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Int.Arch,Occup.Environ. Ill th 38,69-75(1976)
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Short Communications
identification of Two Urine Metabolites of Vinyl Chloride by GC-MS-investigations
G. MULLER', K. NORPOTH1'. and R. ECKARQ2
1 Institut fQr Staublungenforsehur.g ur.d Arr-eitsmedizir. der Universit&t, Westring 10, D-440O Munster. Federal Republic of Germany
^Institut fur Pharmakologie ur.d Toxikoiogie der Universitar, D-4400 Munster, Federal Republic of Germany
Summary. Thiodiacetic acid and S-(cnrboxyn'.otb.yl) cysteine are found in the urine of rats after a 48h exposure to 1COO ppm vinyl chloride. The structure of both compounds could bo clarified by GC-M5 investiqatiors. Chloroethylene oxide, chlocoacetaldehyde and chlorcacetic acid are assumed to be intermedi ates in vinyl chloride metabolism. Compounds which can be transformed to one of these alkylating agents m vivo should also lead to renal excretion of thiodiacetic acid and S-(carboxvmethyi)cysteine. Key words-. Vinyl chloride - Thiodiacetic acid - S-(carboxyrtethyl! cysteine Urinary metabolites.
Recently we described the de*. ordination of thiodiacetic acid and S-(carboxymethyl)cysteine in urine specimens after vinyl chlor ide (VC) exposure 112j , The structure of these VC metabolites was clarified by GC-MS investigations on the dimethyl ester of thiodiacetic acid and on the N-rrifiuoroacetyl-n-butyl ester of S-(carboxymcthyl)cysteine. The results of our investigations and the methods used are reported here.
MATERIAI" 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.
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R&S 114918
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Chemicals
Vinyl chloride of 99.991 purity was purchased from Messer-Griesheim GmbH, D-46QO Dortmund 1 , thiodiacetic acid (98% purity) and S-(uarboxymethyiJcysteine from Janssen Pharmaceutics, Dpt. Aldrich Europe, B-2340 Beerse, Belgium. Diazomethane was gen erated from N-methyl-N-nitroso-p-toiuene sulfonamide according to de 3ocr and Backer Tlj . All other substances including triflucroacetic acid anhydride were purchased at analytical grade from E. Merck AG, D-G100 Darmstadt.
Procedures
Inhalation Experiments. Several rats were exposed over 48 h to - -100C ppm vinyl chloride in the air in an inhalation chamber of
60 * 50 * 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 WX3, 50-100 mesh, He, of Serve Feinbiochemica GmbH & 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 Soer and Backer [ij. After concentration to 1 ml this solution was injected into GC-MS. A fraction containing 'S-(carboxymethyl 1 cysmine 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 v/as methylated in 10 ml of methanol HC1 and then butylated with 10 ml of butanol HC1 according to Gehrke and Stalling ._3j . The solution was filtered and evaporated to dryness and then treated with 1 ml trifluoro acetic acid anhydride and 4 ml dichloromethane at room temperature (see 3}). After evaporation to dryness the residue v/as dissolved in 1 ml dichloromethane for CC-MS measurements.
GC-y.s 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 v.'ith a steel column of 1.8 m length and 3 mm diameter containing 3% SE 3o at Supeicoport 100/120. vho carrier gas was helium adjusted at a flow of 30 ml/ mm. The oven mperature was 135C and the .emperature of the injection
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Fig. 1 (a) Masr. spectroqr.i;;-.-, of thiodiucetic acid diemethylestor. A = standard; B = derivative of ar. urine taboiite of vinyl chloride, (b) Mass spectro grams of N-f luoroao tyl-S- (carboxymethyli-I.-cystcir.e dibutvlestor. A = standard; 3 - derivative of an urine met`.oolite of vinyl chloride
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R&S 114920
TtilUe I Proposed MS fragmentation patterns of derivatives of S- (caruoxymethy1) cysteine and thiodiacetic acid found in the urine of VC-treated rats
N-Fluoroacetvl-S- (carboxymethyl) -L-cysteir.cdibutylester
Thiodiacetic acid dimethyiester
Molecular
337
Molecular
173
m-c4h9
31-1
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240
m-coc4:-:9-c-k8
230
M-COC4H9-KOC4Hg
212
M-COOC;j-CF 3CONH2 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 7o eV.'
RESULTS
In Figure 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 173 and 387, 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-butyl ester of S-(carboxvmethyl)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 chloroerhylene oxide [6,10,1l]. After chloroacetic acid has been found as a vinyl chloride metabolite [7,15] chloroacetaldehyde was discussed as an alkylating precursor produced by spontaneous rearrangement of chloroethylene oxide [6,11j. This compound which shows mutagenic effects [j0,1l], could also be detected [5j. The detection of S-(carboxymethyl)cysteine and thiodiacetic acid in the urine of rats after exposure to vinyl
72
transforma [17] . Aijotl of chlcroc'thio.ne is t ive biotrai the en^re follo|A
C1C - C = CHH
/
C1C - C = O --
C1C - C COOK
GSH-3-transit
intermediate
As demons chloroacetal pound lowers Furthermore in the metab urine of pat agent Ifosfa boxymethyl)c l 4] . The exp product of t foreign comp
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chloride supports pne findings of Yllner who observed the bio.transformation of c.nloroacetic acid no these compounds in rr; :e
[171. Another metabolic pathway including neither the prcduc-.ion of chlcroethylene oxide nor any conjugation reac -.on with gluta thione is proposed by Green and Kathway [i1[. Supposing cxidat-, ive biotransformaticn of vinyl chloride to chloroouhyiene>oxide the entire metabolism of the carcinogen can be outlined as follows:
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COH
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lOP.17"
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* .cct:cspond :^d and :vsteina, `. C: Cl u:th-
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vinyl
GSK-S-alky1trar.1 f eras':
C1C - COH h2 h2
v C1C - C COOK
GSK-S-alkyltransferase
H2M - CH - CCOH
V ir mediate
desaminase CrH`-2
sII
H2C - COOK
H,C - COOH
`t
decarboxvlase
IIi
S
K2C - CGOH
As demonstrated by this metabolic scheme it is assumed that cnlc-e-acetaldehyde can be reduced to chloroethanol. This com pound lowers for its part the liver content of glutathione L8j. Furthermore it seems probable that an epoxidhydrase is involved in the metabolism of vinyl chloride i_2~ . Our studies on the urine of patients treated with the cancer chemotherapeutic agent Ifosfamide1 have shown that thiodiacetic acid and S-(carboxymctr.yl) cysteine appear as urine netabol ites of this drug too [14]. The explanation is that haiogonated acetaldehyde arises as product of the biotransformation of at least three groups of foreign compounds:
* Test drug of the ASTA-Werke AG, D-4312 Bracfcwoce
73
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chloroothane [jSj. 2. Free vinyl chloride, vinyl bromide and vinylidene chloride. 3. Kaloethyl compounds, which can b= dehaloethylated by oxidat ive E- and O-dealkylation, e.g. Ifosfamide1, cyclophosphamide, BCN'C, CCNU, and 2,2 ' -bis (chloroethyl) ether pi 3] .
Recently Watanabe et al. came to the conclusion that S-(2chlorocthyl-)cysteine and its acetylatod analogue, found by Green and Hathwny P], are not real metabolites of vinyl chlor
ide but artifacts of the derivati/.ation procedure Pi6^ . The
authors assume that these artifacts may arise from S- i 2-hydroxyethyl)-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 jj 6] . Thus it seems clear that vinyl chloride is metabolized via two different path ways, one of them leading to a hydroxymercapturic acid. Katanabe et al. discuss the initial formation of a chloroethyl con jugate in the animal followed by hydrolysis to the correspond ing hydroxyethyl compounds before excretion 6j . The possi bility, however, may be taken into consideration, that a {re ductive step As involved an the metabolism of a chloride-free conjugation product as it has been discussed concerning the formation of hydroxymercapturic acid from acrolein JjT . Our findings show that there is another metabolic pathway of vinyl chloride leadin' to chiodiacetic acid and this compound has also Le-an found by Green and Hathway [4j and by Watanabe et al. P6j. S-(carboxymethyl)-L-cystoine, a real precursor of thiodiacetic acid, accumulates only in the case of highly dos-rd treat ment P4]. We found near her this compound nor any chiodiacetic acid in the urine of r^ts 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'no metabolite analysis fl 4j. The aspect if specificity, how ever, needs further investigation.
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1
, 'I
j* Vt`*A` ---
* ^!*\m.**,*`^S"
> ^,aV1 ,f" ...
1 2 tri.; oridov oxjdaii. article,
hat s {2inyl ehlar i'* . The - , 2-h* ;ro>:v; uer cor,ta ride trotted 1U f.0C^3 'civ ;:l_ path'id * w a t a l^-hyl conor rcopor.d-
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kungen der Vtnyichloriuinhaiaticn auf Rcgulatior.srachanismen des Leber-
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Received Juno II, 1976 / Accepted August 16, 1975
3 5-562
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