid the column was then washed using distilled water until neutral reaction was found in the eluate. The collected effluent was evaporated to dryness under reduced pressure at 40C. The residue was dissolved in 2 ml of methanol and methyl ated by diazomethane according to de Boer and Backer [ij. After concentration to 1 ml this solution was injected into GC-MS. A fraction containing S-(carboxymethyl)cysteine was fixed by Dowex 50 WX8, 50-100 mesh, H, after washing the resin with dis tilled water. This fraction could be eluted by 1 n acetic acid. The effluent was then evaporated to dryness and the residue was methylated in 10 ml of methanol KC1 and then butylated with 10 ml of butanol HC1 according to Cehrke and Stalling i_3j - The solution was filtered and evaporated to dryness and then treated with 1 ml trifiuoro acetic acid anhydride and 4 ml diehloromethane at room temperature (see p]). After evaporation to dryness the residue was dissolved in 1 ml dichloromethane for GC-MS measurements.
cc-KS Analysis. A mass spectrometer MAT 112 of the Varian/MAT GmbH, D-2800 Bremen, was used in combination with a Varian 1400 gas chromatograph equipped with a steel column of 1.8 m length and 3 mm diameter containing 3% SE 30 at Supelcoport 100/120. Vhe carrier gas was heliun adjusted at .a flow of 30 ml/rain. The oven .! mperavure was 135C and the temperature of the injection
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rig.t (a) mass spectrogram-: of ehlodlact:c acid dio.ncthylcsier. A atari.-.rd; 0 derivative of or urine metabolize of vinyl chloride, (a) Mass s; ->ttrograins of N-fluoroae*'-.yl-S- (carboxyr.ethyl 1-i-tystoire dlbitylestc r. a standArd: B derivative of an urir.c net-jollte of vinyl -hiorldo
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Table 1 Proposed MS fragmentation patterns of derivatives of S-tcarboxynethyl) cysteine end thiodiacetic acid found in the urine of VC-created rats
N-Fluoroacetyl-S-(carboxymethyl)-L-cysteinedibutylester
Thiodiacetic acid diir.ethylester
Molecular
387
Molecular
178
K-C4Hg
3N
M-CH3O8.
K6
M-COOC4Hg m-cf3 CO nh2 H-S-CH2 COOC4H9
2i,6 27< 240 .
H-COCCH3 tl-HCOOCH,
119 IIS
H-COC4K&-C.Ke M-C0C4 Hj-E0C4 Hg H-C00C4 K9-CF3CONH 2
230 212 200
port 155C. The ion source of the mass spectrometer was ad justed by 270C. The emission stream was 1.5 mA and the ioniz ing energy 70 eV.
RESULTS
In Figurt 1 the mass spectra of two urine compounds obtained with rats after VC exposure (1000 ppm/8 h) are demonstrated. The molecule peaks indicate me values of 1~S and 887, respect ively. The fragmentation patterns (shown in Table 1) correspond -to the structure of the dimethyl ester of thiodiacetic acid and the N-trifluoroacetyl-n-butvl ester of S-(carboxymethyl)cysteine, respectively. They are identical with the patterns obtained after injection of the corresponding derivatives of the auth entic substances.
DISCUSSION
It is assumed by several authors that the carcinogenic effect of vinyl chloride is due to its oxidative biotransformation to chloroethylene oxide .6,10,11]. After chloroacetic acid has been found as a vinyl chloride metabolite [7,15] chloroacetaldehyde was discussed as an .-.Ikylatiny precursor produced by spontaneous rearrangement of chloroethylene oxide [jS, 11 j . This compound which shows mutagenic effects 00,11], could also be .detected [5] . The detec .-.ion of S-(carboxymethyl) cysteine and thiodiacetic acid in the urine of rats after exposure to vinyl
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[l 7] . Anoth-;; of chloroethy thione is pre ive biotransi the entire me follows:
cic - c ch2-- H
c.c v CIC - C COOK H2
CSH-S-a'.i transtar.
v intermediate -----
As demonsti chloroacetaldc pound lowers I Furthermore it in the metabo) urine of patii agent Ifosiam.i boxymethyl)cyt [l 4] . The explt product of thc foreign compo-.
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chloride supports fhe findings of Yllr.er who observed the bio transformation of chloroacetic acid to these compounds in rice [17]. Another metabolic pathway including neither the production of chloroethylene oxide nor any conjugation reaction with gluta thione is proposed by Green and Hathwav [ll]. Supposing oxidat ive biotransformation of vinyl chloride to chloroethylene oxide the entire metabolism of the carcinogen can be outlined'^es follows:
reductase
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R,C - COOH *
H?C - COOH
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As demonstrated,by this metabolic scheme it is assumed that chloroacetaldchyde can be reduced to chloroet'nanol. This com pound lowers for its part the liver content of glutathione [8] . Furthermore it seems probable that an epoxidhydrase is involved in the metabolism of vinyl chloride f2] . Our studies on the urine of patients treated with the cancer chemotherapeutic agent Ifosfamide1 have shown that thiodiacetic acid and S-(carboxymethyl)cysteine appear as urine metabolites of this drug too [14]. The explanation is that halogenated acetaldehyde arises as product of the biotransformation of at least three groups of foreign compounds:
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1. Free ethyl halides as 1,2 aicnioroetimue i_iaj anu
chloroethane Ql 8j.
2. Free vinyl chloride, vinyl bromide and vinylidene chloride.
3. Haloethyl compounds, which can be dehaloethvlated by oxidat
ive N- and O-dealkylation, e.g. Ifosfamide1, cyclophosphamide,
BCNU, CCNli, and 2,2 '-bis (chloroethyl) ether [13].
Recently Watanabe et al. came to the conclusion that S--(2--
chloroethyl-)cysteine and its acetylated analogue, found by .
Green and Hathvay [4], are not real metabolites of vinyl chlor
ide but artifacts of the derivatisation procedure [16]. The
authors assume that these artifacts may arise from S-(2-hydroxy-
ethyl)-cysteine and its acetylated derivative. The latter com
pound could be isolated from the urine of vinyl chloride treated
rats and identified by MS investigations l6j. Thus it seems
clear that vinyl chloride is metabolized via two different path
ways, one of them leading to a hydroxymercapturic acid. Wata
nabe et al. discuss the initial formation of a chloroethyl con
jugate in the animal followed by hydrolysis to the correspond
ing hydroxvethyl compounds before excretion L1!* The possi
bility, however, may be taken into consideration, that a Re
ductive step is involved in the metabolism of a chloride-free
conjugation product as it has been discussed concerning the
formation of hydroxymercapturic acid from acrolein f9]. Our
findings show that there is another metabolic pathway of vinyl
chloride ieadinr to thiodiacetio acid and this compound has
also been found by Green and Hathway [4] and by Watanabe et al.
[j6l. S-(carboxymethyl)-L-cvsteine, a real precursor of thiodia-
cetic acid, accumulates only in the case of highly dosed treat
ment [_! 4j . We found neither this compound nor any thiodiacetic
acid in the urine of rats not treated with vinyl chloride.
In a previous paper we discussed the possibility to develop
biological exposure controls on vinyl chloride workers based on
ur'ne metabolite analysis
T^e aspect of specificity, how
ever, needs further investigation.
HErmSNCES
1. Dc- Boer, Th.J., Becker, H.J.: Diazomethane. Org.Synth. 22' 16 (1950) 2. Iio.lt, H.M., Kappus, H., Bolt, W.: Untersuchur.gen zum Stoffuechsel des
Vinylchlorids unter dem Blickpunkt der chemischer. Xar.zerogenese. Verh. Deutsrh.Ges.Arbeitsmed., 16. Jahrestagur.g, K61n, 5.-3. Mai 1976. Stutt gart: Centner (in press) 3. Gehrke, C.W., Stalling, D.L.: Quantitative analysis of the twenty amino acids by gas-liquid chromatography. Separation Science 2_, 101-138 (1967) 4. Green, T., Hathway, D.E.: The biological fate in rats of vinyl chloride
relation to its oncogenicity. Chru.Biol.Interactions J_l_, 545-562 i1975)
5. Gdthe, R. with j,4-i from the -
G. Grain:, H. gonicity as a I'ur.c20(3-2017
7. Grigoresci industrin
8. Jonnson, i Hi.. -l-.ux.J
9. Kaye, C.F esters an.-
10. KdlAVeil 1. Mutagenic and cr.lor:
11 . Me Cat;!-., . chloroacei (ethylene chloride , (1975!
12. Muller, C. citlorj.is.
13. Mu lie:, C. 14. Norpoch, i
Untersurhc kunyen de> stof tv.echi 5.-3. Aiai 15. Kadwau, Z. the isolar Arch.exp. I 16. Watauahe, chloride . c,:J. 22, 17. Vllner, S. C->] . ct tt. IB. Yllner, S. Acta phare. 19. yllner, S. pharmacol.
Received Juno
74
SPI-03161
J 1,1,2 tri-
..loride. / oxidatr.o- ; i-.amide,
.hal S- (2ioanci by vinyl chlor: 6~ . The o- (2-hy iroxyiutter convoride treated
it seerrs ffertnt path:Oid. Kataroothyl conaorrespond:;o possihat a rc-
de- free mna the
. Oar ay of vinyl our.d has e.nabe et al. r of thiodialosed traatniodiac'.. ;ic ioride. to develop
- based on .(city, how-
!
j
i i
i
j
i }i j 1
|
\ I : ! 1
i'950. . ' -.cl clos sc-. vc-rh.
.976. stutt-
5 Gdthe, R., Cailemar.n, C.J., Ehrenberg, L., Kachr- -ter, C.A.: Trapping
vith 3,4-dichlor''benzene-thiol of reactive metabolites droned in vitro
from the cnrr.inoct-r vinyl chloride. Aabio _3- 234-237 (19741 Greira, H., Bonse. G., Racivav, 2., Reichert, D., Henschler, .3.: Muta
genicity it: vitro and potential carcinogenicity cf chlorinated ethylenes
as a function of metabolic oxirane foraation. Biochex.Pharmacol. 24,
7.013-2017 (1975)
7. Grigorescu, I., Toba, G.K.: Oiorura di vinyl. -Aspecte de toxicclogie
industrialia. P.ev.chim.rom. J_7, 499-501 (1968)
6. Johns 'n, M.K.: Studies on glutathione S-alkyltra-sferase of the rat.
Biochom.J. 95, 44-56 (1966)
9. Kaye, C.F.: Biosynthesis of mercspturic acids from ally! alcohol, allyl
esters and acrolein. Biochem.J. 134, 1093-1101 (1973)
10. Malaveille, C., Bartsch, H., Barbir., A., Camus, A.M., Montesano, R.:
Mutagenicity of vinyl chloride, chicroet'nylenoxido, chloroo.cetaldehyde
and chloroethanol. Biccherr..3iophys.Res.Coram. 63, 363-370 (15*5)
11. Me Cam., J., Simmon, V., Streitwieser, D., Aaes, B.N.: Mutagenicity of
chloroacetaldehycie, a po'--ihie metacolic product of 1,2-dichloroethane
(ethylene dichloride', chicroethar.oi (ethylene chiorohydrin! , vinyl
chloride and cyclophosphamide. Proc. Nat. Acad. Sc (.USA 72^, 3150-3192
(1975)
12. Muller, G., Norpoth, K.: Bcstimnung cveier Orinmetabolite des Vinyi-
chlorids. Naturwiss. 62, 541 (1975)
13. Muller, G., Norpoth, K., Eckarc, R.: In preparation
14. Norpoth, K., Miiller, G., Vi 11ing, U., Gottschalk, D., Gottschalk, J.:
Untersuchungen Obcr den Si -ifvechsel des Vinvlchicrids und Coer Kir-
kungen der Vinylchioridinhalation euf Regulationsmechanismer. des lOeber-
stoffwechsels. Verh.Deutsch.Ges.Arceitsmed., 16. Jahrescagur.g, Kdln,
5.-8. Mai 1975. Stuttgart: Gentner (in press)
15. Radwan, Z., Henschler, D.: Uptake and metabolism o: vinyl chloride in
the isolated perfused rat liver preparation. Naunyn Schmiedobergs
Arch.exp.Path.Pharmek.Suppl. 237, R. loo (1975! 16. Watanabe, P.G. , Me Gowan, C.R., Gehrir.g, P.J.: Fate of [14c_. vinyl
chloride after single oral administration in rats. Toxicol.Appi.Pharma
col. 36, 339-352 (1976)
17. Yllncr, S.: Metabolism of chloroacetate-l-i4C in the mouse. Acta pharma-
col. et tox:col. 30, 69-oO :197>)
18. Yllner, S.: '.'.tabclism of 1. 1,2-trichioroethar.e-1,2-'4C in the mouse.
Acta pharmacoi. et toxiccl. jo, 249-256 (1971) 19. Yllner, S.: Metabolism of 1,2-cichlorocthane-'"C in the mouse. Acta
pharmacol. et toxicol.
257-265 (1971)
twenty ar.ir.o 101-135 (IV 7) invl chloride , -34 5-5A2
Received June 11, 1970 / Accepted Aucmsz 16, 1975
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