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revink CF sjamenn/ niska produkSweden. 3:321-332. Evaluation of Evaluation of 211-215. graphs on the ance: IARC, Evaluation of lining; Crude Evaluation of yon, France: ed Inert Gas Sjofarten (in al petroleum ers. APMIS; slo: Institute Sympo- sstudy of entas, Oslo. fiU America* Journal of Mwtrial Madldii 27:545-571 (1995) \ Relationship Between Locations of Chromosome Breaks Induced by Vinyl Chloride Monomer and Lymphocytosis ? s' - o / - Aleksandra Fu6ld, PhD, Vlasta Hitrec, PhD, Vera Garaj-Vrhovac, PhD, Danica Barkovic, md, and Dragan Kubelka, BSc The distribution of vinyl chloride monomer (VCM)-induced chromosome breaks was studied in cultured lymphocytes of subjects occupationally exposed to this gas. In the examined subjects, the mean group value of chromosome aberrations is 6.5% and for sister chromatid exchange (SCE) frequencies, the mean value per cell is 7.9. These values are significantly higher than in the control population. Occupational exposure to VCM caused lymphocytosis together with disturbances of mitogenic activity in lym phocytes stimulated by phytohaemagglutinin. The results of G-banding showed that sites of chromosome breakpoints caused by VCM can be related to the lymphatic tissue disorders. 1995 Wiley-Liss, Inc. Key words: vinyl chloride monomer, localization of chromosome breaks, lymphocytosis, SCE, G-banding, occupational exposure INTRODUCTION Vinyl chloride monomer is a chemical used in the plastics industry. Today thousands of workers employed in this industry are exposed to its influence. Exposure to this gas causes multisystem disorders including acroosteolysis, thrombocytopenia, portal fibrosis, hepatic and pulmonary dysfunction, immune complex disorder, pri marily angiosarcoma of the liver, lung cancer, brain tumors, and malignant mela noma [Creech and Johnson, 1974; Tabershaw and Gaffey, 1974; Waxweiler et al., 1976; Buffler et al., 1979; Doll, 1988; Ward, 1976] together with increased frequen cies of SCE, chromosome aberrations, and micronuclei [Ducatman, 1975; Anderson, 1981; Sinues et al., 1991; Fueic et al., 1990a]. It has been suggested by several investigators that chromosome breakpoints caused by radiation and some chemical agents cluster on certain parts of chromo somes [Funes-Cravioto et al., 1974; Casperson et al., 1972; Holmberg and Jonasson, Institute for Medical Research and Occupational Health, University of Zagreb. Zagreb, Croatia (A F . V.G.. D.K.). Pediatric Clinic, Cytogenetic Laboratory, University of Zagreb, Zagreb, Croatia (V.H.). DiNA-Petrochemical Industry. Krk, Croatia (D.B.). Address reprint requests to Dr. Fuii5, Institute for Medical Research and Occupational Health University of Zagreb. 2 Ksaverska Cesta, P.O. Box 291. 41001 Zagreb, Croatia. Accepted for publication May 31, 1994. 1995 Wiley-Liss, Inc. ASI 000013944 1973; Bauchinger and Gotz, 1979; Kano and Little, 1986], This phenomenon could be connected with increasing evidences that some nonrandom chromosome changes are involved in certain cancers [Yunis and Soreng, 1984; Sandberg, 1991; Mitelman et al., 1978; Barrios et al., 1988], According to our previous results [Fuic et al., 1990b], a nonrandom pattern of chromosomal abnormalities is also present in lym phocytes obtained from subjects occupationally exposed to VCM. In the present study, chromosome aberrations in 20 subjects occupationally exposed to VCM were localized by G-banding and correlated with SCE frequencies, mitotic activity, and total blood counts. MATERIALS ANO METHODS Twenty male workers aged 30-50 years from the plastics industry were chosen for cytogenetic examination. They had been employed in a polyvinyl chloride plant for 8 years, on average. Those with recent X-ray exposure and drug treatment were excluded from this study. Exposure to VCM was measured continuously by gas chromatography. The VCM concentration in the working environment was 1 ppm, periodically reaching 300 ppm for a short period, due to technological process. Lymphocyte cultures were initiated immediately after blood samples were col lected and then cultured for 48 hr. Cultures of lymphocytes were stimulated by phytohemagglutinin (Wellcome). Eight ml of RPMI 1640 (Gipco) containing 20% calf serum was added to 0.5 ml samples of whole blood. Fixation of the cultures and preparation of the slides were carried out according to the conventional method. Complete metaphases stained by Giemsa were used for analysis. Two hundred cells per person were scored [IAEA, 1986]. Sister-chromatid exchange frequencies were measured on slides from cultures of lymphocytes to which 10 p.g/ml bromodeoxyuridine was added. The cultures were harvested after 72 hr (Parry, et al., 1985]. Mitotic activity was measured on slides prepared for SCE. The first, second and third metaphase were differentiated by the level of incorporated bromodeoxyuridine. Five hundred cells per person were counted [Lamberti et al., 1983]. G-banding was performed by treatment with trypsin and staining with Giemsa. The chromosomes were identified and classified according to the 1SHCN [1985]. A total of 1,200 metaphases were analyzed. The total blood counts were analyzed automatically on Coulter Electronica 186. The presence of lymphocytosis was analyzed in 100 subjects from the same plastics industry as the rest of the examinees and in the same working environment. A control population of 100 healthy subjects, aged 30-50 years, was analyzed for blood count. The control population for cytogenetic analysis consisted of 20 male subjects aged 30-50 years with the same smoking habits as the examinees and without recent exposure to ionizing radiation and drugs. Frequencies of chromosome aberrations are compared as Poisson rates, and distributions of mitotic activity by Xs;2-test. Blood count data were analyzed using standard descriptive statistics. Since the Kolmogorov-Smimov test does not show statistically significant deviation from log-normal distribution, the difference between control and experimental group was tested using the one-tailed t-test on logarithms of AS1 000013945 i could hanges telman u<5id et al., ent in lym- upationally requencies. ere chosen oride plant ment were aphy. The y reaching ; were collulated by ining 20% iltures and 1 method, idred cells tures twere econd and tyuridine. i Giemsa. .1985], A mica 186. ie plastics A control od count. subjects >ut recent ites, and Fed using lot show between rithms of Locations of Chromosome Breaks Induced by VCM 567 TABLE I. Comparison of Frequencies of Chromosome Aberration, Sister-Chromatid Exchange Frequencies, and Mitotic Activity Between VCM Exposed and Control Subjects Chromosome aberration (%) SCE Mitotic activity <%) Subject Employment Acentric Chromatic Chromosome Dicenmc Per fyr) frag breaks breaks chromosome Ring tot cell Range Ml M2 M3 19 25 34 -t 4 53 6 10 74 8 1l 99 10 10 11 14 12 12 13 2 14 14 15 9 16 10 17 9 18 9 19 5 20 8 Mean group values for the 20 controls 0.5 1 l 2 2 ! 0.5 1 0.5 1 0.5 0.7 5 6 4 1 5 6 6.5 6 2 7 2 45 6.5 6.5 3 7.5 6 6 7 2 11 0.5 2 1.5 I l 15 1.5 0,5 0.5 2 05 1 0.5 05 on 6* 5 8 4-9 14.1 23 5 62.2 9* 7.4 4-13 6.8 12.5 80,2 4 6.7 4-10 7 2 14 4 78.3 l 3 4.4 4-6 10 5 27.8 61 5 6.5* 5.3 4-8 11 8 25 3 52 4 7* 6.4 4-11 15 9 21 5 52.5 05 9* 8 4-15 7.3 25.2 67 4 6* 8,7 4-16 6.8 39,7 53 4 2 5,7 4-12 13.6 40 9 45 4 8-5* 7.7 4-15 9 1 25 5 65 2 6* 95 5-17 9 8 25 3 64 8 8* 9,4 4-18 8,9 23 2 67.8 8* 8.5 4-21 10,8 16.3 72 8 0.5 8* 8.6 4-22 98 25,3 64 8 6* 9 4-21 10.5 16.9 72.5 8* 9.9 4-18 7.1 17.6 75 2 7.5* 96 4-14 7 2 23.7 69 7* 9,1 4-16 9 4 26,4 69 7 5* 8.5 4-18 9.1 34,7 63 3 9.3 4-21 7.6 20.3 72 2-6 5.9 4-7 7.7 34.9 53 6 *p <0.01; Ml. M2, and M3, p <0.001. observed values. Statistical analysis were performed on a PC 386 computer with the STATGRAPHICS statistical package. RESULTS The investigated exposed subjects had a minimum of 2 years of steady daily exposure to VCM, and their histories contained no other known exposure signifi cantly different from that of the control group. The examined workers have higher values than the control population in both the chromosome aberration assay and SCE (Table I). The quantitatively dominant type of aberration is chromatid break, but dicentric and ring chromosomes are also present. Sister-chromatid exchange frequencies are also increased comparing to the control values. The individual range is 4-22 per cell in the examined group and 4-7 per cell in the control group. The distribution of the values of mitotic activity in the examined population differs significantly from that in the control population (Table II). The values for the second mitosis (M2) are significantly lower than expected, while the number for the third mitosis (M3) is significantly elevated. The G-banding technique shows that bands which correspond with chromosome breaks are located on chromosomes 1,2, 3, 5, 6, 7, 8,9, 10, 11, 12, 13, and 14 (Fig. ASI 000013946 568 Fuitf et al. TABLE II. Band Localization of Chromosome Aberrations Caused by VCM and Related Diseases Band Type of aberration Related disease lp34-32 tptt lq21 Iq23 lq25 Iq32 Chromatid gap Chromatid gap Chromatid break Chromatid gap Chromatid break Chromosome gap Deletion Chromatid gap Chromatid gap Chromatid break T-ALL MDS, AML, MPD CHL, ML ML, AML. ALL ALL MPD ML 2p21 2p23 2q3l Chromatid gap Chromatid gap Translocation t(2;12) (q31;p!2) random PML, MH, T-ML random 3p21 Chromatid gap ML Mr 3q21 Chromatid gap AML, MDS, MPD 5q31 Chromatid gap AML, ALL Chromatid break 6q2I (14-27) chromatid gap ML, ALL. ATL. HCL. PLL 7pll-ql1 7pl3 Translocation t(7:l4) (p!3;pll) Deletion ALL, ML ALL, AML, MPD 8q22 Chromatid gap MPD, ML, AML-M2 9p21-22 9qlI--qlI 9q34 Deletion Chromatid gap Chromosome break Chromosome break ALL AML, MPD, CHL ALL, ML lOqll Chromatid break random Ilq23 Chromosome break ALL, AML. MDS AML-M5, AML-M4m5, ML 12pl2 translation t(2;I2) (q31;p!2) 13ql2 Chromatid gap MPD, MDS I4q 11 I4q24 Translation t(7;14) (pl3-qll) Chromatid gap ATL. T-CRLL, T-PLL B-CLL, ML ALL. acute lymphoblastic leukemia (LI-L3 in FAB classification); AML, acute lyeloid leukemia (subdassified FAB MI-M7); ATL, adult T-cell leukemia/lymphoma; CLL, chronic lymphocytic leukemia; CML. chronic myeloid leukemia; HCL, hairy cell leukemia; MDS, myelodisplastic syndrome; MH, malignant histiocytosis; ML. malignant lymphoma, subclassification as 8- or T-cell type; MPD, myelo proliferative disorder, PLL. prolymphocytic leukemia. 1). Banded chromosome analysis reveals that the breakpoints very often coincide with some specific cancer breakpoints, such as acute myeloid leukemia, acute lympho blastic leukemia, and myeloproliferative disorders [Mitelman et al., 1991]. Blood test analysis show statistically significant increase in the percentage of lymphocytes in the VCM exposed examinees comparing to control. The individual range is 28-67, and the mean group value is 41.3. By contrast, the control subjects ranged from 22 to 42, with a mean group value of 30.2. ASI 000013947 Locations of Chromosome Breaks Induced by VCM 569 JD L 'D HCL, PLL D M2 Subjct Fig. 1 Percentage of cells in [he first (Ml), second (M2) and third (M3) division in exposed subjects and in the control group (MK1, MK2, MIG). DISCUSSION L M4m5. ML -PLL emia (sub- leukemia: ome; MH. JD, myelo- cide with lympho* entage of ndividual I subjects After metabolic activation, VCM, a chemical used in the plastics industry, is a strong carcinogen and mutagenic agent in mammals. However, the study of the consequences of occupational exposure to VCM has been largely restricted to mor tality and epidemiological studies of malignancies or severe health disorders. Too little attention has been paid to health disorders caused by exposure to low concen trations of this gas. The metabolic products of VCM, chloroacetaldehyde and chloroethylenoxide, are known to be alkylating agents which lead to the depurination of DNA molecule [Barbin and Bartsch, 1989] and probably to an unbalanced nucleoid pool in the cell. Another phenomenon caused by alkylation is the neighboring base effect, which is the result of specific, nucleotide sequences in the regulation of gene expression. There are adenine and thymidine-rich sequences which affect the initiation and ter mination of transcriptions that are dependent upon their base sequence. Modification of these or other similar sequences by alkylation could have profound effects on the cell [Briscoe and Cotter, 1985]. It is now well established that human tumors, particularly leukemias and lym phomas, are characterized by nonrandom chromosomal abnormalities such as trans locations and deletions. Nonrandom chromosomal translocations are among those that define regions of the genome of oncogenic potential. At the same time, nonran dom chromosomal deletions reflect the loss of genetic information at a specific site in the genome and suggest the presence of tumor-suppressor genes. Some neoplasias originate from exposure to various mutagenic agents from the environment. The specifically vulnerable chromosomal regions for some mutagens have already been described [Morad et al., 1973; Barrios et al., 1988; Mitelman et l 570 Fuiil et al. al., 1978], Additionally, elevated SCE frequencies in subjects exposed to chemical mutagens exemplify the danger of the carcinogenic action of the mutagenic agent, since sites of origin of SCE frequencies coincide with fragile sites [Glover and Stein, 1987] , which can result in unequal chromosomal recombinations and rearrangements [Lattetal., 1986]. The biomonitoring results in our study confirm previous findings about the mutagenic activity of VCM. Breaks on chromosomes caused by VCM cluster on specific cancer breakpoints [Fudid et al., 1990b], which may be a predisposing factor to the induction of chromosomal rearrangements. The breakpoints described in our study are often related to malignancies of B lymphocytes, which correlates with the findings of Ward et al. [1976], who described an increase in the circulating B lymphocytes in VCM-exposed subjects. An increased sensitivity of B lymphocytes to mutagens was also found in a population of gasoline pump mechanics [Hogstedt et al., 1991]. Blood test analyses confirm that a population occupationally exposed to VCM exhibit disorders of the lymphopoietic system; of the 100 subjects in this study, 75% exhibited an increase in the percentage of lymphocytes together with disturbances of mitotic activity. It is important to emphasize that none of the subjects suffered from any chronic disease or expressed symptoms typical of VCM disease. Thus, lympho cytosis might be hidden for a long period of latency without visible health disorders. The epidemiologic study of Smulevich et al. [1988] revealed an elevated num ber of subjects suffering from leukemias and lymphomas after occupational exposure to VCM. 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