Document bv2Nr3m4mvOmV84r0zXxeEM3

EXO-MEDICAL RESEARCH DOCUMENT DESCRIPTION FORM 63 68 Duplicate in all cards:--> / 974 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 1 20 21 40 41 ('rafHe. $ (LiAj-kLjz./n/irY (L.J- UJALM-T/b'k,!*rpe (LA L. 77 78 --(JJL 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. ,0 ,,,, 61 62 -ff~a;s** lIliHi Lf. Lb/cJ>Jo,-o n-F 21 Re~A~oJ~/ue-hhe^ C6. N\eJ~r^holi'fe- CJiHcJ RUrXt- // /n // 22 23 24 Source (Journal, Vol., Number, Pages, Date ) 12 61 62 31 32 Brief Summary 12 10 SUMMARY: 61 62 61 62 63 64 f ^Trapping With 3,4-Dichloro- benzenethiol of Reactive fie O P-4. ^ Metabolites Formed in vitro From >/ the Carcinogen Vinyl Chloride IL^j j AMBIO Vol. 3 No. 6, pp 234-236 (1974) 000012B Many mutagenic and carcinogenic compounds are known to be biologically i active only after metabolic transformation in the body. In many cases, transformations to reactive metabolites occur by means of the microsomal detoxification system of the liver. In order to facilitate the identification of such metabolites, formed in vitro from potentially mutagenic or carcinogenic compounds, the use of suitable thiols as trapping agents is proposed. The thiols must be acidic to ensure a sufficient concentration of negatively charged thiolate anions, highly reactive as nucleophiles. Furthermore, the choice of lipophilic, chlorine-containing aromatic thiols facilitates detection of reaction 1 . JJl products by means of gas chromatography and mass spectrometry! The present work describes the use of 3,4-dichlorobenzenethiol to trap i reactive metabolites formed from vinyl chloride (I) (Figure 1), recently reported h \r>to be carcinogenic for mice at low doses (50-ppm) in air and to cause liver ':V cancer in persons working in PVC-plants. Vinyl chloride has also been shown R&S 107415 mutagenic in in vitro systems in the presence of liver preparations. Cell-free liver microsome preparations were exposed to vinyl chloride in the presence of air. The reactive metabolites were trapped by means of the <r: 3bove thiol, added to the liver preparation or, alternatively, exposed in a separate vessel to the gas mixture leaving the liver preparation. One of the products formed from the trapping agents was identified as 3,4-dichtoro- phenylthioacetaldehyde by means of combined gas chromatography--mass spectrometry. The results are consistent with the formation of chloroethylene oxide (II) as a reactive metabolite of vinyl chloride, but can also be interpreted as indicating the formation of chloroacetaldehyde (III). This is known to be cs formed from chloroethylene oxide by a slow intramolecular rearrangement. Its formation along other routes under the m vitro conditions used, is \ considered unlikely but cannot so far be wholly excluded. It has recently been shown in this labo ratory (I) that the ilnvumentcd carcino genic effects in m.m 12. 3) of long-term exposure to relatively low concentrations in air of \inyl chloride (I) is reflected also in its mutagenic action at higher concentration in in ciirn systems on Yo7ir.oneiia responding to simple allocating agents Under the conditions used, its mutagenic activity was clearly demon strable only after metabolic activation with liver homogenates, prepared accord ing to Ames ct at (4). An obvious inicrpretation of 'these results involves ihe assumption that vinvl chloride, like aro matic hydrocarbons (S) i-, subject io epoxidatiun through the niicmsom.il de toxification enzymes with formation m this case of chloroctlnlcnc ovule (II) as a reaeuve metabolite (I, 6) Ibis highly reactive epoxide lias heen synthesized from ethylene oxide hi several routes (7. k), In a pure slate u is slowly rearranged to chioroaccialdclivdc (III) and it Indroly/es in water to glycol aldehyde (S). Ihe present .nvestigations were perfuimed in urdcr to verify the above hypothesis bv using a simple nucleophile. 3 4-Jichlorobcn/eneihiol as a trapping agent for reactive metabolites. Such a thiol should be ucniic enough to ensure a Milfinent concentration of thiolate anions already under neutral conditions, a necessary requeue for a rapid reaction with alkylating species. Die dichloropiicnyt group aisu gives pronounced lipo philic properties to the expected reaction products and facilitates their detection by gas chromatography (GC) and by com bined gas chromatography-- mass spec trometry (GC-MS) techniques. In a typical experiment, an air - vinyl chloride mixture was passed through a tuhe containing the supernatant of a rat liver homogenate freed from nuclei, mitochondria and other large particles by centrifugation and fortified with all necessary cofactors for the mixed func tion oxygenase system according to Ames ct at (4), and in addition fortified with Cl Figure 1. Vinyl chloride (1) is translormed r vitro by the microsomal mixed functiooxygenase detoxication system to a volatile reactive metabolite, most probably chloroeir.y. ene oxide (llhtrapped by 3,4-dichlorobenyen-.thiol as the aldehyde IV, or the as yet nv detected derivative V. Alternatively, II re arranges spontaneously to chloroacetaldehyor (lit) which with the thiol likewise gives IV. 3.4-dichlorobcii/enethio1 Ihe .muiuir ihiol used did not inhibit die niitnwom.:; activity, as indicated by the capacity M the system to convert p-mtmnnisol " yellow-colored p-nitrophenolaie (h> i some experiments, rat liver micros,mi^ freed from cytosol by further scrurif. cation were substituted for the muz. some cvtosol mixture (10), By R Golhe, C J Calleman, L Ehrenberg and C A Wachtmeister, Environmental Toxicology Unit and Department of Radiobioiogy, Wallenberg Laboratory, University of Stockholm, Lilia Frescati, S-104 0S Stockholm, Sweden 4 4d * rf > i Ti> -- l - ^ i .$ .fc< . * + * , . W * ' *<.< ^ 4 __________* k * m < * - * * ^ . J rt, After two hours of exposure to the gas agent in methanol. After two hours, the oxide (II) and chloroacetaldehyde (III) stream, the reaction mixture was extract reagent solution was worked up as above as metabolites of vinyl chloride under the ed .vith hexane. The acid fraction, solu and analyzed by GC.-MS. The ion m/c = in \'iiro conditions used. ble in bicarbonate solution, and the neu 220 was not detectable in the mass The reaction of chloroethylene oxide tral fraction of the hexane extract, re chromatogram, confirming absence of (II) with thiolate should give the com spectively. were fractionated by thin- the sulfide IV. The ions m/e = 248 and pounds IV and V as major products. layer chromatography (TLC). Authentic 191. however, were present, indicating Only the compound IV, 3,4-dichloro- samples of possible reaction products that the compound of molecular weight phenylthioacetaldehyde, can be detected from the postulated metabolite (I!) with 248 mentioned above is formed directly by the present method, the thiol were prepared from chloro- from vinyl chloride and the thiol under Chloroacetaldehyde (III), if formed by acctaldchyde dimethyl acetal and chloro- the present experimental conditions. isomerization of II or via other routes, acctic acid methyl ester, respectively, The results presented here are in ac would react directly with the thiolate to and were chromatographed simultane ously as reference substances. The ap propriate zones were collected and the cordance with a formation of either or both of the compounds chloroethylene form IV. The present results hence give support extracts were analyzed by GC-MS. The neutral fraction among several peaks gave a peak of the same retention time on GC as 3.4-dichlorophenxithioacet- aldchyde (IV). characterized by its ma-s spectrum which was identical with the SOME FACTS ABOUT VINYL CHLORIDE \ mass spectrum of the reference sample concerning molecular ion (M+, m/c = 220), isotope distribution pattern, and Vinyl chloride at room temperature is a colorless gas with a' sweet odor. It has been used as a propeiiant for various spray products, but its main Use is as a base peak. In addition, a second, strong peak was ohxened. the mass spectrum (M~, m/e = 24S) of which likewise in I dicated a compound containing the di~ chlorophensIthio-group. The question of structure of this compound is so far un settled and will he discussed in another context. monomer (VCM) for polyvinyl chloride <PVC> resin synthesis. The world pro duction of PVC in 1972 is estimated to have been about 16 000 million pounds, with the USA as a leading PVC producer (4300 million pounds). In Sweden about 110 000 tons of PVC are produced each year, in two plants. VCM causes acroosteolysis. Raynaud's phenomenon, sclerodermia and dis turbances of liver function among VC'PVC workmen. VCM was recently de monstrated to be carcinogenic in rats, mice and hamsters. 20 ppm, the lowest tested dose, is carcinogenic in rats and mice, showing angiosarcoma of liver and other organs, lungadcnomas and other tumots. In the winter of 197---1974 The bicarbonate soluble fraction of the hexane extract was methylated and then worked up analogous!). By means of the GC-MS technique, the presence of 3.4- i dichlorophcnslthioacetic acid methyl *1 ester was established. ' - J. . In experiments of a modified tsp'e. no thiol was added to the liver homogenate tube. Instead, the air - vinyl chloride VCM was also strongly associated to angiosaicoma of the live! among workmen in the VC/PVC industry. To date (October 15, 1974), 26 cases of angiosarcoma associated with VCM have been discovered in the world. The mean latency time is about 20 years. Twenty-one of these cases occurred in the PVC svnihetizing industry, one case in a VCM production industry, three in PVC converting industries (compounding, moulding and so forth) and one case in a plant wheic spray cans were filled with VC. In Sweden two eases of angiosarcoma of the liver arc known among a total risk population at the plant in question of about 620 men. The esposure levels in the plant in question aie believed to have been fairly high in the past, at least occasionally, X CM is thus to be considered mixture was passed through a second tube containing 3.4-dichlorobcn/cnethii 4 dissolved in methanol to trap cxelusivclv the volatile metabolites of sin>I chloride. Also m tills case, the appropriate TLC fractions from the tlnol tube were shown by GC-MS analysis to contain the sulfide itV and the unknown product of mole cular weight 248. as a human and animal cjrcinogcn, I he hygienic standauls were temporarily lowcicd in the spring of 1974 in the USA to 50 ppm scihng value for a 15-minuic period and in Sweden to 20 ppm time-weighted average and 50 ppm ceiling value foi a corresponding period. In October both the USA and Sweden set -landards in the work environment of one ppm time-weighted average and five ppm ceiling value for a 15-minute period, which will be in effect from January I. 1975. Respiratory masks will be manda tory for workers exposed above that limn from January 1. 1976. Until that date, respiratory masks should be used when levels exceed 20 ppm tn Sweden and 25 ppm in the USA. In a final control experiment an air- Bo Hohnbcrg vinyl chloride mixture was blown directly through a tube containing the trapping to a metabolization of vinyl chloride to Vinyl chloride metabolization in vitro in to TLC as above. 3,4-dichlorophcnyl- chioroethylene oxide (II) only if other the presence of thiolutc A tube containing thioucctaldchydc (IV) being applied as rout'es to chloroacctaldchyde are not vinyl chloride (Aerc'Ol Packing Co, Val- a reference.. The appropriate zone was operating. A metabolic pathway for vinyl lentuna. Sweden) was held at --20C in extracted with ethyl acetate and injected chloride in the rat has recently been an ice bath. Air was blown through the into the GC-MS unit. The peak with the proposed, involving 2-chloroethanol as a tube at a rate of 10 ml/min. The result same retention time (1,5 min) on GC as primary metabolite, subsequently osi-. ing gas mixture was led through a similar the reference sulfide IV gave a mass di/cd via the alcoholc dehydrogenase tube kept at 37C and containing mag spectrum which displayed the same mole route to chloroacetaldehyde and further nesium chloride (80 nmoles). glucose-6- cular ion (Mt. m/e = 220; 2 Cl) and i via chloroacctic acid (6). Such a route phosphate (50 nmoles), nicotinamide base peak (M --CHO. m/e = 191: 2 Cl) would, however, be improbable under the adenin dinuclcotide phosphate (NADP) as the mass spectrum of IV. Also the in vitro conditions used here, in some (40 //moles), the 9000 X g supernatant lower part of the spectra showed simi experiments involving isolated liver (3 ml) from a liver homogenate accord larities. somewhat obscured by the low microsomes. ing to Ames et al (4) from male rates signal-to-noise ratio in case of the un A final interpretation of the connec (Spraguc-Dawiey, Aniicimcx) and 3,4- known. tions between the metabolism of vinyl dichlorobcnzcnethiol (1 mg) in a final Most remaining peaks of the gas chloride and its mutagenic and carcino volume of 10 ml, 0.1 M with respect to chromatograms were characterized hy genic properties must await an unam a phosphate buffer (pH 7.4). their mass spectra. Fragmentation biguous identification of reactive meta After 2 hours' exposure to the gas patterns and isotope distribution indi bolites and a thorough characterization stream, the proteins were denaturated cative of the 3.4-dichlorophenylthio- of their reaction patterns, in vitro and with hexane (50 ml). The hexane phase group was given hy the corresponding in vivo (11). Such studies are underway, was centrifuged (10 000 rpm) for 10 min. disulfide, hy a few minor peaks and hy a EXPERIMENTAL Reagents 3,4-dichlorophcnylthioacetaldchydc dimethyl acetal was prepared from 3.4-dichlorobenzenethiol (Aldrich-Europe) by reaction with chloroacetaldehyde dimethyl acetal (Merck-Schuchardt) in lutidine at 60'C overnight. The product was isolated and characterized by its proton magnetic resonance, infrared and mass spectra. The corresponding aldehyde (IV) was not isolat The supernatant was shaken with sodium hydrogen carbonate solution (8 percent. 50 ml). The carhonate phase was stirred for 24 hours with dichloromcthanc (0.5 ml), dimethyl sulfate (0.5 ml) and tetrahutyl ammonium hydroxide (25 mg). The reaction mixture was extracted with hexane, the hexane extract was evaporat ed to a small volume and subjected to purification by TLC (Merck Silicagel large peak (retention time 8.4 min), which displayed a probable molecular ion M1, m/e = 248. Trapping of volatile vinyl chloride meta bolites A rat liver microsomal suspension was prepared and washed in 0.2 M potas sium phosphate buffer pH 7.5 as de scribed by Arrhenius (10). The microsome incubation system was prepared, with minor modifications, as recum- ed but obtained in solution after hydrolysis, HF: hexane-ethyl acetate 9;I), whereby mended hy the same author; To 10 ml in the absence of air, of the above acetal with aqueous sulfuric acid (2 M) for 100 hours at 20*C and extraction with hexane. 3.4-dichiorophenylthioacetic acid methyl ester was prepared and characterized anal ogously from methyl chloroacetate and the thiol. Mass spectrometry A Hewlett-Packard 5930 A mass spectrometer combined with an HP 5933""A datasystem and connected with an 3.4-dichlorophenyIthioacetic acid methyl ester was chromatographed as a refer ence substance on the same plate, sepa rated from the region containing the unknown sample through a free area. The appropriate zone was collected and extracted with ethyl acetate. The pres ence of 3.4-dichlorophenylthioacetic acid methyl ester was established by com final volume of 0.2 M phosphate buffer: 5 ml microsomal suspension correspond ing to 40--70 mg protein. 0.5 "moles NADP. 50 "moles g!ucose-6-phospl:ate. 600 "moles nicotinamide, and 1.25 Rom berg Units glucose-6-phosphatc dehydro genase. An air-vinyl chloride mixture as de scribed above was passed via the bottom HP gas chromatograph 5700 A. with a parison with the authentic reference of a tube containing the microsomc in split to a flame ionization detector, was used for characterization of reaction pro ducts from the in vitro experiments de scribed below. The 160X0,19 cm (inner dia meter) glass column was filled with 2 per cent polycthene on acid washed and silanized Chromosorb W 100/120 mesh. The carrier gas (helium) flow was held at 30 ml/min. The column temperature was 130*C for 4 min and then programmed 8*C/min to sample. The mass spectrum from the appropriate GC-pcak was identical with that of the reference sulfide with respect to molecular ion (M~. m/e = 250). iso tope distribution and base peak (m/e = 191). The hexane phase from the initial extraction was further extracted with bicarhonate solution until free from cubation system, kept at 37C. to a second tube containing 3,4-dichlorobcnzcncthioi (5 nig) in methanol (5 mil. kept at 20C After two hours, the meth anol solution was worked up as above, Tlte neutral fraction was subjected to purification by TLC. The presence i : u,c aldehyde IV and the compound. M ` = 248, in the appropriate fractions was con 200-C. thiol. The neutral extract was subjected firmed hy GC-MS. R&S 107417 leferences and Notes U Rannug. A Johansson. C Ramcl and C A Wachtmcisicr. Atnhin 3. 194 11974), I. J M Creech and N M Johnson. Jniinuil nj Occuiniltuitul Methane 16. 15(1 (19741. i. B Holmberg and G Molina, IVoik PnvtHiitniennil Health, in fire". 4. B N Ames. VV E Dursion. E Yamasaki and F D Lee. /`loeeedinys at the iXtirtontil Atudeni' of Science',. USA 70. 2281 11547.3). 5. J W Daly. D M Jenna and B VVukop. Lxfierienna 28, 1129 (1972). 6. P J Gchrtng. Annul' of ihe AVh Ynik Attidini' nf Stiencej. in inc'i. 7. Ch Walling and P S Frederick, Jnurnai at American Clivnucat Society 84. 3326 (19621. 8. H Gross and J Freiberg, Journal jur Pntkti'che Chennc 3IJ, 506 (1969k 9. V G Zannom. in finidiiniennil' nf Dine Menduih'in and thuy /)iv. ini'innii. U N l.a Do. H G Mandel and E I. Way. cd' (Williams and Williams Go. Baltimore. 1971), pp 506^569. 10. la Arrhenius, C'ltcinntd tlintnyieul /menu non' I, 161 (1969 70). 11. L Hhrenbcrg. K D llicscbe. S Oslcrman-Golkar and I Wennberg. Miihinun Kc'ctuch 24. S3 (19741. 12. Thanks arc due <o Mm Lilly Johansson for preparation of the microsomc incubation -y-icms and to Mr Olof Radmnrk for run ning the mass spectrometry. This work was financialls supported by (he Swedish Xaiurai Science Research Council and by The Swedish Work Ensironmcm Fund. 13. Received Sepiembcr 25. 1974.