Document ByGeg407rmNXEoq2M8YRMb5xJ

r &S 109806 BIO-MEDICAL RESEARCH DOCUMENT DESCRIPTION FORM 53 O#0 68 69 76 Duplicate in all cards: --> I 0&&ggS2 year as-1961- File number [Right justify (Numeric only] 77 78 TTCl Sub-Index Code Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded by one blank space 1 20 21 feLnnu.G LI JLoH-aAjSSOAJ. {U/VLU-rfiEizTiBe A, 40 41 A'/imeL , (L............... . 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. 12 I ~) hp e-n 11'7Tu O-G- ULm./! dMlortcJe*- 61 62 21 hAeSk~Ci^bah'c-* filctiUtChton 22 23 24 Source (Journal, Vol., Number, Pages, Date) 12 Am Bin l)oi 3 a)o.s~ ** --*,,r ____ //V - /??': 61 62 31 32 Brief Summary 12 10 SUMMARY: 61 62 61 62 63 64 k a C w too o00 Nl \ TfUT- The Mutagenicity of Vinyl Chloride After Metabolic Activation Rannug, A Johansson, C Ramel and C A Wachtmeister, Environmental Toxicology Unit, Wallenberg Laboratory, University of Stockholm, S-104 OS Stockholm, Sweden Vinyl chloride has recently been shown to cause a malignant liver tumor disease In man after occupational exposure In PVC plants. This actualizes the problem of whether such hazards could be avoided or at least diminished in the future by a screening for mutagenicity of chemicals used In industries. The basis for such a screening procedure Is the close correlation between carcinogenic and mutagenic effects of chemicals. Experiments with Salmonella. bacteria showed that the carcinogenic hazard of vinyl chloride could have been traced by means of mutagenicity tests. The data indicate that vinyl chloride is not mutagenic per so but becomes mutagenic after a metabolic activation In the liver. direct carcinogenic compounds. In order to overcome that problem, combined test methods have been worked out w-ith microorganisms and mammals. The mi croorganisms are used for the actual mutagenicity testing, but they are ex posed to those metabolites of the com pound which are formed in an intact mammal (host mediated assay) (13) or in mammalian liver microsomal systems In vitro (12, 14). Vinyl chloride has attracted a consider makes use of the close correlation be The present investigation of the gen able amount of public attention because tween the carcinogenic and mutagenic etic effect of vinyl chloride has been of the recent discovery of its carcinogenic properties of chemicals (4). This corre performed on Salmonella typhimurium. action in man. In several countries cases lation, as do other data, points to the The mutagenicity of the compound was of the rare malignant disease angiosar fact that changes in DNA are at least an analyzed by means of reverse mutations coma of the liver have been reported essential cause of cancer induction. The in the histidine locus. The effect of among workers who have been exposed testing for mutagenicity is much cheaper metabolic activation was studied by add to vinyt chloride in plastic industries (1). and far less time consuming, and further ing rat liver microsomal systems to the Maltoni has furthermore reported an more, the results can in many cases be bacterial cultures according to the meth increase of angiosarcoma in rats after interpreted in molecular terms with re od worked out hy Ames (14), exposure to 250 ppm vinyl chloride (2). spect to the changes occurring in DNA. The objective of the presen tiga- Later results indicate carcinogenic effects This obviously is of importance for a tion has primarily been to vinyl at still lower doses (3). The hazard of better understanding of the biological chloride as an example of a compound vinyl chloride primarily concerns workers action of suspect compounds. carcinogenic to man, in order to study in the plastic industries, who get a parti One major obstacle in using muta the applicability of mutagenicity testing cularly high exposure in different con genicity tests with microorganisms for for the detection of carcinogens in the nections, but this occupational health a preliminary screening of carcinogenic human environment We are likely to be problem may have ramifications for the effects is the fact that many compounds confronted with this kind of problem general public. Vinyl chloride has, for behave as carcinogens only after an ac at an accelerated rate in the future and instance, been used as a propellant for tivation in the mammalian body. This the necessity of finding a sensible solu hair sprays and pesticides. Moreover, the activation, or in other cases deactivation, tion to the screening of carcinogenic polymerized product of vinyl chloride, of a foreign compound, is principally hazards of chemicals is imminent. Con I PVC plastic, is used ail over the world performed by the liver and involves oxi sidering the vinyl chloride problem,' one for a variety of products, among other dation, reduction, hydrolysis and con could raise the following question: Had things for the wrapping of various food jugation. A wide variety of oxidative re this genetic test method been available I stuffs. It seems inevitable that the pop actions eg C- and N-oxygenation, O-, and been used for screening of vinyt ulation is exposed to the monomer via N-, and S-dealkylation and epoxidation chloride when it was introduced for in such sources, although the doses are most are mediated by the drug metabolizing dustrial production, would such a screen likely small. system located in the membranes of the ing have given a warning of the carcino The carcinogenic effects of vinyl chlo endoplasmatic reticulum of the liver genicity of this compound? ride focus attention on the general and fundamental problem of how such haz ards can be avoided or at least diminish ed in the future--and above all how car cinogenic chemicals such as vinyl chlo ride can be spotted experimentally in the first place. Considering the vast number of chemicals used in industries, it is hardly realistic to rely on cancer induc tion tests on mice with measurement of tumor frequency; such tests take at least two years. The only feasible procedure at present seems to be a system which cells (3, 6, 7, 8). This mixed-function oxygenase mechanism can utilize reduced nicotinamide adenine dinuclcotide phos phate (NADPH) as an electron donor (9). These reactions also take place in vitro after the addition of NADPH-generating system to microsomal fractions (10, It, 12). Although microorganisms are particularly suitable from a practical genetic point of view for mutagenicity testing, they do not perform the same metabolic activation as mammals and they will therefore not pick up such in MATERIALS AND METHODS Four histidine-requiring strains of Salmo nella typhimurium (TA 1535. TA 1536. TA 1537, and TA 1538) were used for this investigation. These strains have been described in detail by Ames er al (15). They are used in a back-mutation system, where reversion to histidine independ ence occurs either as a result of base-pair substitution (TA 1535) or base-pair ad dition or deletion (TA 1536*--TA 153-S). Cultures were grown overnight in com- IS4 ' AMBtO, NO. 5 w plctc medium (Difco, Antibiotic medium ments and will therefore be described in given directly to the bacteria with gase 3). Platings with the soft agar technique some detail. After plating of the bacteria ous vinyl chloride brings about a higher were made on complete medium (CA with and whithout microsomal systems exposure dose than a treatment with vi. plates) and on minimal medium (MA the petri dishes were placed in a closed nyl chloride previously dissolved in 'I plates) described by Vogel and Bonner vessel (standard vacuum desiccator, water or in the liver microsomal sus (16) with supplements according to Ames (17) . The soft agar consisted of 0.9 per cent NaCl and 0.6 percent agar. Undiluted-bacteria (0.1 ml) were added to soft agar (2 ml) and poured onto MA volume 10 1) equipped with two teflon tubes for inlet and outlet of gas. The heavy vinyl chloride gas was pressed into the glass vessel via one of the tubes which ended near the bottom. This addi pension. In another `set of experiments (Table 2 and Figure I), the treatment was given with 20 percent gaseous vinyl chloride. The results were in good accordance with plates and 0.1 ml of appropriate dilutions tion caused an expulsion through the the previous ones, that is, a significant correspondingly onto CA plates. Dilu other tube (at the top) of a corresponding increase of mutants was obtained with tions were made in 0.9 percent NaCI. volume of air, which could be measured. liver microsomal systems. Without liver Colonies on MA plates were counted The atmospheric concentration of vinyl after two days' incubation at 37 C, giv chloride measured in this way was. ing the number of mutants. The number of viable cells (surviving cells) were counted on CA plates after one day's incubation. during the main experiments 20 percent. When alt vinyl chloride had been added, the tubes were dosed and a magnetic stirrer was started to ensure homoge Figur 1. TA 1535 (bssa substitution) traafad In -in atmaapbar* at air containing 20 peccant .(v/v) vinyl chlorida (VC). Experiment la Tibia 2 Male rats (Sprague-Dawley, Antici- neous distribution of the gas. Three mex) maintained on normal diet were starved 16--20 hours before they were sacrificed by decapitation and the liver was homogenized. The homogenate was centrifuged at 9000 Xg for ten minutes. The supernatant containing the microsomes was mixed with a NADPH-gen- different times of exposure to the gas were used, 30. 60 and 90 minutes. Two experiments with TA 1535 were made in this way. An experiment with all four strains TA 1535--TA 1538 was finally performed with a treatment time of 90 minutes. erating system consisting of NADP, glucose-6-phosphate, phosphate buffer (pH 7.4), MgCI, and KC1 in the pro RESULTS The results of the preliminary experi portions given by Ames tt al (14). When ments with TA 1535 and liver micro microsomal systems are mentioned in somes is presented in Table 1. As can be the text below or in the tables it includes seen, no mutagenic effect could be traced the 9000Xg supernatant containing the after the treatment with vinyl chloride microsomes and the NADPH-generating dissolved in water or in the microsomal I system. The negative control without suspension. After an exposure to 11 per NADP is therefore designated "micro cent vinyl chloride gas, on the other somal systems -NADP". 2-aminoanthra- hand, there was a significant increase of cene was used as a positive control. This mutants. It should be pointed out that compound induces mutations after mi the survival of the bacteria was not crosomal activation in all the strains affected in any detectable way by the used (14). treatment andtherefore the results cannot The vinyl chloride used (AB Aerosol be due to a selective survival. The fact Packing Co, Vallentuna, Sweden) has that only the treatment with vinyl chlo been analyzed with gas chromatography ride gas had any effect on the mutation and mass spectrometry for impurities rate indicates that a continuous treatment ---- VINYL CHLORIDE + MICROSOMES ---- CONTROLMICROSOMES by S Jensen (18). The purity was very high and only trace amounts of isopropa nol were found. When nothing else is stated the procedure of Ames tt al (14) was followed in the experiments. Three different types of treatment with vinyl chloride were used in preliminary Tabto 1. Tha *11act on TA ISIS (bain substitution) aftw Mmnl typis at tnatnwnt wttn vinyl cMtttda (VC) al tha pmm al llvnr microsomia. Sarlis 1--Z and W respectively nn per formed slmuttanaoua/y. Statistical analyse* with t-test. Slgnlifcsnca levels: ' * 041<p<jS; *-0.001<p<0.01; -- pCO.OOI. experiments with TA 1535. A water solution of vinyl chloride was prepared by bubbling vinyl chloride gas through water in a test tube for a couple of min utes. This solution was added to the soft agar together with the microsomal system and bacteria before plating. An Series Type of treatment Number of viable cells per plate Xl0`* Mean value of 2 plates Number of mutants per plate Mean value of 5 plates Number of mutants per 10 surviving cells SE attempt was also made to dissolve vinyl chloride directly in the microsomal sus 1 pension by bubbling it through for half VC dissolved in water 19.2 a minute prior to the addition to soft 2 Control 18.0 agar. In the' third procedure the bacteria were first plated together with the mi 3 VC dissolved in.microsoma) crosomal system and then treated 75 suspension 4.1 27.2 6.6 0.4 minutes in a vinyl chloride-containing atmosphere (11 percent v/v) at room tem 4 11% VC in atmosphere 4.5 78.2 17.6 2.6" perature (23 C). This last procedure was adopted in the following main experi 5 Control 4.3 23.0 5.3 0.2 AM8IO, 1074 195 R&S 109808 r i n. Table 2. TA ISIS (but substitution) IniM In i atmoaphara oI *<r containing 20 panant (v/v) gaaaoua vinyl dtlorida (VC). Tba vKact maaaurad aa number ol mutant* par 10* aurriving can*. Statutlcal analyse*. aaa Table 1. Type of treatment EXPERIMENT I Number of Number of viable cells mutants. per plate Mean value xio'* of 5 plates Mean value of 3 plates. * Number of mutants per 10'* surviving cells 5 E EXPERIMENT II Number of viable cells per plate X to** Mean value of 3 plates. Number of mutants. Mean value of 5 plates Number of mutants per 10 survhing cells S E Time of treatment (minutes) VC+ microsomal system VC Control + microsomal system VC-(-microsomal system VC Control + microsomal system VC+microsomal system VC Control -1- microsomal system 4.2 3.1 4.1 4.1 3.4 4.1 4.0 3.7 4.1 69.2 19.2 45.0 72.2 . 18.4 39.4 us 22.8 37.4 16.3 2.0 6.2 0.7 I0.9 l.l 17.$1,3*** 5.40,8 9.60.4 29.0t.l*** 7.1 1.0 9.0 1.0 3.8 2.0 3.7 3.9 2.3 3.9 4.2 3.6 3.9 49.8 10.6 25.0 51.6 18.8 20.4 77.2 22.6 I3.21.0** 5.40.9 6.70.9 13,4 1,0*** 8.1 0.6 5,20.7 18.40.9***` 6.2 0.9 -- 30 60 90 `The number or mutants from "Control+microsomal system" in this series had to be excluded because of infection. However, there is a coed agreement between the control scries within both experiments I and II, and therefore the comparison of the series with 90 minutes treatment has been made versus the pooled controls for 30 and 60 minutes of experiment II. microsomes, vinyl chloride- did not ex hibit any mutagenic effect. Concerning the survival of the cells, as in the pre vious experiment there was no sign of a toxic effect by vinyl chloride together with liver microsomal systems. It may be pointed out, however, that the data in dicate some toxic effect on the bacteria by vinyl chloride itself without liver mi crosomes. In order to elucidate the activation of vinyl chloride by the liver microsomes, a series without NADP was included in this experiment The results, presented separately in Table 3 together with the relevant data from Table 2, show that a mutagenic effect was only obtained with the liver microsomal system and NADP, but not without NADP. It should be mentioned that because of lack of space in the container used for the treatment, -the series measuring the survival of the bacteria had to be omitted for the treatment with the liver micro somal system without NADP. However, on the basis of the survival rate measured in the other experimental scries, it can be concluded that the number of mutants per plate should give a sufficiently accu rate estimation of the mutation rate. In particular, the similarity of mutation fre quencies between the series with liver microsomal systems without _NADP as compared to the series wjjtf'only vinyl chloride is indicative fupithe inter pretation of the mechanism of activa tion of vinyl chloride. As mentioned above, this lack of mutagenic effect without NADP indicates that in the test system used, an active metabolism by the microsomes is required for the muta genic effect. An experiment with all four strains TA 1535--TA 1538 was finally perform ed, in order to throw some light on the molecular mechanism behind the mu tagenic effect of vinyl chloride. The re sults are shown in Table 4. As in the previous experiments a mutagenic effect was obtained with TA 1535, responding to base pair substitutions, but no increase of mutations was caused in TA 1536-- TA 1538. responding to frame shift mutations. DISCUSSION From the experiments reported above, it can be concluded that vinyl chloride induces point mutations, but only after a metabolic activation. It can further more be established that vinyl chloride causes base pair substitution, but appar ently no insertion or deletion of base pairs in DNA. The latter kind of effect is hardly to be expected with a com pound like vinyl chloride. It is evident that vinyl chloride re sembles many other carcinogens in the respect that it acts as a mutagen via a metabolite, which seems to be formed in ^l^su the liver. It is reasonable R^Bsume that the carcinogenic action is also de pendent on the same metabolite. Alkenes. cycloalkenes as well as a variety of aromatic compounds are known to be metabolized, eg in the rat liver, via intermediate epoxides, formed in the NADPH-dependent oxygenation by microsomes (5, 19). Furthermore, tri chloroethylene is metabolized in the rat to trichloroacetic acid and triehloroethanol. which compounds are assumed to be formed via an intramolecular rearrange ment to trichloroacetaldchvde of a pri mary metabolite, trichloroethylene oxide (5). The most plausible primary meta bolite from vinyl chloride (1) in the present system hence would be chloroethylene oxide (II). This compound has been synthesized by several methods from ethylene oxide (30. 31) and is de scribed as a liquid, bp 65--67-' C (31>. which is rapidly hydrolysed by water and even at room temperature slowly re arranges to chloroacetaldchyde (III). Cl CH - CH, l Cl CH.-CHO 111 H>\Cr--;CH, Cr \0/ II 196 AMMO. VOL 3 NO. J Tu^ls 3, TA 1935 (bts substUutfon) trsstsd in *n ibnospher* of alf containing 20 poreant 1W) gM04M vinyl chforid* (VC). Number of mutant* por piste, main valua 'of 5 platas S E Type of treatment Time of treatment References and Notes 1, J 1. Creech and M N John^m. Journal of Occupational Median* 15. 150 (19741. 2. C Multoni and G t efcminc. Rcmhconti delta Clow* %h Scien:e ft\n he. inutemu- 30 minutes 60 minutes 90 minutes tithe e natural! Scr, H l.VL fas*;. 5 (1974). .1, Technology review, V*h Ssicntwt 52, 5JS Experiment 1 119741. 4, C Miller and i A Miller, in Chcnucul Mutagens, Principles and Methods for VC+microsomal system VC-microsomal system --NADP VC Control-i-microsomal system 69.2-8.6 20.6-3.5 19.2 2.1 45.0-4.7 Experiment II 72.2 = 5.3 18.6 = 0,7 18.4 = 2.6 39.4 = 1.7 115.8 -.4.2 30.8=1.3 22.8 = 3.1 37.4=4.3 their Detection. Vol 1. A Hollaendcr Ed, (Plenum Press. New York--`London, I97|| pp H.WII9. 5. O V Parke, The Biochemistry of Foreign Compounds (Pcrgamon Press. Oxford 1968). 5. H G Mandcl. in Fundamentals of Drug Metabolism and Drug' Disposition, B N* La Du. H G Mandcl. E L Way Eds. VC--microsomal system VC+ microsomal system --NADP VC Control + microsomal system 49.8-3.9 12.4=1.9 10.6=1.8 25.0=3.5 51.63.9 16.4=0.6 18.8=1.5 20.4 2.7 77.2=3.6 20.2=12 22.6=3.4 (Wavcrly Press. Inc. Baltimore, 1971) pp 149--186. 7. B B Brodie, J Axelrod. J R Cooper, L Gandett, B N La Du. C Miioma and 5 Udenfr.cnd. Science 121. 60.1 (1955). 5. O Hayaishi. Proceedings of the Fourth International Congress of Biochemistry - / V B, 33. p 31 (1964). 9, E Arrhenius, Xenohioticu 1, 487 (1971), . Chloroethylcne oxide can be expected to react as a bifunctional alkylating agent. Moreover, it belongs to the group mutagenic property of this compound would have been undetected if only wa ter solutions had been tested. 10. G C Mueller and J A Miller. Journal of Biological Chemistry ISO. 1125 (1949). 11. E Arrhenius, Chemico-Biological Inters actions l. 361 (1969 70). (2, H V Mailing, Mutation Research 13. 425 of highly reactive ^^chlorinated aliphatic ethers, among which, eg, chloromethyl methyl ether (CMME) and bistchloromethyl) ether are recognized as strong carcinogens (22). Mutagenicity tests of the metabolites discussed are under way. The fact that vinyl chloride needs a metabolic activation before it acquires a mutagenic property focuses attention on the necessity of using a test system which takes into consideration and as far a? possible mimics the metabolism per formed in the mammalian body. The liver microsomal system with Salmonella is a very useful tool in this respect. It should, however, be pointed out that the way of distributing the compound is essential. False negative results can easily be brought about by a low water solu bility and a high volatility as in the There is an increasing wealth of data which indicate that potential carcinogens can at least be pointed out by means of mutagenicity tests (4. 14) and the pre sent findings with vinyl chloride are in good accordance with such a view. The question posed above, whether the car cinogenic effect of vinyl chloride could have been indicated earlier by means of mutagenicity tests, can therefore be answered with **ycs*\ The actual se quence of events with this compound was, however, the reverse--the carcino genic effect was discovered before any mutagenicity tests had been performed. This is clearly unsatisfactory. If a rea listic testing procedure for chemicals with respect to mutagenic and carcinogenic, effects is to be built up, it seems that cooperation between governmental au thorities. scientists and industry is essen (19711. 13. M G Gabridge and M S Legator. Pro ceedings of the Sotiety for Experimental Biology and Medicine 130, S31 (1969). 14. B N Ames, W E Durston. E Yamasaki and F D" Lee. Proceedings of the Motional Academy of Sciences. USA 75. 22S1 (1973). 15. BN Ames. F D Lee and W Durston. Proceedings of the Motional Academy of Sciences, USA 75, 782 (1973). 16. Hi Vogel and D M Bonner. Journal of Biological Chemistry 215. 97 (1956), 17. B N Ames, in Chemical Mutagens Prin ciples and Methods for their Detection, Vol 2. A Hollaender. ed. (Plenum Press. New York--London. 1971) pp 267--282. IS. Personal communication. $ Jcnwn, Wal lenberg Laboratory. Environmental Toxi cology Unit. University of Stockholm. Sweden. 19. J W Daly. D M Jcrina and 6 Witkop. Expertentia 25. (129 (1972). 2(). Ch Walling and P S Frederick. Journal of American Chemical Society 54, 3326 (1962). present case with vinyl chloride. The tial. 21. H Gross and i Freiberg. Journal fdr Praktische Chemte 311. 506 (1969). Table 4, Strains TA 1535 (basa substitution) and TA 1535-1535 (frama shift) treated for 90 minutes bi an atmosphere of air containing 20 percent (v/v) gaseous vinyl chloride (VC), Statistical analyses, sea Table 1, 22. B K J Lcong, H N Macfarland and W H Reese, Archives of Environmental Health 22, 663 (1971). 23. This investigation has been supported by grants from the Swedish Board for Tech nical Development. The authors are grate ful to Dr B N Ames, University of Cali Type of treatment Strain Number of viable cells per plate XlO"* Number of mutants per plate Mean value Number of mutants per 10* surviving cells. = 5 E fornia. Berkeley, for supplying the S typhimunum strains and for valuable ad vice and to Drs E Arrhenius, $ Jensen and D Jcnvtcn for helpful discussions. 24. Received June 13. 1974. Mean value of 5 plates of 3 plates VC+microsomal system Control microsomal system TA 1535 TA 1536 TA 1537 TA 1538 TA 1535 TA 1536 TA 1537 TA 1538 4,1 1.2 1.7 3.7 4.3 1.1 1.9 4.0 41.2 0.8 13.6 20.4 15.2 0.8 14.4 22,2 10.1 0.4"* 0.7 ; 0.3 7.9l.l 5.5 = 0.6 3.5 j 0.7 0.8 0.4 7.7 J-0.9 5.60.l R&S 109870 AMBIO. 1974