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All lsent does not creating new Mutation Research, 242 (1990) 265-270 Elsevier MUTGEN 01602 265 Mutagenicity olNinyl chlpridein man: comparison of chromosome aberrations with micronucleus and sister-chromatid exchange frequencies Aleksandra Fuid, Djurdja Horvat and Boris Dimitrovid 1 Institute for Medical Research and Occupational Health Zagreb and 1 ` Vimlplasuka', Medical Department, Zadar (Yugoslavia) (Received 22 February 1990) (Revision received 1 June 1990) (Accepted 11 June 1990) Keywords: Vinyl chloride; 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 is unfortunately still a matter of extreme interest, carcinogenic and mutagenic substance (Anderson because there are a great number of factories with et al., 1980, 1981; Purchase et al., 1976, 1978; old technology that provide no real protection for Hansteen et al., 1978; Green and Hathway, 1978; employees. Maltoni and Lefemine, 1975; Ducatman et al., Vinyl chloride monomer is a substance that 1975; Funes-Cravioto et al,, 1975). During the requires activation by metabolic enzymes in the past 20 years, the accumulated knowledge about liver (Bartsch and Montesano, 1975). Its metabo hazards for people employed in the plastic in lites, chlorethyleneoxide and chloracetaldehyde, as dustry has made it necessary to develop a technol alkylating agents, react with amino acids and DNA ogy with closed systems which even use robots. In (Green and Hathway, 1978; Osterman-Golkar et the most developed countries these workers now al., 1977). r run much the same risk as the entire population of coming into contact with VCM through polluted In order to show and compare the conse quences of the action of VCM on lymphocyte air, food or water. The mutagenic activity of VCM chromosomes in workers exposed to VCM in our study we used three methods: the chromosome aberration assay, the micronucleus assay and the Correspondence: Dr. A. Fudid, Institute for Medical Research and Occupational Health, Mole Pijade 158, Zagreb (Yugo slavia). sister-chromatid exchange method. The influence of smoking on the mutagenic activity of VCM was also investigated. 0165-1218/90/J03.50 1990 Elsevier Science Publishers B.V. (Biomedical Division) UCC - 110887 s TABLE 1 COMPARISON OF FREQUENCIES OF CHROMOSOME ABERRATIONS, MICRONUCLEI AND SISTER-CHROMATID EXCHANGES BETWEEN EXPOSED AND CONTROL SUBJECTS Subject Employ ment (years) Ciga rettes/ day Micronuclei (%) Total 0 1 12 2 10 3 15 4 15 5 12 6 12 7 25 8 16 99 10 15 11 3 12 IS 13 25 14 15 IS 22 16 15 17 15 IS 22 19 14 20 0 30 0 20 0 0 20 20 20 0 20 15 0 0 0 30 0 15 Mean group values Mean group values for the 20 controls 2.1 3.6 3.7 4.9 6.4 9.4 10.3 10.5 9.9 11.2 11.5 12.3 14.6 15 16.2 16.3 20.9 23 26.9 12.1 6.5 6.6 2.3 97.9 96.4 96.3 95.1 93.6 90.6 89.7 89.5 89.1 88.6 88.7 87.7 84.4 85 83.8 83.7 79.1 77 73.1 87.8 6.5 98.4 2.8 1 2.1 3.6 2.8 4.9 5.6 7.6 10.3 10.5 9.17 9 9.6 5.4 14.6 14 16.2 13.6 16.8 12 20.2 9.45 5.2 5.8 1.7 2 0 0 0.9 0 0.7 1 0 0 0.8 2.2 0.9 2,7 0 1 0 2.7 1.5 8 6.7 1.5 2.1 0.7 0.9 34 00 00 00 00 00 0.7 0 00 00 00 00 00 4.1 0 00 00 00 00 1.1 1.5 30 00 0.4 0.07 1.06 0.3 0.05 0 0.2 Chromosome aberrations (%) SCE Total Chromatid Chromosome Dicentric Acentric Per breaks breaks chrom. frag. cell Range 4 5.5 9 8.5 11.2 6.2 10.8 10 10.5 8 4 8.5 9 5.5 13 12 8.5 9.5 10.5 2 2.5 6 5.5 5 2.8 8.1 5.5 6 6 1.5 7 6 2 3.5 8 6.5 7 8.5 1 1.5 1.5 1.5 2 2.8 1.2 2 2.5 0.5 1 1 1 1.5 3 2 1 1.5 . 1-5 0.5 0.5 9.1 5-17 0.5 1 9 4-14 0 1.5 11 5-23 0 1.5 7.6 4-15 1.4 2.8 11.1 5-23 0 0.6 8.5 5-13 0 1.5 6.4 4-11 0 2.5 8.1 4-14 0 2 12.3 4-26 0.5 1 13.1 4-24 0.5 1 8.6 4-11 0.5 0 11.5 5-27 0.5 1,5 10 4-20 0.5 1.5 6.3 4-10 1.5 5 6.3 4-13 0.5 1.5 8.9 4-16 0.5 0.5 10.7 4-14 0.5 0.5 8 4-15 0 0.5 9.3 4-14 8.5 5.2 2.6 2.1 1.5 0.6 0.4 0.4 1.4 1.1 9.2 4-27 1.9 1.1 0.7 0.2 0 0.15 5.9 0- 7 0.3 0.4 0.2 0.2 1.1 The frequencies of abnormal cells in the group exposed to VCM are significantly greater than in controls (P < 0.001). 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-stimulated blood lymphocytes. Into 0.5-mI 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 cytochalasin B at a final concentration of 3 /tg/ml was added after 44 h to the lymphocyte cultures according to the method of Fenech and Morley (1985). Micro nucleus slides were made by fixation in meth anol: acetic 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 micronuclei per cell. Bromodeoxyuridine in a concentration of 10 pg/ml 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 r-test (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 < 0.001). The mean group value for micronuclei is 12.2% with a range of 2.1-26.9%. With increasing numbers of micronuclei 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 2 3 4 5 6 7 8 9 10 20 30 20 20 20 20 20 15 30 15 Employment (years) 12 15 12 16 9 15 15 25 15 15 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 ucc - 110889 mosomes and acentric fragments are also present. The results of the micronucleus and chromosome aberration assays are comparable. Increasing numbers of micronuclei per binucleated cell and increased numbers of cells with more than one micronucleus are followed by a higher percentage of chromosome aberrations or more severe chro mosomal damages. Sister-chromatid exchange frequencies are also increased, with a mean group value of 9.2 per cell. The individual range of sister-chromatid exchange frequencies is 4-27 per cell. Table 2 shows the influence of smoking on the results of the sisterchromatid exchange assay compared with non smoking subjects. Discussion During the last few decades it has been shown that VCM, as a mutagenic and carcinogenic sub stance, causes liver damages, acro-osteolysis, brain, lung and skin cancer in workers chronically ex posed to this gas (Bartsch and Montesano, 1975; Smulevich et al,, 1988). There is a strong supposition that the modifica tion of the DNA structure by VCM and the observed mutagenic properties are due to a pro cess of depurination (Green and Hathway, 1978). The gap so produced might be filled by various bases resulting in `mispairing' during DNA repli cation and there is a high accordance between the severe damaging effect of DNA depurination and mutagenicity (Roberts, 1975; Loveless, 1966; Hathway, 1977). In our study wc examined 19 workers from a polyvinyl plant with an average duration of em ployment of 15 years. We used a battery of 3 tests (chromosome aberration, micronucleus and sisterchromatid exchange assay) to analyze the conse quences of the action of this dangerous gas on lymphocyte chromosomes. The subjects in our study showed an increased percentage of chromosome aberrations, micro nuclei and increased values of sister-chromatid exchanges. Compared with the control all results were statistically highly significant (P< 0.001). These results may be due to the relatively long period of employment in this plant and the con tinuous exposure since it is known that the forma tion of the 3 major urinary metabolites of VCM in the organism involves glutathione utilization and this process seriously depletes the glutathione pool, despite the compensating mechanism. It is pro posed that the fundamental role of glutathione in the body may be to protect the tissue against electrophilic attack by drug metabolites and other alkylating agents. In that way chronic exposure to a high concentration of VCM lowers the body's ability to protect itself against attack by reactive metabolites (Green and Hathway, 1977). The results of the micronucleus assay show that the mean group value is 12% with an individual range of 2.1-26.9%. The appearance of more than one micronucleus per binucleated cell is related with the results of chromosome aberrations. If the number of cells with more than one micronucleus increases, the percentage of chromosome aberra tions increases simultaneously or chromosome damages are more severe. Although the results of the micronucleus and chromosome aberration as says did correlate it was evident that the values of chromosome aberrations ranged between 3% and 13% while at the same time the values of the micronucleus assay ranged between 2.1% and 26.9%. From this observation we can suppose that micronuclei could be a result not only of lost genetic material but also of some yet unknown mechanism which affects other cell processes such as the spindle action. The distribution of types of chromosomal aber rations shows that 61% are chromatid breaks, which is characteristic for the action of most chemical agents. At the same time, chromosome breaks, acentric fragments and dicentric chro mosomes are also present in lower percentages. As the third method we used the sister-chro matid exchange assay. The mean group value of sister-chromatid exchange frequencies per cell was 9.2. The mean individual values ranged between 6.3 and 13.1 per cell. It is important to mention that apart from objective differences in exposure depending on the working microenvironment there exist various environmental, biological and physi ological influences which affect the frequencies of sister-chromatid exchanges in human peripheral lymphocytes (Tucker et al., 1988). On the other hand, the molecular mechanism of sister-chro matid exchange induction is not clear. Our current . 110890 J 269 of VCM in knowledge suggests that the mechanisms of forma working conditions of people employed in the ization and tion of sister-chromatid exchanges and aberrations plastic industry, since cytogenetic monitoring thione pool. are different (Wolf, 1982), and hence, sister-chro studies have shown that no biologically significant It is pro- matid exchange frequencies and chromosome increases in the frequencies of chromosomal aber itathione in aberrations cannot be correlated. The conclusion rations are found in workers in plants where mod sue against from our results is that sister-chromatid exchanges em technology is used compared with control s and other and chromosome aberrations cannot be used to populations. exposure to the body's predict one from the other, moreover such find ings are in agreement with some other authors References by reactive | (Galloway et al., 1986; Hansteen et al., 1978). ') I Comparing the results of sister-chromatid ex Anderson, D., C.R. Richardson, T.M. Weight, I.F.H. Purchase and W.G.F. Adams (1980) Chromosomal analysis in vinyl / show that I change frequencies and the micronucleus assay, it chloride exposed workers, results from analysis 18 and 42 individual | could be observed that values differ. This phe months after an initial sampling, Mutation Res., 79, 151-- more than [ nomenon has already been described by some 162. ! is related ' authors (Madle et al., 1986; Lin-Lee et al., 1984) Anderson, D., C.R. Richardson, I.F.H, Purchase, H.J. Evans ions. If the and it is explained by different mechanisms in the icronucleus , formation of genetic alterations. 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 me aberra- j The adverse health effects of smoking have tion Res., 82, 137-144. iromosome been documented in hundreds of studies during Antweiler, H. (1976) Studies on the metabolism of vinyl chlo s results of :rration asie values of en 3% and 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 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 I.J. Selikoff (1975) Vinyl chloride exposure and human chromosome aberration. Mu # the provides an inductive agent for both initiating and tation Res., 31, 163-168. and ippose that lly of lost ; unknown 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 Fenech, M., and A.A, Morley (198S) 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. Osterman-Golkar (1975) Chro mosome abenations in workers exposed to vinyl chloride, jesses such lymphocytes of smoking subjects compared with Lancet, i, 459. omal aber:id breaks, a of most 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. Galloway, S.M., P.K. Berry, W.W. Nichols. S. Wolman. 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. iromosome Our data confirm the findings of previous stud Green, T., and D.E. Hathway (1975) The biological fate in rats uric chroentages. sister-chrop value of ies that humans professionally exposed to high concentrations of VCM during several years of employment demonstrate increased values of chromosome aberrations, increased numbers of 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, Chem.-Biol. Interact., 22, 211-224. er cell was micronuclei and high frequencies of sister-chro Hansteen, I.L., L. Hillestad, L. Thiss-Evensen and S.S. Heldaas d between 0 mention 1 exposure ment there 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 (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. and physi- says documents that they are good indicators of IAEA (1986) Biological dosimetry. Chromosome aben-ation juenciesT of peripheral the other iister-chro- 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. analysis for dose assessment. Technical Reports Series No. 260, International Atomic Energy Agency, Vienna, pp. 59-63. Lin-Lee, V.W., J.A. Heddle, C.F. Arlett and B. Broughton (1984) Genetic effects of specific DNA lesions in mam >ur current It is our duty to use those data to improve the malian cells, Mutation Res., 127, 139-147. ucc 110891 270 Loveless, A. (1966) Genetic and Allied Effects of Alkylating Agents, Butterwonbs, London. Madle, E., A. Korte and B. Beech (1986) Species differences in mutagenicity testing. II. Sister-chromatid exchange and micronucleus induction in rats, mice and Chinese hamsters treated with cyclophosphamide. Mutagenesis, 1, 419-422. Malioni, C., and G. Lefemine (1975) Carcinogenicity bioassays of vinyl chloride: current results, Ann. N.Y. Acad. Sci., 246, 195-218. Osterman-Golkar, S,, D. Hultmark, D. Segerbach, C.J. Colleman, R. GSthe, L. Ehrenberg and C.A. Wachtmeister (1977) Alkylation of DNA and proteins in mice exposed to vinyl chloride, Biochem. Biophys. Res. Commun., 76, 259-266. Pavli,1. (1970) StatistiCka teorija i primjena, Tehnidka Knjiga, Zagreb, pp. 233-237. Purchase, I.F.H., C. Richardson and D. Anderson (1976) Chro mosomal effects in peripheral lymphocytes, Proc. R. Soc. Med., 69, 290-291. Purchase, I.F.H., C.R. Richardson, D. Anderson, G.M. Paddle and W.G.F. Adams (1978) Chromosomal analysis in vinyl chloride exposed workers, Mutation Res., 57, 325-334. Roberts, JJ. (1975) Nucleic acid modifications and cancer, in: E.J. Ambrose and FJ.C. Roe (Eds.), Biology of Cancer, Halstead, Chichester. Smulevich, V.B., I.V. Fedotova and V.S. Filatova (1988) In creasing evidence of the rise of cancer in workers exposed to vinyl chloride, Br. J. Ind. Med., 45, 93-97. Tucker, J.D., L.K. Ashworth, G.R. Johnston, N.A. Allen and A.V. Carrano (1988) Variation in the human lymphocyte sister-chromatid exchange frequency: results of a long-term longitudinal study, Mutation Res., 204, 435-444. Wolff, S. (1982) Chromosome aberrations, sister-chromatid exchanges and the lesions that produce them, in: S. Wolf (Ed.), Sister Chromatid Exchange, Wiley, New York, pp. 41-57. UCC - 110892