Document bB4D18b6gdJaqQDwjpM8KgoR6

R&S 000635 The fViutagsnicity of Vinyl ChSorid:i ,0 After EVletaboSsc Activation (Ssi; By U Rannug, A Johansson, C Ramcl and C A Wachlmeister, Environmental Toxicology Unit. Wallenberg Laboratory, University of Stockholm, S-1Q4 05 Stockholm, Sweden AMBIO Vol. 3, No. 5, pp 194-197 (1974). Vinyl chloride has recently been shown to cause a malignant liver tumor disease In man after occupational exposure in PVC Dlants. This actualizes the problem of whether such hazards could be avoided or at least diminished in the future by a screening for mutagenicity ol 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 se but becomes mutagenic alter a metabolic activation in the liver. Vinyl chloride has attracted a consider able amount of public attention because of the recent discovery of its carcinogenic action in man. In several countries cases of the rare malignant disease angiosar coma of the liver have been reported among workers who have been exposed to vinyl chloride in plastic industries (1). Malloni has turlhermorc reported an increase of .mgiusareuma m rats atter exposure to 250 ppm vinyl chloride (2). Later results indicate carcinogenic etfects at still lower doses (3). The hazard of vinyl chloride primarily concerns workers in the plastic industries, who get a parti cularly high exposure in different con nections, but this occupational health problem may have ramifications for the P general public. Vinyl chloride has, for instance, been used as a propellant for hair sprays and pesticides. Moreover, the polymerized product of vinyl chloride. PVC plastic, is used all over the world for a variety of products, among other things for the wrapping of various food stuffs. It seems inevitable that the pop ulation is exposed to the monomer via such sources, although the doses arc most likely small. The carcinogenic effects of vinyl chlo 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 ntimhcr of chemicals used in industries, it is hardly realislic 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 *t present seems to be a system which makes use of the close correlation be tween the carcinogenic and mutagenic properties of chemicals (4). This corre lation, as do other data, points to the fact that changes in DNA are at least an essential cause of cancer induction. The testing for mutagenicity is much cheaper and far less time consuming, and further more, the results can in many cases he interpreted terms wit!, spect to the changes occurring in DNA. This obviously is of importance tor a better understanding of the biological action of suspect compounds. One major obstacle in using muta genicity tests with microorganisms for a preliminary screening of carcinogenic effects is the fact that many compounds behave as carcinogens only after an ac tivation in the mammalian body. This activation, or in other cases deactivation, of a foreign compound, is principally performed by the liver and involves oxi dation, reduction, hydrolysis and con jugation. A wide variety of oxidative re actions eg C- and N*oxygenation, O-, N-, and S-dca!kvlation and epoxidation arc mediated by the drug metabolizing system located in the membranes of the cndoplasmatic reticulum of the liver cells (5, 6, 7, 8). This mixed-function oxygenase mechanism can utilize reduced nicotinamide adenine dinucleotide phos phate (NADPH) as an electron donor (9). These reactions also take place in vitro after the addition ol NADPH-gcn- Crating system to microsomal fractions (10. II, 12). Although microorganisms arc 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 direct carcinogenic compounds. In order to overcome that problem, combined lest methods have been worked out with microorganisms and mammals. The mi croorganisms arc used for the actual mutagenicity testing, but they are ex posed to those metabolites of the com pound which arc formed in an intact mammal (host mediated assayi (13) or in mammalian liver microsomal systems in vitro (12, 14). The present investigation of the gen etic effect of vinyl chloride has been performed on Salmonella typhimurium. The mutagenicity of the compound was analyzed by means of reverse mutations in the histidine locus. The effect of metabolic activation was studied by add ing rat liver microsomal system; to the bacterial cultures according r-- iv.~ moth -----t. v, liku- Jwuv vjf rtiliLh I if/, The objective of the present investiga tion has primarily been to use vinyl chloride as an example of a compound carcinogenic to man, in order to study the applicability of mutagenicity testing for the detection of carcinogens in the human environment. We are likely to be confronted with this kind of problem at an accelerated rale in the future and the necessity of finding a sensible solu tion to the screening of carcinogenic hazards of chemicals is imminent. Con sidering the vinyl chloride problem, one could raise the following question; Had this genetic test method been available and been used for screening of vinyl chloride when it was introduced for in dustrial production, would such a screen ing have given a warning of the carcino genicity of this compound? MATERIALS AND METHODS Four histidine-requiring strains of Salmo nella typlumurium (TA 1535, TA 1536, TA 1537. and TA 1538) were used for this investigation. These strains have been described in detail by Ames et a! (15). They arc used in a back-muiation 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 153S). Cultures were grown overnight in corn- 194 iumn vni x mo s R&S 000636 ^plete medium (Difco, Antibiotic medium 3). Platings with the soft agar technique were made on complete medium (CA plates) and on minimal medium (MA plates) described by Vogel and Bonner (16) with supplements according to Ames (17). The soft agar consisted of 0.9 per cent NaCI and 0.6 percent agar. Undiluted bacteria (0.1 ml) were added to soft agar (2 ml) and poured onto MA plates and 0.1 ml of appropriate dilutions correspondingly onto CA plates. Dilu tions were made in 0.9 percent NaCI, Colonics on MA plates were counted after two days' incubation at 37; C, giv ing the number of mutants. The number of viable cells (surviving1 cells) were counted on CA plates after one day's incubation. Male rats (Sprague-Dawley, Anticimex) 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-gencrat'mg system consisting of NADP. glucosc-6-phosphatc, phosphate buffer (pH 7.4), MgCU and KCI in the pro portions given by Ames ct at (14). When microsomal systems arc mentioned in the text below or in the tables it includes the 9000Xg supernatant containing the microsomes and the NADPH-generating system. The negative control without NADP is therefore designated "mtcro- SOmM -- WArtP" P.aminnanrhn- cenc was Used as a positive control. This compound induces mutations after mi crosomal activation in all the strains used (14). The vinyl chloride used (AB Aerosol Packing Co, Vallcntuna. Sweden) has been analyzed with gas chromatography and mass spectrometry for impurities by S Jensen (18). The purity was very high and only trace amounts of isopropa~noI were found. When nothing else is stated the procedure of Ames / at (14) was followed in the experiments. Three different types of treatment with vinyl chloride were used in preliminary 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 attempt was also made to dissolve vinyl chloride directly in the microsomal sus.pension by bubbling it through for half a minute prior to the addition to soft agar. In the third procedure the bacteria were first plated together with the mi crosomal system and then treated 75 minutes in a vinyl chloride-containing atmosphere (11 percent v/v) at room tem perature (23 C). This last procedure was adopted in the following main experi ments and will therefore be described in some detail. After plating of the bacterta' with and whithout microsomal systems the petn dishes were placed in a elosed vessel (standard vacuum desiccator, volume 1U I) 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 tion caused an expulsion through the other tube (at the top) of a corresponding volume of air, which could be measured. The atmospheric concentration ot vinyl chloride measured in this way was during the main experiments 20 percent. When all vinyl chloride had been added, the tubes were closed and a magnetic stirrer was started to ensure homoge neous distribution of the gas. Three 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 ali four strains TA 1535--TA 1538 was finally performed with a treatment time of 90 minutes. given directly to the bacteria with gase ous vinyl chloride brings about a higher exposure dose than a treatment with vinvl chloride previously dissolved in water or in the liver microsomal sus pension. In another set of experiments (Table 2 and Figure 1). the treatment was given with 20 percent gaseous vinyl chloride. The results were in good accordance with the previous ones, that ts, a signdicant increase of mutants was obtained with liver microsomal systems. Without liver Figure l. TA 1535 (base substitution) treated In an atmosphere of air containing 20 percent (v/v) vinyl chloride (VC). Experiment \ In Tablo 2e RESULTS The results of the preliminary experi ments with TA 1535 and liver micro somes is presented in Table 1. As can be seen, no mutagenic effect could be traced alter the treatment with vinyl chloride dissolved in water or in the microsomal suspension. After an exposure to 11 per cent vinyl chloride gas, on the other hand, tiicre was a significant inctease of mutants. It should be pointed out that the survival of the bacteria was not affected in any detectable way by the treatment and therefore the results cannot be due to a selective survival. The fact that only the treatment with vinyl chlo ride gas had any effect on the mutation rate indicates that a continuous treatment - - VINYL CHLORIDE + MICROSOMES -- CONTROL* MICROSOMES Toble- lj -THe-eflect cn-TA ttt$.(base *ubslltutlon) after different types of treslment with vinyl chloride (VC) at the presence ol Uver rtucrosomes. Series and 3--5 respectively ware per* formed simultaneously. Statistical analyses with t-test. Significance levels; * 0.01 <p<0.05; 0.001<p<0,C1; ** p<0.001. Series Type of treatment Number of viable cells per plate xio-* Mean value of 2 plates Number of mutants per plate Mean value or 5 plates Number of mutants per 10 surviving cells S E 1 VC dissolved in water 2 Control 3 VC dissolved in microsomal suspension 4,1 4 11% VC in atmosphere 4.5 5 Control 4.3 19.2 18.0 27.2 6.6 0.4 78.2 17.62.6** 23.0 5.30.2 ,9$ Table 2. TX 1535 (base subslHutlon) (rested In sn atmosphere ot air containing 20 percent (v/e) psscojs vinyl chloride (VC). The eltect measured as number ot mutants per 10* surviving cells. Statistical analyses, see Table 1. EXPERIMENTl EXPERIMENT 11 Type of treatment Number of Number of viable cells mutants. per plate Mean value X 10*' of 5 plates Mean value of 3 plates. Number of mutant* per 10 " surviving ceils^S E Number of viable cells per plate X 10'* Mean value of 3 plates. Number of mutants. Mean >aluc of 5 plates Number of mutants per 10' sunmng cells S E Time of treatment (minutes) VC-Fmicrosomal system VC Control + microsomal system VC-f- microsomal system VC Control + microsomal system VC+microsomal system VC Control-)-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 1 W.8 22.8 37.4 16.32.0 6.20.7 10.91.1 17.81.3*** 5.40.8 9.60.4 29,0l.t*** 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 13.21.0** 30 10.6 5.40.9 25.0 6.70.9 51.6 I3.4 1.0*** 60 18.8 8.1 0.6 20.4 5.2 0.7 77.2 18.40.9***` 90 22.6 6.20.9 `The number of mutants from "Control + microsomal system" in this scries had to be excluded because of infection. However, there is a good agreement between the control scries within both experiments 1 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. V I R&S 000637 microsomes, vinyl chloride did not ex*uMifu*. tmy muc.-a.i^ciii_c c.ntc. u. Z^u'.i_i_L__u__ i` iiii^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"TabIe 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 scries measuring the survival of the bacteria had to be omitted for the treatment with the liver microsomal system without NADP. However, on the basis of the survival rate measured in the other experimental series, 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 with only vinyl chloride is indicative for the inter pretation of the mechanism of activauuil of vinyl chloride. As mentioned above, this lack ol mutagenic ettect 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 153S 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 vi.ivl chloride re sembles many other carcinogens in the respect that it acts as a mutagen via a metabolite, which seems to be formed in the liver. It is reasonable to assume ilisit ilic CtilCuiOgiuC dCtiGtt is wC' pendent on the same metabolite. Alkcnes, cycloalkenes as well as a variety of aromatic compounds arc known to be metabolized, eg in the rat liver, via intermediate epoxides, formed in the NADPH-dcpcndent oxygenation by microsomes (5, 19), Furthermore, tri chloroethylene is metabolized in the rat to trichloroacetic acid and trichloroctha- nol, which compounds are assumed to be formed via an intramolecular rearrange ment to trichloroacetaldehyde of a pri mary metabolite, trichloroethylene oxide (5). The most plausible primary meta bolite from vinyl chloride (l) in the present system hence would be chloro- ethylene oxide (ID. This compound has been synthesized by several methods from ethylene oxide (20. 21) and is de scribed as a liquid, bp 65--671 C (21), which is rapidly hydrolysed by water and even at room temperature slowly re arranges to chloroacetaldehyde (III). Cl CH - CH, H\ i CICH.-CHO Ill n .... . . ....... ` 196 \ v Tbl* 3. TA 1535 (ba lubifltuflon) freafed In an ofmosaftere ot sir containing 20 percent (*/v) 5seoui vin/J chloride (VC). Number cf mutants per plate, mean value ol 5 plates i S E Type of treatment Experiment I Time of treatment 30 minutes 60 minutes 90 minutes VC+ microsomal system VCq-microsomal system -- NADP VC Controlt microsomal system 69.2 = 8.6 20.6-3.5 19.2 = 2.1 45.0=4.7 Experiment II 72.2 = 5.3 18.60.7 18.4-2.6 39.4 1.7 115.8 = 4.2 30.8=1.3 22.8 = 3.1 37.4 = 4.3 VC+microsomai system VC-Fmicrosomal system -- NADP VC Control-)-microsomal system 49.8-3.9 12.4-1.9 10.6=1.8 25.0-3.5 51.6=3,9 16.4 j-0.6 18.8-1.5 20.4 2.7 77.23.6 20.2 = 2.2 22.6 3.4 - Cbloroethylcnc oxide can be expected to react as a ^functional alkylating agent. Moreover, it belongs to the group of highly reactive n-chlorinatcd aliphatic ethers, among which. r,e, chloromcthyl methyl ether (CMME) and bislchloromethyl) 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 lakes into consideration ana as tar as 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 ^present case with vinyl chloride. The mutagenic property of this compound would have been undetected if only wa ter solutions had been tested. 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 arc in good accordance with such a view. The question pc,.ed above, whether the car cinogenic effect of vinyl chloride could have been indicated earlier by means of mutagenicity tests, can therefore be answered with "yes". The actual se quence ot events with this comnound was. however, the reverse--the carcino genic ctfect 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 tial. Table 4. Strains TA 1535 (base substitution) and Ta 1536-1533 (frame shift) treated for 90 minutes In an atmosphere ot air containing 20 percent (v/v) gaseous vinyl chloride (VC). Statistical analyses, see Table 1. Type of treatment Strain '* Number of viable cells per plate xio'* Mean value of 3 plates Number of mutants per plate Mean value of 5 plates Number of mutants per 10' surviving cells.=S E References and Notes 1 J L Creech .mJ M N Johnson, Journal of Occupational Wlutu'iitr 16. |?0 2. C Mjliorn and G L*. C-urne. Ucudiconn del'icJ Civile di t` I'iuln, 'inift'iim- iicce e 1121!hizj11 Scf. X I.VI, f,iNj. 1 tN,4) 3. Technology rcxicw. Ac'w Scientist 62. 548 0974). 4. E. C Miller and J A Miller, in Chemical ,Mutagens. Principles ami Medunls for their Detection. Vol 1. A HolhcnJtr hd. (Plenum Press, New York--London, 1771) pp S3--U9, 5. D V Parke, The Biuchcmutrv of Fmei^n Compounds (Pcrgamon Press. Oxford 1968). 6. H G Mandcl. in Fundamental! of Dm* Metabolism and Drug Dnpuutum. B N La Du. H G Monde!, L L \k.iv Lds, (Waserty Press. Inc. Baltimore, 1971) pp 149--IriG- 7. B D Brodic. J Axelrod. J R Cooper. L Gandeu, B N La Du. C Mitona and S Udcnfricnd. Science 121. 6<H (19?^). 8. 0 Hayaishi, Pioceedm%s of we Fourth International Congress of Biochemistry / U B. 33. p 31 (1964). 9. E Arrhenius, Xenobtottcfl 1. 4N7 l!9'|), 10. G C Mueller and J A Miller. Journal of Biological Chemists ISO. 1125 MQ40), 11. E Arrhentus. Chemico-Bmhrcual Inter* actions 1. 361 (1969 70). 12. H V Mailing:. Mutation Hcsearch 13. 425 0971), 13. M G Gabndge and M S Locator, Pro~ ceed.nzs of (he Societv lot F.spetmu nta! Btoio^y and Medicine 130. S3l 09n9i 14. B N Ames, W E DurniOn, E \amaaki and F D Lee. Ptoceetltncs of the National Academy of Sciences, USA 70. 2281 (W"3 15. B N Ames. F D Lee and W E Dur*>ton, Pro<, Ot the National Academy of Sciences. USA 70, 7$2 (1973). 16. H J Vogel and D M Bonner. Journal of Biolocnal Cnemtstiv 2IS, 97 i|V*6l 17 o v *;*, :n Vol 2, A llollaendcr. ed. (Plenum Press. New York--London. 1971) pp 2t>7---282, IS. Personal communication, S Jcnvcn. Wal lenberg Laboratory, Environmental Toxi cology Unit. University of Stockholm. Sweden. 19 J W Daly, D M lerina and B Wukop. E.xpcncntia 28, 1129 (1972). 20. Ch Walling and P S Frederick. Journal of Amertcan Chemical Soaetv 84. 3326 (1962). 21. H Gross and J Freiberg, Journal fur Praktischc Chcnue 311, 506 (1969). 22. B K J Leone, H N Macfartond and W H ^Recse, 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 fornia. Berkeley, for supplying the S tvphtmurium strains and for valuahle ad vice and to Drs E Arrhenius, S Jensen and D Jensscn for helpful discussions. 24. Received June 13, 1974. VC+ microsomal system Control + microsomal system '. TA 1535 TA 1536 TA 1*47 TA l.-3 TA 1535 TA 1536 TA 1537 TA I53S 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 7,9 l.l 5.50.6 3.50,7 0.8 0.4 7,7=0.9 5.6O.I R&S 000638 AMBIO. 1974 197