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utaiion Research, 243 (1990) 95-99 scvier 95 MUTLET 0282 o Localization of breaks induced by vinyl chloride in the human chromosomes of lymphocytes Aleksandra Fucic, Durda Horvat and Boris Dimitrovic Institute for Medical Research and Medical Health, Mose Ptjade 15S, Zagreb i Yugoslavia/ (Accepted 14 August 1989) Keywords. Vinyl chloride: Mutation. Fragile site Summary A group of 67 workers occupationally exposed to VCM was examined for the presence and distribution of breaks along the chromosomal length. Breaks induced by VCM are not randomly distributed as had been expected in a normal population. According to our results there exist highly sensitive and highly resistant ^facations along the chromosomes to the actions of VCM. The link between the highly sensitive segments of ^Jffomosomes, fragile sites and the activation of oncogenes is discussed. Effects on health of vinyl chloride monomer (VCM) (Petty et ah, 1930) were reported for the first time in 1930. Since then there have been numerous reports which associated exposure to VCM with lung cancer, brain tumors, cancer in the digestive system, primarily angiosarcoma of the liver and malignant melanoma (Creech and Johnson, 1974; Monson et ah, 1974; Takershaw and Gaffey, 1974; Waxweiler et ah, 1976; Buffier et ah, 1979; Storetvedt et ah, 1984). Nowadays the link between carcinogenesis and chromosome changes is a well-known and accepted fact. Vinyl chloride monomer is used in the plastics industry. Not only are the thousands of workers employed in this industry all over the world affected by being Correspondence: Dr. Boris Dimitrovic, `Vinilplastika', Medical Department, Lole Ribara 12. Zadar (Yugoslavia). exposed to its influence, but also the entire popula tion as a result of numberless plastic products which contain some percentage of the chemically uncoupled monomer in everyday life. In past years there have been numerous reports from many countries which demonstrated a significant excess of chromosomal aberrations among workers ex posed to VCM (Ducatman et ah, 1975; FunesCravioto et ah, 1974; Anderson et ah, 1981). Ex periments have shown that VCM metabolites in duce mutations in microbial systems and in mam malian cells (Bartsch and Montesano, 1975; Rannug et ah, 1974), There are rapidly accumulating data which suggest that some non-random chromo somal changes involved in certain cancers have break points that coincide with chromosomal fragile sites (DeBraekeleer, 1985; LeBeau and Rowley, 1984; Yunis and Soreng, 1984). According to some recent analyses (DeBraekeleer, 1985; Heim -7992/90/S 03.50 (? 1990 Elsevier Science Publishers B.V. (Biomedical Division) % and Mitelman, 1987) of the distribution of chro mosomal breaks, the majority of the bands which contain fragile sites are involved in structural aber rations in cancer and some of them are associated with specific chromosomal structure changes in specific types of cancer. The aim of our study was to show that chromosomal breaks in workers ex posed to VCM are not randomly distributed along the chromosomal length. Such data can be used in evaluating the link between a mutagenic substance and, depending on the fragment of chromosome which is affected, the type of malignant transfor mations which could appear in the organism. Material and methods For this study 67 workers in the plastic industry were chosen for cytogenetic examination. The workers examined had been employed in the polyvinyl chloride plant for 15 years on average. Exact measurements of the exposure to VCM were performed continuously. The VCM concentration in the working environment was 5 ppm. Due to the technological process the concentration periodical ly reached 2000 ppm for short periods of time. Lymphocyte cultures were initiated immediately after blood samples were collected and cultured for 48 h. Fixation of the cultures and preparation of the slides were carried out according to the conven tional method. Complete metaphases stained by Giemsa were used for analyses. 2000 cells per per son were scored for chromatid and bichromatid chromosomal breaks. To estimate the number and locations of breaks each chromosome was divided into 5 (submetacentric chromosome) and 6 segments (metacentric nI a13 chromosome). This scheme of division of the human metaphase chromosomes into segments w as taken from the study of Funes-Cravioto et al. (1974) (Fig. 1). The breaks within Al, A2, A3, B, C and D chromosome groups were taken into ac count. The distribution of breaks was based on the study of 14 400 analyzed cells. The distribution of the units of length of the metaphase chromosomes was based on the reports from the Denver Conference (1963). For statistical analyses of observed and expected frequencies the `chi-square' criterion was used (Paxlie, 1970). Results In our study we analyzed the distribution of chromatid and bichromatid chromosome breaks in the population of workers employed in the poly vinyl chloride plant. During their work the workers were exposed to concentrations of VCM varying from 5 to 2000 ppm. In 14 400 complete meta phases we counted 626 breaks in chromosomes Al, A2, A3, and groups of chromosomes B, C, and D. The results of our study revealed (Table 1) that the distribution of breaks along the chromosome arms in the lymphocytes of the population exposed to VCM was not identical to the expected random distribution of breaks in the lymphocytes of an unexposed normal population. The comparison of the distribution of breaks within the groups and segments of chromosomes w ith the distribution ex pected on the basis of the relative length of metaphase chromosomes showed statistically sig nificant values on the 1st segment of chromosome Al (Pc0.001), 1st (/3<0,05), 5th and 6th (fed.001) segment of chromosome A2, 1st La KXJ 1.1 sj Lj_L lKXi Jl.sJ BC HHUHI] O Fig. 1. Scheme of the division of the human metaphase chromosomes into segments. ASI 00004945 f I TABLE 1 LOCALIZATION AND FREQUENCY OF OBSERVED BREAKS ALONG THE LENGTH OF INDIVIDUAL CHROMOSOMES OR GROUPS OF CHROMOSOMES INDUCED BY VCM COMPARED TO THE EXPECTED VALUES IN A NORMAL POPULATION Group of chromosomes Al Chromosome segment i 2 3 Observed values 56 55 31 Expecied values 21.9 21.9 21.9 Probability of breakage < <0.001 < <0.001 0.10 A2 1 2 3 4 5 6 1 8.1 <0.05 5 8.1 0.30 6 8.1 0.80 8 12.2 0.20 28 12.2 < <0.001 45 12.2 < <0.001 A3 1 2 3 S 17.4 <0.05 17 17.4 0.90 5 17.4 <0.01 B1 2 3 4 5 C1 2 3 4 5 3 13.2 <0.01 5 13.2 <0.05 32 21.7 <0.05 48 21.7 < <0.001 37 21.7 <0.01 13 45.8 < <0.001 21 45.8 < <0.001 42 60.9 <0.05 83 60.9 <0.01 37 60.9 <0.01 D1 2 3 4 0 12.7 < <0.001 13 22.2 0.10 20 22.2 0.70 8 22.2 <0.01 626.0 626.0 CP<0.05) and 3rd segment of chromosome A3, on all segments of the chromosomes of groups B and C and on 1st and 4th segment of the chromosomes from group D. A statistical larger number of breaks compared wiht that expected was oberved on the 1st and 2nd arm of chromosome Al, the 5th and 6th segment of chromosome A2, the 3rd, 4th and 5th segment of chromosomes in group B and the 3rd, 4th and 5th segment in the chromosomes from group C and statistically significant smaller number of observ ed breaks on the 1st segment of chromosome A2, the 1st and 3rd segment of chromosome A3, the 1st and 2nd segment of chromosomes from group B, the 1st and 2nd segment of chromosomes from group C and the 1st and 4th segment of chromosomes from group D. Discussion The evidence for random distribution of spon taneous breaks in human lymphocytes is well ASX 00004946 98 known from an extensive study by Funes-Cravioto et al. (1975) who analyzed spontaneous breaks in the lymphocytes of newborns. Newborns were chosen because they represent a population which can give us much more reliable data than a popula tion of adults exposed to numberless chemical substances during their lifetime. The results of studies which consider the question of the distribu tion of breaks along the chromosomal length pro voked by the influence of different chemical substances are opposed to these findings. Such studies show that the distribution of breaks is not random and that some parts of chromosomes are more or less affected (Funes-Cravioto et al., 1975; Hampel et al., 1966; Glover et al., 1984). In earlier papers such locations w'ere called `hot and cold spots' but nowadays they are connected with fragile sites and even non-random changes in cer tain cancers (DeBraekeleer, 1985; LeBeau and Rowley, 1984; Yunis, 1983). In our study we examined a group of 67 workers occupationally exposed to VCM for the presence of chromosomal aberrations and their distribution along the chromosomal length. This investigation show-ed that the breaks induced by VCM were located in a non-random fashion i.e. there were groups of chromosomes with a disproportionally high frequency of breaks as well as groups of chromosomes where damage was below the ex pected values. The highest frequency of breaks compared to that expected in the normal unex posed population was located on the 1st and 2nd segment of chromosome Al, the 5th and 6th seg ment of chromosome A2, the 5th segment of chromosomes from group B and the 4th and 5th segment of chromosomes from group C. Locations with lower frequencies than expected were the 1st and 2nd segment of the chromosomes from group C and the 1st segment of the chromosomes from group D. The analyses of the locations of breaks along the chromosomal length allowed us to judge the mechanism of action of different chemical sub stances depending on the chemical organization of each chromosome. According to our results there exist locations highly sensitive and highly resistant to the actions of VCM along the chromosomes (Fig. 2). Some banding methods indicate that fragile sites are points on chromosomes which tend to break non-randomly when exposed to specific chemical agents. Their expression is mediated by perturba tions of the nucleotide pool (Sutherland and Hecht, 1984). If we correlate this definition of fragile sites w ith the mechanism of action of VCM metabolites on DNA, we can see that the DNA is depurinated by reaction with this mutagenic substance (Roberts, 1975). The unbalanced pool of nucleotides is a condition which may lead to the ex pression of specific fragile sites connected with disturbances in the concentrations of purines. On the other hand, fragile sites are frequently located at breakpoints of chromosomal rearrangements found in tumor karyotypes (LeBeau and Rowley, 1986; Hecht and Glover, 1984; Yunis and Soreng, 1984; DeBraekeleer et al., 1985; Hecht and Hecht, 1986; Glover et al., 1986). From such a point of view there is a correlation between specific fragile sites and cancer breakpoints and there is also a possibility that the expression of fragile sites is a predisposing factor in the occurrence of cancer in some people. The distribution of breaks in the chromosomes induced by VCM show's us that the most sensitive parts of the chromosomes are the terminal segments of chromosome A1, chromosome A2 and the terminal segments of chromosomes from | 1 without ttotrstmly *igrficaf comparing to cipecttd taghty lomriivc t*gm*t* Fig. 2. Scheme of the distribution of breaks along chromosomal length. ASI 00004947 1i .. * $ groups B and C. For some types of cancer maps already exist with cancer-specific breakpoints and fragile sites (Heim and Mitelman, 1987; Yunis and Soreng, 1984). The carcinogenic action of VCM is described in numerous studies (Creech and John son, 1974; Monson et al., 1974; Takershaw et at., 1974). If non-randomly distributed breaks along chromosomes induced by VCM represent a specific family of fragile sites induced by an unbalanced pool of purines and if we link these regions with regions which carry genes related to cancer (on cogenes) or if they are predisposing factors for chromosomal rearrangements it may be possible to find confirmation of occupationally connected malignant transformations at DNA level for dif ferent chemical substances. Acknowledgements The authors acknowledge the excellent technical assitance of Mrs. Jasminka Kapetan and Mrs. Jadranka Racic. differences Anderson, D., C.R. Richardson, I.F.H. Purchase, H.J. Evans and M.L. 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