Document MJb5xwv7yJKJqMwZXgQjybQeL

4AJ4Y/ S M VV// 0tS Mutation Research 242 (1990) 265-270 Elsevier \) c rsis ry)0. r^6T^/C I T\) MUTGEN 01602 / QM iZ.c> V /^03/S..;9 77^ /V ft ^ Mutagenicity of vinyl chloride in man: comparison of chromosome aberrations with micronucleus and sister-chromatid exchange frequencies ^ Aleksandra Fucid, Djurdja Horvat and Boris Dimitrovic 1 Institutefor Medical Research and Occupational Health, Zagreb and 1 ` Vmilplastika\ Medical Department, Zadar (Yugoslavia) (Received 22 February 195(0) (Revision received 1 June 1990) (Accepted 11 June 1990) Keywords: Vinyl chioride; Micronucleus assay; Chromosome aberration'assay; Sister-chromatid exchanges; Smokcrs'/non-Smokers' human lymphocytes Summary The mutagenic effects of vinyl chloride monomer in man were studied in the lymphocyte culture with 3 methods: the chromosome aberration' assay, the micronucleus assay and the sister-chromatid exchange method. Compared with control, values obtained by these tests are increased in workers occupationally exposed to vinyl chloride. In relation to non-smokers, smokers exposed to vinyl chloride show significant increases in sister-chromatid exchange frequencies. The problem of correlating the results of the chro mosome aberration assay with micronucleus and sister-chromatid exchange frequencies is discussed. Vinyl chloride monomer (VCM) is a well-known carcinogenic and mutagenic substance (Anderson et al., 1980, 1981; Purchase et al., 1976, 1978; Hansteen et al., 1978; Green and Hathway, 1978; Maltoni and Lefemine, 1975; Ducatman et al., 1975; Funes-Cravioto et aL, 1975). During the past 20 years, the accumulated knowledge about hazards for people employed in the plastic in dustry has made it necessary to develop a technol ogy with closed systems which even use robots. In the most devdoped countries these workers now run much the same risk as the entire population of coming into contact with VCM through polluted air, food or water. The mutagenic activity of VCM Correspondence: Dr. A. Fu5<, Institute for Medical Research and Occupational Health, MoSe Fijade 158, Zagreb (Yugo slavia). is unfortunately still a matter of extreme interest, because there are a great number of factories with old technology that provide no real protection for employees. Vinyl chloride monomer is a substance that requires activation by metabolic enzymes in the liver (Bartsch and Montesano, 1975). Its metabo lites, chlorethyleneoxide and chloracetaldehyde, as alkylating agents, react with amino adds and DNA (Green and Hathway, 1978; Osterman-Golkar et al., 1977). In order to show and compare the conse quences of the action of VCM on lymphocyte chromosomes in workers exposed to VCM in our study we used three methods: the chromosome aberration assay, the micronucleus assay and the sister-chromatid exchange method. The influence of smoking on the mutagenic activity of VCM was also investigated. 0165-1218/90/503.50 1990 Elsevier Science Publishers B.V, (Biomedical Division) ii >i i i F i 1 | i i NGC36368 Material and methods Nineteen workers from the plastic industry were chosen for cytogenetic examination. As a control group we chose 20 male subjects from the general population, 40-50 years old. The examined workers had been employed in the polyvinyl chlo ride plant for 15 years on average. Those with recent X-ray exposure and drug treatment were excluded from the study. Exact measurements of exposure to VCM were performed continuously. The VCM concentration in the working environ ment was 50 ppm. Due to the technological pro cess the concentration, could periodically reach 2000 ppm for a short period of time. The chromosome aberration assay was carried out on cultures of phytohemagglutinin-sdmulated blood lymphocytes. Into 0.5-ml samples of whole blood 8 ml of F-10 medium (Gibco) containing 20% of calf serum was added. Lymphocytes were incubated at 37 C for 48 h. After 45 h colchicine was added. Fixation of the cultures and prepara tion of slides were carried out according to con ventional methods .(IAEA, 1986). Two hundred well-spread and complete metaphases were analyzed for every person and results are pre sented as percentages. For micronucleus preparations cytoctalasin B at a final concentration of 3 p.g/ml was added after 44 h to the lymphocyte cultures according to the method of Fenech and Morley (1985). Micronucleus slides were made by fixation in methanokacetic acid (3:1) without hypotonic treat ment. In the micronucleus test 1000 binucleated cells per person were analyzed. Results are pre sented as the distribution of the percentage of cells with 1, 2, 3 and more micronudei per cell. Bromodeoxyuridine in a concentration of 10 /ig/roJ was added to the culture medium for sis ter-chromatid exchange preparations. The cultures were harvested at 72 h. The sister-chromatid ex changes were compared with the percentage of chromosomal breakage and the micronucleus frequency from the same blood sample. Smoking habits of the individuals were examined. The results are statistically compared by Stu dent's Mest (Pavlid, 1970). 267 Results We examined 19 workers from the plastic in dustry and 20 control individuals from the general population. The individual and mean group re sults are presented in Tables 1 and 2. The values of chromosome aberrations, micronuclei and sis ter-chromatid exchange frequencies in workers ex posed to VCM show statistically significant in creases compared with the control group (P < 0.002). The mean group value for micronuclei is 12.2% with a range of 2.1-26.9%. With increasing numbers of micronucled per binucleated cell the number of cells with more than one micronucleus increases. The mean group value for chromosome aberra tions is 8.5%. Chromatid breaks are the predomi nant type of aberration and they represent 61.2% of all breaks. Chromosome breaks, dicentric chro- TABLE 2 VINYL' CHLORIDE EXPOSURE AND SISTER-CHRO MATID EXCHANGE FREQUENCIES IN SMOKING AND NON-SMOKING SUBJECTS Subject Cigarettes/ day Smokers 1 20 2 30 . 3 20 4 * 20 5 20 6 20 7 20 8 15 9 30 10 15 Employment (years) 12 15 12 16 9 15 15 25 15 IS SCE/cell 9.1 11 11.1 8.1 12.3 13.1 11.5 10 10.7 9.3 Range 5-17 5-23 5-23 4-14 4-26 4-24 5-27 4-20 4-14 4-14 Mean for smokers 10.61.4 4-27 Non-smokers 10 20 30 40 50 60 70 80 90 10 1.5 12 25 3 15 22 15 22 9 4-14 7.6 4-15 8.5 5-13 6.4 4-11 8.6 4-14 6.3 4-10 6.3 4-13 8.9 4-16 8 4-15 Mean for non-smokers 7.8 1.07 4-16 I NGC36369 knowledge suggests that the mechanisms of forma* tion of sister-chromatid exchanges and aberrations are different (Wolf, 1982), and hence, sister-chro matid exchange frequencies and chromosome aberrations cannot be correlated. The conclusion from our results is that sister-chromatid exchanges and chromosome aberrations cannot be used to predict one from the other, moreover such find ings are in agreement with some other authors (Galloway et aL, 1986; Hansteen et al., 1978). Comparing the results of sister-chromatid ex change frequencies and the micronucleus assay, it could be observed that values differ. This phe nomenon has already been described by some authors (Madle et al., 1986; Lin-Lee ct aL, 1984) and it is explained by different mechanisms in the formation of genetic alterations.' The adverse health effects of smoking have been documented in hundreds of studies during the last decades. The exact mechanism by which cigarette smoking leads to the formation of cancer is still unknown. The variety of biologically active compounds inhaled during smoking obviously provides an inductive agent for both initiating and promoting steps of malignant growth. It is also' interesting that VCM is present in the cigarette smoke (Antweiler, 1976). The present data show increased sister-chromatid exchange frequencies in lymphocytes of smoking subjects compared with non-smokers. The mean group and individual val ues of sister-chromatid exchanges are lower in non-smokers than in smokers. Duration of em ployment has no influence on the results. Our data confirm the findings of previous stud ies that humans professionally exposed to high concentrations of VCM during several years of employment demonstrate increased values, of chromosome aberrations, increased numbers of micronuclei and high frequencies of sister-chro matid exchanges. Sister-chromatid exchanges per cell are even more elevated in subjects who smoke. The positive correlation between the results of the micronucleus and the chromosome aberration as says documents that they are good indicators of exposure to chemical agents and that the results of the micronucleus assay, which is a cheaper and simpler method, can be used for screening pur poses. It is our duty to use those data to improve the 269 working conditions of people employed in the plastic industry, since cytogenetic monitoring studies have shown that no biologically significant increases in the frequencies of chromosomal aber rations are found in workers in plants where mod ern technology is used compared with control populations. References Anderson, D., C.R. Richardson, T.M. Weight, I.F.H. Purchase and W.G.F. Adams (1980) Chromosomal analysis in vinyl chloride exposed workers, results from analysis 18 and 42 months after an initial sampling, Mutation Res., 79, 151162. Anderson, D., C.R. Richardson, I.F.H. Purchase, HJ. Evans and M.L O'Riordan (1981) Chromosomal analysis in vinyl chloride exposed workers: comparison of the standard tech nique with the sister-chromatid exchange technique, Muta tion Res., 82,137-144. Antweiler, H. (1976) Studies on the metabolism of vinyl chlo ride, Environ. Health Perspect., 17, 217. Bartsch, H., and R_ Montesano (1975) Mutagenic and carcino genic effects of vinyL chloride. Mutation Res., 32, 93--114. Ducatman, A., K. Hirschom and IJ. Sdikoff (1975) Vinyl chloride exposure and human chromosome aberration. Mu tation Res., 31, 163-168. Fenech, Mn and AA. Morley. (1985). Solution to the kinetic problem in micronucleus assay, Cytobios, 43, 233-246. Funes-Cravioto, F., B. Lambert, J. Lindsten, L. Ehrenberg, A.T. Natarajan and S. Ostennan-Golkar (1975) Chro mosome aberrations in workers exposed to vinyl chloride. Lancet, i, 459. Galloway, S.M., P.K. Berry, W.W. Nichols, S. WoUnan, K.A. Soper, P.D. Stolley and P. Archer (1986) Chromosome aberrations in individuals occupationally exposed to ethyl ene oxide and in a large control population. Mutation Res., 170, 55-79. Green, T., and D.E Hathway (1975) The biological fate in rats of vinyl chloride in relation to its oncogenicity, Chem.-Biol. Interact., 11, 545-562. Green, T., and D.E Hathway (1978) Interactions of vinyl chloride with rat liver DNA in vivo, Cbem.-BioL Interact., 22, 211-224. Hansteen, I.L., L. Hillestad, L. Thlss-Evensen and S.S. Heldaas (1978) Effects of vinylchloride in man, a cytogenetic follow up study, Mutation Res^ 51, 271-278. Hathway. D.E. (1977) Comparative mammalian metabolism of vinyl chloride and vinylidene chloride in relation to onco genic potential. Environ. Health Perspect., 21, 55-59. IAEA (1986) Biological dosimetry. Chromosome aberration analysis for dose assessment, Technical Reports Series No. 260, International Atomic Energy Agency, Vienna, pp. 59-63. Lin-Lee, V.W., JA. Heddle, GF. Arlett and B. Broughton (1984) Genetic effects of specific DNA lesions in mam malian cells, Mutation Res., 127,139-147. [ i i IMGC36370