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Biotransformation in rats of vinyl chloride vis-a-vis its oncogenicity
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Early warning that one year's intermittent inhalational exposure of
Wistar rats to 3% (v/v) of vinyl chloride elicited epidermoid and
mucoepidermoid carcinomas in the para-auricular region of surviving
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animals has been confirmed in more detailed and extensive investigations ,
which revealed, in addition to Zymbal gland carcinomas, liver angiosarcomas
and nephroblastomas in Sprague-Dawley rats that had been exposed to
atmospheric concentrations as low as 250 ppm. Vinyl chloride also induced
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liver angiosarcomas in Swiss mice at 500 ppm . This latter work established
a direct relationship between the dose level and length of treatment with
vinyl chloride and neoplastic response. Those observations and the
world-wide record by June 1974 of 24 cases of liver angiosarcoma amongst
workers, who had been engaged upon vinyl chloride/polyvinyl chloride
manufacture, demand a better understanding of the biological fate of
vinyl chloride in mammals in relation to its established oncogenicity.
Moreover, angiosarcoma induction by chronic intraperitoneal (vinyl
chloride) injections implies that vinyl chloride, and not a product of
its photolysis, is the causative agent.
r!4 1
Excretion data showed that the main eliminative route for-- C_l vinyl chloride after oral, intravenous or intraperitoneal administration to rats is pulmonary (Table 1) (see also ref 4); both unchanged vinyl chloride and vinyl chloride-related CO^ are excreted through the lungs and other ti_l4 Cij metabolites via the kidneys. A marked change in
2 excretion pattern that accompanies a nearly two thousand-fold difference in intragastric dose level is due to a saturable drug metabolism and to an highly efficient pulmonary excretion of original substance that leaves the relatively low concentration of vinyl chloride material remaining in circulating blood for hepatic biotransformation. For groups of animals that had been orally dosed vinyl chloride within a dose-level range of 1-450 mgkg \ the pulmonary output of unchanged substance (expressed in terms of % of the dose) is proportional
to the logarithm of the reciprocal dose (Fig 1). Excretion patterns after
i.v. and i.p. injections are predictable from the characteristics of excretion after oral administration (Table 1). Whilst pulmonary excretion of unchanged vinyl chloride after oral dosing is complete within 3-5 h, pulmonary elimination of CO^ and renal excretion of vinyl chloride metabolites occupies 3 days. In comparison, 99% of a small i.v. dose was excreted unchanged within 1 h of injection; 80% within 2 min. Since the rate of elimination of single oral doses of ( Cj vinyl chloride is unaffected by up to 60 days' chronic dosing with the unlabelled substance at 3, 30 and 300 mgkg ^ day \ excretion data for an acute dose apply also to the chronic situation.
Distribution studies by whole-animal authoradiography agree wi deductions made from the excretion data, but small localizations of are visible in the para-auricular region of appropriate sections, possibly in the Zymbal glands.
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Of the vinyl chloride in the circulating blood, which escapes the highly efficient pulmonary arterial-alveolar transfer and is metabolized, thiodiglycollic acid, S-(2-chloroethyl) cysteine, N-acetyl-S-(2-chloroethyl) cysteine and urea are major urinary metabolites, chloroacetic acid and glutamic acid comprise the minor metabolites, and methionine and serine are trace metabolites. Families of related [ CJ metabolites were separated from the urine of t^cj vinyl chloride-treated rats by anion-
exchange chromatography, and residues from successively displaced fractions were derivativized, purified by t.l.c. and preparative GC and examined by
5 GC-mass spectrometry . Accurate mass measurements of the principal ions and NMR spectroscopy aided allocations of structure, and where possible, mass spectra were compared with those of authentic substances.
The halogen atom of vinyl chloride is stable to nucleophilic reagents,
and glutathione reacts directly iri vivo with
vinyl chloride in an
anti-Markownikov manner to give S-(2-chloroethyl) cysteine (a) (Fig 2),
which is further metabolized into the mercapturic acid, N-acetyl-S-
(2-chloroethyl) cysteine (b). Retention of chlorine in metabolites (a)
and (b) would have been realized only through the reactions that have
been mentioned. Associative reaction with molecular 0^ typically involves
the bonding of both olefinic C atoms at the transition state. We envisage a singlet oxygen-bonded form^ in dynamic equilibrium with a cyclic
peroxide form. Chloroacetic acid (c) would be formed through rearrangement of a singlet oxygen-bonded transition state, and (c) is metabolized further into thiodiglycollic acid (d) and glutamic acid (e), whereas
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formaldehyde (f), and through it CO^ (g) and urea (h), is formed by
dismutation of a cyclic peroxide transition state. Evidence for formaldehyde*
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14 ri4 -)
formation rests on the production of CO^ and
CJ urea, and on the detection
of j^c] methionine (i) and L^^clserine (j), themselves established
formaldehyde metabolites. Formation of formaldehyde and CO^ along this
metabolic pathway finds confirmation in their production ini vitro amongst the reaction products of a known cyclic peroxide of vinyl chloride 7 * 8 .
Another reaction that seemed feasible would involve addition of a thiol to vinyl chloride followed by addition of the resulting 2-chlorothioethane to another molecule of vinyl chloride to yield di-(2-chloroethyl) sulphide, which would hydrolyse to thiodiglycol, and which might be expected to yield thiodiglycollic acid by subsequent oxidation. Whilst present work does not exclude di-(2-chloroethyl) sulphide formation, it is unlikely that this intermediate is a major source of thiodiglycollic acid (see refs. 9,10). In comparison, our observation that thiodiglycollic acid is the hitherto undiscovered major metabolite (more than 60% of the dose) of chloroacetic acid in rats strongly supports its genesis from vinyl chloride by this metabolic pathway.
Although present work does not exclude the occurrence ini vivo chloroethylene oxide, it provides no evidence for its formation, despite the fact that rearrangement of this substance to chloroacetaldehyde 14 would account for chloroacetic acid production. However, if the epoxide were formed even as a bonded transition state, the ensuing reaction of the epoxide or its rearrangement product with glutathione would afford
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products , which would differ structurally from the ones, which have been established rigorously for metabolites (a) and (b). Moreover, chloroethylene oxide is not prepared direct from vinyl chloride in vitro^.
The metabolism of vinyl chloride is important to its oncogenicity. Thus, (i) formation of all three major urinary metabolites, thiodiglycollic acid S-(2-chloroethyl) cysteine and N-acetyl-S-(2-chloroethyl) cysteine, utilizes glutathione, and under certain circumstances, vinyl chloride exposure would deplete the glutathione pool seriously, despite compensating mechanisms. Besides significant blocking of non-protein sulphydryl groups m the blood of vi. nyl chlori.de operati.ves 13 , some decrease in the availability of hepatic non-protein sulphydryl groups has been found in
14 treated rats . Since a fundamental role for glutathione in the body may be to protect against electrophilic attack by drug metabolites and other alkylating agents 15 , chronic exposure to an high dose level of vinyl chloride would lower the body's protection against attack by reactive metabolites. (ii) Biotransformation of vinyl chloride involves intermediary metabolism, and formation of glutamic acid implies C^-alkylation of an oxaloacetate intermediate, whilst thiodiglycollate production involves double C2~alkylation of cysteine-derived hydrogen sulphide^. In addition,well-established
metabolic pathways for formaldehyde afford very small amounts of methionine
and serine ^ The fact that this study provides ample evidence for
and
C2 alkylation of intermediary metabolites suggests that this sort of chemical reaction may lead to the transcriptional changes of the RNA, DMA or other macromolecular cell constituents, which would appear to be responsible
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for vinyl chloride-induced neoplasm. However, alternative reactive metabolites may contribute to the physico-chemical mechanism of carcinogenesis and for example, di-(2-chloroethyl) sulphide may be important.
It may be significant that vinyl chloride has been shown to react in vivo with the same sort of nucleophilic protein constituents, with which N-hydroxy-2-fluorenylacetamide sulphate (the ultimate carcinogen about which
18 most is known) reacts iii vitro and in vivo . Those reactions are held to be especially important to the oncogenicity of 2-fluorenylacetamide through its reaction with nucleic acid.19
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D E Hathway and T Green
Imperial Chemical Industries, Central Toxicology Laboratories, Alderley Park, Cheshire, SK10 4TJ, UK
Received March
, 1975
*9 101
Viola, P.L., Proc. 10th int. Cancer Res. Congr., Houston Texas, May 22-29 (1970); Viola, P.L., Bigotti, A., and Caputo, A., Cancer Res., 31 516-522 (1971).
!Maltoni, C., Int. Congr. Ser. no. 322, Advances in tumour prevention, detection and characterization, vol. _2_, 8pp. Cancer detection and prevention. Proc. 2nd int. Svmp. on Cancer Detection and Prevention, Bologna, April 9-12 (Excerpta-Medica, Amsterdam 1973); Maltoni, C., and Lefemine, G., Rc. Accad, naz. Ser. 8, 56, 1-11 (1974); Maltoni, C., and Lefemine, G., Enyir. Res., 7, 387-405 (1974).
Maltoni, C., in Wld. Hlth., Int. Agency Res. Cancer, Int. Techn. Rep. no. 74/005. Report of a Working Group on Vinyl chloride, Lyon, June 24, 25 (1974).
4 Schaumann, 0., Medizin Chem., 2, 139-147 (1934).
Green, T., and Hathway, D.E., Chem.-Biol. Interactions, in the press ^Alder, R. W. , Baker, R., and Brown, J. M., 'Mechanism in Organic Chemistry1
(Wiley-Interscience) London, 1971) p. 295 t seq 7
Lederer, M., Angew. Chem., 71, 162 (1959)
Razuvaev, G.A. , and Mmsker, K.S., J. gen. Chem. U.S.S.R., 28, 957-964 (1958). 9
Roberts, J. J., and Warwick, G. P. , Biochem, Pharmac., 12, 1329-1334 (1963) ^Davison, C., Rozman, R. S., and Smith, P. K., Biochem. Pharmac., 7, 65-74
(1961).
Zief, M., and Schramm, C. H., Chemv. Tnd., 660, 661 (1964).
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12 Johnson, M. K. , Biochem, Pharmac., 16, 185-199 (1967)
13 Gabor, S., Radu, M., Preda, N., Abrudean, S., Ivanof, L., Anca, Z., and Valaczkay, C., Ig. Microbiol. Epidem., Buc., 13, 409-418 (1964)
14 Hefner, R. E., Watanabe, P. G., and Gehring, P. J., Ann. N.Y. Acad. Sci. in the press
^Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R., and Brodie, B, B., J. Pharmac. exp. Ther., 187, 211-217 (1973).
16 Kun, E., in 'Metabolic Pathways' (edit, by Greenberg, D.M.) Vol. _2, p. 248 et seq (Academic Press, New York, 1961).
17 Hathway, D. E., 'Biotransformations', in 'Foreign Compound Metabolism in Mammals'(edit, by Hathway, D. E. ) Vol. 2, p. 247 (The Chemical Society, London, 1972).
18Miller, J. A., and Miller, E. C., Jerusalem Symp. on Quantum Chem. & Biochem. (edit, by Bergmann, E. D, , and Pullman, B.) vol. l_j_ 237-261 (The Israeli Academy of Sciences and Humanities, Jerusalem, 1969).
^Kriek, E., Biochim. biophys. Acta, 355, 177-203 (1974).
O ->l o>
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TABLE ] Excretion pf radioactivity in rata, given a tingle dose of [_14c] vinyl chlon 'e
[U^ vinyl chloride per kg.
L14 1 Cj vinyl chloride per kg in orn oil solution, and another 4 mis were each dosed similarly with 450 mg of Four rats were each injected in the femoral vein v-`th 250 pg f r_14cl vinyl chloride per kg in X-<"-hydrnxyethyl) lactaaiide.
Four rats were each injected i.p. with 25U VS of
similarly with 450 mg ot
vinyl chloride.
vinyl chloride per kg n H-10-hydroxycthyl) lactaiide, v'd another 4 ann.ils were each injected
Size of
dose
Time 00
Intrag astric
______Exhale d air,
Vinyl chloride
cr
Urine
Faeces
Radioactivity Excreted (Z of dose}3
Intravenous
__ Exhale d air 'Vinyl
chloride
Urine
Faeces
Intrapur i tonen1
f 'ihal f d air____
^VITjyl
C02~..
chijride
Urine
Faeces
250 ug kg'1
0-24 24-43 40-72 Total
3.7+1.2 3.7+1.2
12.6+1.1 0.9
13.5+1.3
71.5+5.0 3.3 0.3
75.1+4.2
2.8+2.5 1.6 0.2 4.6+3.0
99.0+0.3 0.1 99.0+0.8 0.1
4. 50 mg kg"I
0-24 24-48 48-72 Total
91.9+2.5 91.9+2.5
0.6 0.1
0. 7
4.5+2.3 0.4
0.8
0.3
0.1
5.4+2.2 0. 7
0.5 0.5
0.1 0.1
43.2+4.6
10.3+2,2 0.7
41.5+4.8 1 .6
1. 6 0.2
43.
11.0+1. 2
43.1+4.7 1.8
96. ^4.1
0.7
2.S+0.9 0.1
0.1
96. '*4.1
0. 7
2.6+0.9 0. 1
aValues shown are the means + S.D. of those means.
Pulmonary e xcre tio n o f unchanged v in y l
a
i-l
Uo
&
<CoOu
-a U-l O M
Dose-1
g3o3
(/) Fig. 1 Linear-log plot of the pulmonary excretion of unchanged vinyl O chloride against the reciorocal doses -J --------- ~------------------------------------ --------------------------O) Tt Five groups of 3 rats were dosed i.g. with vinyl chloride at dose 1_.:'s
in the range of 1 to 450 mgkg ; each point represents 3 independent valu
si9zoi. ssa
C!CH2-CH2SCH2-CHC02H
cich2-ch2sch2-chco2h
NH-j (a)
NHAc (b)
-{s [ch2ch2ci] A 2"T'J2l
r s[ch2co2h]2 M)
ho2c ch2ci (c)
ho2c [ch2]2chco2h
NH,
(e)
------- ~ (CH50) -----2 (0
CO, (g>
MeSCH2*CH2-CHC02H
(i) m2 '
hoh2c-chco2h nh2 (j>
NHoloCO
(h)
If
metabolism of vinyl chloride in rats