Document jgaBrLgZwNVGv7EkKJ1nM7Rvk

December 1990 Appearing monllih Vol. 242. No. 4 Completing Vol. 242 MUHCAV ZA2 (4) K-171I-(100) |l990] ----------- MUTATION RESEARCH International journal on mutagenesis, chromosome breakage and related subjects Editor-in-Chief: F.H. Sobels. Leiden Board of Managing Editors J. Ashby. Macclesfield: C. Hranecsehi. Modena (DNAging); S.M. Galloway. It'cat Point. PA (Mutation Research Letters); J.M. Gentile. Holland. Ml (Mutation Research Letters); 13.W, Glieknian. Toronto, Out.; P.C. llanawalt, Stanford. CA (DNA Repair); P.II.M. Lohman. Leiden (DNA Repair); K. Sankaranarayanan. Leiden; P.J. dc Serres. Research Triangle Park. NC: R.B. Sctlow. Upton, A'Y (DNAging); M.D. Shelby, Research Triangle Park. NO, T. Sugimura, Tokyo (DNAging); H. Takebe. Kyoto (DNA Repair); J. Vijg, Rijswijk (DNAging); fi. Vogel, Leiden: J.S. Wassoni, Oak Ridge, TN t i Genetic Toxicology Testing and Biomonitoring of Environmental or Occupational Exposure R & S 150884 A. form or by any blither. Elsevier oduett liability, rn the matenaI on of diagnoses .aiion docs not >ufaciurcr, ;opyright o! the 'iillcci any sums 2 of the Dutch opyright Act of Inc. Concern U ts given on the tele for copying th a copy of the code appears in the author. All t does not ating new Mutation Research, 242 (1990) 265-270 Elsevier MUTGEN 01602 /<" /?//- (/trv) 265 Mutagenicity of vinyl chloride in man: comparison of chromosome aberrations with micronucleus and sister-chromatid exchange frequencies Alcksandra Fui, Djurdja Horvat and Boris Dimitrovic 1 institutefor Medical Research and Occupational Health, Zagreb and 1 `lUntlpUtsttka*, Medical Department. Zadar {Yugoslavia) (Received 22 February 1990) (Revision received 1 June 1990) (Accepted 11 June 1990) Keywords: Vinyl chloride; Micronucicus 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 arc 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 el a!., 1976, 1978; Hanstccn et al., 1978; Green and Hathway, 1978; Maltoni and Lefemine, 1975; Ducatman ct 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 developed 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. Fui, Institute for Maiic.il Research and Occupational Health, Mote Fijade 158, Zagreb (Yugoslavia). is unfortunately still a matter of extreme interest, because there are 3 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 acids and DN A (Green and Hathway, 1978; Ostcrman-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. 0165-1218/9(5/503.50 O 1990 Elsevier Science Publishers B.V. (Biomedical Division) R&S150885 SB momnd&n *- -,r - v,;>.*~- 266 98809 TABLE1 COMPARISON OF FREQUENCIES OF CHROMOSOME ABERRATIONS. MICRONUCLEI AND SISTER-CHROMATID EXCHANGES BETWEEN EXPOSED AND CONTROL SUBJECTS Subject Employment (years) Ciga rettes/ day Micronuclei (%) Toial 0 1 12 20 2 10 0 3 IS 30 4 IS 0 5 12 20 6 12 0 7 25 s 16 0 20 9 9 20 10 IS u3 20 0 12 IS 20 13 25 15 14 15 0 IS 22 0 IS 15 . 0 17 15 IS 22 30 0 19 14 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 S.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 l 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 Chromosome aberrations (%) SCE 3- 4 Total Chromatid Chromosome Dicentric Acentric Per Range breaks breaks chtom. frag. cell 00 42 00 5.5 2.5 00 96 00 8.5 5.i 0 0 11.2 5 0.7 0 6.2 2.8 0 0 10.8 8.1 0 0 to 5.5 0 0 . 10.5 6 00 86 00 4 1.5 4.1 0 8.5 7 00 96 00 5.5 2 0 0 13 3.5 0 0 12 8 u 1.5 8.5 6.5 30 9.5 7 0 0 10.5 8.5 1 1.5 1.5 1.5 2 2.8 1.2 2 2.5 0.5 1 1 1 1.5 1 2 i 1.5 1.5 0.5 05 9,1 5-17 0.5 l 9 4-14 0 1.5 11 5-33 0 1.5 7.6 4-15 1.4 2.8 11.1 5-23 0 0.6 B.S 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-H 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 s 4-15 0 0.5 9.3 4-14 0.4 0.07 8.5 5.2 1.06 0.3. 2.6 2.t 1.5 0.6 0.4 0.4 1.4 i.i 9.2 4-27 1.9 0.05 '0 l.t 0.7 0.2 0 0.15 5.9 0- 7 0.2 0.3 0.4 0.2 0.2 U The frequencies of abnormal cells in the group exposed to VCM are significantly greater than in controls (P < 0.001). 1 ti'pii;. r 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-ml samples of whole blood 8 ml of F-10 medium (Gibco) containing 20% of calf scrum 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 jig/ml was added after 44 h to the lymphocyte cultures according to the method of Fcncch and Morley (1985). Micro nucleus slides were made by fixation in methanoltacctic 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 micronuclci is 12.2% with a range of 2.1-26.9%. With increasing numbers of micronuclci 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 arc 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/cdl 9.1 11 1U 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 S 4-15 Mean for non*smokcrs 7.8 1.07 4-16 R&S150887 268 mosomes and acentric fragments arc also present. The results of the micronuclcus and chromosome aberration assays arc comparable. Increasing numbers of micronuclci per binuclcalcd cell and increased numbers of cells with more than one micronuclcus arc 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, aero-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 we 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, micronuclei 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 ,o 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 micronuclcus assay show that the mean group value is 12% with an individual range of 2.1-26.9%. The appearance of more than one micronuclcus per binuclcalcd'cell is related with the results of chromosome aberrations. If the number or cells with more than one micronuclcus increases, the percentage of chromosome aberra tions increases simultaneously or chromosome damages arc more severe. Although the results of the micronuclcus 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 R&S150888 t * -A* knowledge suggests that the mechanisms of forma tion of sister-chromatid exchanges and aberrations arc 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 cl al., 1986; Hanstcen 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 (Madlc ct al., 1986; Lin-Lee cl 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 initialing 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 arc 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 or 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.P.H. Purchase and W.G.F. Adams (1980) Chromosomal analysis in vinyl chloride exposed workers, results from analysts 18 and 42 months after an initial sampling. Mutation Res.. 79. 151162. Anderson. D.. C.R. Richardson. J.F.M. Purchase, ItJ. Evans and M.L. 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