Document X78ZaG7x3NOdddKeZER85VRVg

Mutation Research, 51 (197S) 371--27S Z EIBviur,'North-HaUand Biomedical Press a 371 EFFECTS OF VINYL CHLORIDE IN MAN A CYTOGENETIC FOLLOW-UP STUDY INGER-LISE HANSTEEN , LEIF HILLESTAD *, EYVIN'D THUS-EVENSEN e and SIPJ STORETVEDT HELDAAS c M Laboratory of Genetics, and b Department of Internal Medicine, Telemark Central Hospital, Olavsgt. 26, 3900 Porsgrunn and ` Medical Health L'nit, Sorsk Hydro, Porsgrunn Fabrihker, 3900 Porsgrunn (Xorway) (Received S December 1977) (Revision received 6 February 1978) (Accepted 7 March 197S) Summary Cytogenetic studies were performed on 39 workers from a PVC plant in 1974. 16 healthy men without any connection with the plant were chosen as controls. The cytogenetic study was repeated for 37 of the 39 workers 2--2.5 years later. During this time interval the workers had only had a minimal exposure to VCM. This repeated study was performed with 32 matched con trols from the office employees in the factory. Breaks, gaps and stable rearrangements were scored in 100 metaphases per person from 48-h lymphocyte cultures. The mean chromosome-breakage frequency for the workers (3.41%) was sig nificantly higher than for the controls (1.79%) in the first investigation. In the repeated study no difference was found in mean chromosome-breakage frequency between the workers and their matched controls. Neither was there any difference between these breakage frequencies and the breakage frequency for the previous control group. These results might indicate a relationship between the reduction in exposure to VCM and the normalized chromosomebreakage frequency. Sister-chromatid exchanges were studied for 16 workers with matched controls in the repeated study. A mean of 7.6 SCEs per cell was found for both workers and controls. Bone-marrow samples.from 4 workers were studied in the first investigation. The mean chromosome-breakage frequency was higher in the bone marrows (4.2%) than that reported for normal bone marrows (0.2, 0.4, 1.7%), and higher than for the corresponding lymphocyte cultures. Abbreviations: BidU. 5-bromodcoxyuxidint: SCE. agur-ehxomfcud xcn*ng; VCM, vinyl chiondt monutner. . - ....- -v; . y.y;:.y;. :. * ,y.y.y..y,y.,y.y,::1::;;: y 1 : y : ::': .y.y'y'.y! 1 x: l - ; .v Y.v*. --, - 3 TY.' 7: " 3:: Y'YYY, .y.'.Y.y.YYy".".Y.v.Y.v.`Y.` ..'..V.V.V.V.'* y.y.y ........\ 7.YY,:.Y.".;.,-.Y,v!:::hYYY,Y\;. Y.Y .r y.y.\ ' '' - ;' Y.Y..- v if?7f::`'yry.>y' -vVVV :. .: " '' -.`rr,yy `` - ............. ucc 023911 I 272 - Introduction Reports on the clastogenic effect of vinyl chloride monomer (VCM) have been published for workers in PVC plants [1--3,5,13,17]. ThePVC workers had been exposed to VCM for years. Yoder et al. [19] found a high chromosomebreakage frequency in agricultural workers during extensive occupational exposure to pesticides, but the chromosome-breakage frequency was back to normal after some months without exposure. We therefore repeated the cyto genetic study of the PVC workers 2--2.5 years after the first examination to see if we could find the same effect. During this time the workers had only mini mal exposure to VCM. The results for both the first and second cytogenetic study are presented in this report. Methods Cytogenetic studies were performed on 39 workers from a PVC plant in 1974. 14 of these were chosen at random. 13 were chosen because they had been heavily exposed to VCM for years. Moreover, chromosome analyses were performed in connection with clinical re-examination on 12 workers whose first health screening revealed abnormalities. At the re-examination all the laboratory tests proved normal. 16 healthy men, who had no connection with the PVC plant, were chosen as controls. Matching for age between the workers and the controls was attempted, but was not entirely successful. The cytogenetic study was repeated for 37 of the 39 workers 2--2.5 years later. This repeated study was performed with 32 matched controls from the office employees at the factory. They were matched for sex, age, ethnic back ground and time of employment. In addition, 6 of the previous control group were re-examined. A medical history, including viral infection, radiation exposure and drug intake, was obtained for ah participants in both studies. Exact measurements of die exposure in the PVC plant were provided from 1974. The estimated values for the previous years indicate that the exposure had been considerable (Table 1). Immediately after the blood samples were collected, lymphocyte cultures were initiated and cultured for 48--53 h according to conventional methods in both the initial and the repeated study. Whenever possible, 100 metaphases were studied for each individual. 20 metaphases, including suspect rearrange ments, were karyotyped. Another 5 were analysed under the microscope. The remaining metaphases were cotinted and scored for breaks and gaps. The system of Hirschom and Cohen [6], which considers all breaking events in the cells, was applied to these data, and the results are reported as breakage frequency per 100 cells. In the repeated study only cells with suspect rear rangements were karyotyped. Chromosome studies were done on bone-marrow samples from 4 workers in the initial study. The sister-chromatid exchange (SCE) technique [11] was used on 16 :.. .. vv ,w *7::ii'.-: `: *. . .: ................ ".\\\V".YV ..V.V.W.Y '"a" v. :t ,, ;;;V.yy.:yy.v.y.v:.y.v.y:.y:::::: i: y.yy v.v,.y.y. . ::::::::::;':Y:Y;Y-.V: -.Y:.Yy.y.yy.y - . v.iv.v.v.vtit'AtvAtvAVAV/.v.-.v.; .y.y i::::::::: .'.yy.y.' *.v y.y.'\y.y.,yy"; .... .. . >;.v ,, :r ,, ....... it'.ttY.'.'Y.'.y.y..`.Y.'.i.: :^;j.y.y.y,,,-t ."y.I"y-;^ j'Kr. ucc 023912 * 273 TABLE 1 air concentration of vcm in the pvc plant Eititnti** in bmd upon the lv*l of production, and the number and typet of autoclav* uacd in the retptcuve tuna penods. Tim* periods Air cone, of VCM in ppm I960-- 1S64 19S5--1959 1980-1967 1968--1971 1973--1974 1974 1975-- Meuund 2000 1000 500 100 80 23 1 workers and their matched controls in the repeated study only. DrdU (2 Mg/ ml) was added to the cultures 24 h after initiation. The cultures were harvested at 72 h. The 33258 Hoechst--Light--Giemsa method described by Goto et al. [4] was used for staining with a high-voltage mercury lamp (Zeiss HBO 200 W/4) as light source. The SCEs were scored in 30 cells from each individual, and the values compared with the breakage frequency found in 48-h cultures from the same blood sample. Results The results for the control groups in 1974 and 1977 are presented in Table 2 together with the results for the workers chosen at random (Group I), for the workers examined on clinical grounds (Group II), and for the heavily exposed workers (Group HI), with their matched controls. The mean breakage fre quency for all the 39 workers was 3.7% in the initial study. After thorough examination of all the subjects, different factors (as radiation and drugs) which may influence the breakage frequency were found for one worker in Group I, for one in Group n, and for two workers in Group III. These workers were omitted. The mean breakage frequency for the remaining 35 workers was then 3.41% compared with the controls with 1.79% (Table 2). The mean breakage frequency for the heavily exposed workers, 3.97% (Table 2), was slightly higher than for the rest of the workers with 3.15% (Table 2). The results were analysed statistically by the Wilcoxon two-sample ranking test [7]. All tests for workers versus controls (Table 3) showed that workers scored significantly higher on a 2.5% significance level in the initial study. In the repeated cytogenetic study 2.5 years later, no significant difference was found between chromosome breakage frequency for the workers and their matched conttols (Table 4). The chromosome-breakage frequencies for each of the three groups of workers were back to normal. No significant difference was found between the control group in 1974 and the matched controls either (Table 4). _ A closer study of the individual values revealed that the worker omitted from Group I had a high breakage frequency at both examinations. He was- found to have carcinoma labii. The rest of the randomly chosen workers with ........... ",V-"..V.V.7V.V.\v \v v v I.'.V 7.W, v.-.y,. A'.v.. r7 ucc 023913 274 uec 023914 TARLE 2 RESULTS OF TUB INITIAL (1274) AND REPEATED (1977) CYTOGENETIC STUDY OF THE FVC WORKERS AND THE TWO CONTROL GROUPS Groups Afs Employment In ycui Breokrge frequency per 100 cells 1974 1977 t, Controls i Hepesled control (6 workers) Workers Group 1 Group II Grpup III X (run.) 42.8 (27--88) 44.9 (23--88) ll.l <26--67) 81.7 (39--63) X (ran|.) 10.3 (1-19.8) 13.7 (4--31) 16.6 (10.6--M) X (SO) 1.79 (1.87) 3.72 (2.12) 2.48 (1.26) 3.97 (1.61) X (SO) 1.68 (1.11) 1.40 (1.43) 1.82 (1.86) Group I, workers chosen at rsodom. Group II. workers sxemlned on cllnkal (rounds. Group 111, workers heevlly exposed. X (SD> 2.33 1.26 (1.09) 1.60 (1.28) 1.91 (1.16) ...T , 1 ." ' : 1 Jl ittSr-. Hy. lliilll ;v-C 111 . !-'s: r I''1 ::ii! I!** ' -ii; .:|=- I;!/i:;i '.'I! : i: "I.: ' Ill; I. 275 TABLE 3 STATISTICAL EVALUATION WITH WILCOXON TWO-SAMPLE RANKING TEST Thi probability percent*** reflaeta the extent to which the two sample* compand arc itatiaticailr identlcaL Samplaa tailed 1974 for total brtekafe frequency Probability percent*** Heavily txpoiad workan varau eontrola The ran of the worker* varaia control* All worker* vanua control* 0.33 2,1 0.48 TABLE 4 STATISTICAL EVALUATION WITH WILCOXON TWO-SAMPLE RANKING TEST The probability parcanta** reflect* the extant to which the two sample* compared an statistically iden tical. Sample* tasted 1977 for total break*** frequency Probability percents** worker* it random venue control* Worker* -- clinical v*su* control* Worker* heavily exposed versus control* All worker* versus consols Control* 1977 versus control* 1974 Group l Group n Group to 20 64 SI 43 41 TABLE 8 SCE AND TOTAL BREAKAGE FREQUENCY IN 18 PVC WORKERS AND THEIR MATCHED CON TROLS No, Worker* Matched control* Bnakiti frequency 46-fa culture SCE pet cell 72-h culture (ranee) Break*** frequency 48-h culture SCE par call 72-h cultun (ran**) 16 6 78 16 4.6 14 4 11 4 23 3 6 3 26 3 36 3 37 3 3 2.8 27 1.5 30 1 39 0 16 0 31 0 X 2.6 10.9 6.6 6.4 6.1 6.2 9.3 7.9 6.8 6.7 - 4,7 7J $.6 6.6 9.9 9.6 9,5 7.6 (2-17)(2-14) ' (3-17) (2-11) (4--16) (3-19) (4--22) (2-13) (4-13) (2-11) (2-17) (1-16) (3--12) (4-21) (3-21) (4--20) (1--21) 4 2 3 3 2 1 3 1 3 0 4 3 2 0 4 0 2.3 7,2 5.6 11.6 8.2 7.6 6,3 SB 5.9 a.s 7.6 5.6 11.A 7.4 8.1 11.1 8.6 7,5 (2-13) (3--10) (8-21) (2-10) (1-15) (3-11) (2-9) (1-15) (3--11) (3-14) (0-11) (5-28) (1--19) (2-16) (3--24) (3-19) (0-24) "ittf."'- ----- -1-.i - `::: ,v.i '.'.'.v.'.::: : 7 :^>xjY'' v.-**:-:-: t;: iv.v.'.v::::v.v..v.v::7:` v.v v.v.v.'.. ..v.v.v.w.*.-.*, I::::::::::.::;:':: :rr ii'.ww.v" v-.v.......... y.r;:::. ; .> - ''* V.'.V.V.'.V.V.'.''.V.V.V.V.V.V.Y.V.Y,V.Y.\iV-TT ' - -.1 " 1 - - - - t:::; .'.v.v.'ev.v.v.v.-.v. v-v.v-v.v.-.v-v.'e-'.v.'--.. -: uce 023915 276 high breakage frequencies in 1974 had lower values in 1977. None of the workers chosen on clinical grounds had higher breakage frequencies than the highest control value in either investigation. The two heavily exposed workers, who were omitted from the statistical evaluation in 1974 because of drug intake, had lower breakage frequencies in 1977 despite their using the same drugs. The rest of the heavily exposed workers also had lower breakage frequencies in 1977, except one. He died of lung cancer four months after the last blood sample was taken. There was no difference in the mean number of SCEs per cell between the workers and their matched controls 2.5 years after exposure (Table 5), just as there was no difference in breakage frequency. By comparing the chromosomebreakage frequency and SCEs for each individual, we found a high breakage frequency with a high number of SCEs (Table 5, No. 18) and a low number of SCEs (Table 5, No. 7). We also found low breakage frequencies with both high and low numbers of SCEs (Table 5, last cases). Bone-marrow samples from 4 workers were studied in the first investigation. A total of 288 cells was examined. The mean breakage frequency was 4.2% with range 0--7. Congenital chromosomal abnormalities were found in 2 of the workers studied. One had 46,XY, with rep (5; 12) (5qter -* 5q34::12qter-* 12ql5) and rep (15; 19) (lSqter-*- 15q22::19) in all cells studied. The other had a normal karyotype in 2/3 of the cells studied, and a deleted Y chromosome in the remaining cells. Discussion The chromosome-breakage frequency for the workers in 1974 was signifi cantly higher than for the controls. It was then concluded that VCM might be the cause of this increased frequency. As the control group was chosen from premises outside the factory, one could not completely exclude that other agents than VCM could he the cause of the increase. When the cytogenetic study was repeated 2--2.5 years later, a control group was therefore selected from the office employees at the factory. The repeated study demonstrated that the chromosome-breakage frequency was back to normal after a significant reduction of exposure of the workers for two years, the same effect as Yoder et al. [19] found for pesticides. There was no significant difference between the chromosome-breakage frequency in the first and second control groups (Table 4). These two observations provide evidence for VCM as the real cause of the chromosome damage found in the first investigation, as also demonstrated by others [1--3,13,17]. In the first part of the study the heavily exposed workers were more signifi cantly different from the controls than were the rest of the workers, but a dose--response relationship could not be established. Low as well as high breakage frequencies were found in heavily exposed workers. Workers with high breakage frequencies during chronic exposure may be at risk and will be followed with special attention. -- At present little is known about the significance of the chromosomal aberra tions incurred by workers exposed to-VCM or other chemicals. In the present .'ii'.'.'.Y"' v.V::V.:::t *v:V:Y:V: ' ;:::;::;_:;' :v,:::: ' - ; _-v.-v.;::::::::::::: tv "" Y.Y.V.Y.V :l`v" v :v.v. ',* V V ,Vv " tV-.VTV:V:`- :-y?:y;vVV" :;j.=. .....i............... . ..... .*.*.*.-*.................................-.... - '.v.'.v.v.v.v.'.; ucc 023916 277 work no relationship between chromosomal aberrations and VCM disease could be traced, except perhaps for one of the heavily exposed workers. This worker, however, was omitted from the statistical evaluation, as only follow-up exami nations can provide the answer to the likelihood of a VCM disease. His chromosome-breakage frequency was back to normal, too, after his removal from exposure. The chromosome-breakage frequency was consistently high in two workers. Cancer was discovered in these two workers. The histological type of the cancers has not been confirmed to have any connection with VCM exposure. A salient point in chromosome studies is whether.the findings in peripheral, blood can also be detected in the tissues and in the germinative apparatus. In this study, 4 bone-marrow samples were subjected to chromosome analysis. A higher total breakage frequency was found in the bone-marrow samples com pared with the lymphocyte cultures from the two workers who were ade quately studied: 4% versus 2% for one, and 7% versus 2.5% for the other. The quality of the bone-marrow preparations was rather poor in the two other bone marrows. The mean breakage frequency for the marrows was 4.2%. Unfortu nately, control samples were not available, but reports on haematologically normal bone marrow in the literature [8^9,14] give mean frequencies of aberra tions of 0.4, 1.7 and 0.2% respectively. No conclusions can be drawn from studies of the 4 bone-marrow samples. At present it can be noted, however, that the mean breakage frequency was higher in the bone marrows from the PVC workers than that reported for normal bone marrows. Breaks and gaps were the predominant aberrations in bone marrow as well as in the lymphocyte cultures. Many papers have recently reported increases in SCEs after acute exposure to different chemicals in cell-culture systems [12,15], in animals [16], and in man [12,18], In the present study the same mean value was found for workers and their matched controls, which establishes a normal mean incidence of SCEs per cell of 7.6. This is in agreement with Morgan and Crossen's [10] finding of 7.9 SCEs per cell in human lymphocyte cultures. A normal incidence of SCEs is what we would expect when the chromo some-breakage frequency is within normal variation for both workers and con trols. It does not necessarily follow, however, that we would have found a higher number of SCEs in the workers-in-1974 during chronic exposure, as in the present investigation we found both high and low numbers of SCEs with the highest breakage frequencies (Table 5). The question about a connection between chromosome damage scored as chromosome-breakage frequency and as number of SCEs per cell will therefore have to be investigated further before we can evaluate the use of SCE in the study of chronic exposure. Acknowledgements We want to thank K.O. Qausen, V. Haugan and K. SkSrdal for skilful tech nical assistance. References " 1 Ducatmaa, A., K. Hinchocn and I-J. Sallkoff, Vinyl ehlond* fxpocurc and human chromosome aber rations* Mutation Rat., 31 (1975) 163--168. y.v ;v.-.v.v:.:::::::;:.. ..::::v.v.v.v v.w.v. ' ;;,; r v:--v v!" iv.v.v,' v.viv.v j..:;;:::':::-:: v.. t liTT : ` `` - ..t.................. . . :V:v.v!:V::; *. : 7:: ;:tt- /.v v,-.v . v ' y,y- ' " ; ... ;;; -. -- - V:"- /.v,... ' : i'.'.'.'.'.y.v.' L UCc 023917 2TS 2 FUig, I,, and A.M. Thiet*. External chromosome studies undertaken on persons and animals with vc illness. Abstract of 2nd Int. Conf, Environ. Mutagens. 1977, p. 219; Mutation Res., 53 (1979) 187. 3 FunesOavioto. F-. B. Lambert, J. Linsten. L, Ehrenberg, A.T. Natarajan and S. OHerman-Golkar. Chromosome aberrations in workers exposed to vinyl chloride. Lancet, I (1975) 459. 4 Goto, K., T. Akematsu, H. Shunazu and T. Sudyama, Simple differential Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanicra of stamina. Chromosoma. 53 (1975) 223--230. 5 Hansteen, J.L-. L, Hillestad and . Thiis*Evensen, Chromosome studies in workers exposed to vuiyl chloride. Abstract of Vtb Annual Meeting of EEMS, (1975) 63;Mutation Res., 38 (1976) 112. 6 Hirschom. 1C., and M.M. Cohen. Drug-induced chromosomal aberrations. Ann. N.Y. Acad. Sci., 151 (1968) 955-987. 7 Hodges J r.. J.L., and E.L. Lehmann. Grundbegreper i sandsynlighetsregning og statlatlkk, 2nd edn., Nyt Nordisk Fodag, Arnold Buaek, Copenhagen, 1972. 8 FCnuutila, S., O, SUnelL P, Lipponen and t. Saarinen. Bone-marrow chromosomee in healthy subjects, Hereditaa. 82 (1976) 29--36. 9 Krogh Jensen. M.. J. Erlksen and B.W. Djernes. Cytogenetic studies in myelomatosis, Scsnd. J. HasmatoL, 14 (1975) 201--209. 10 Morgan, W.F., snd P.E. Crossen, The incidence of sister chromatid exchanges In cultured human lym phocytes, Mutation Res., 42 (1977) 305--312. 11 Perry, P,, snd S. Wolff. New Gitmse method for the differential staining of nstsr chromatids. Nature (London), 251 (1974) 156--158. 12 Perry, P.. snd HJ. Evans, Cytological detection of mutagen--carcinogen exposure by sister chro matid exchange. Nature (London), 258 (1975) 121--125. 13 Purchme, I.F.H., C.R. Richardson and D. Anderson, Chromosomal sad dominant lethal effects of vinyl chloride. Lancet, 11 (1975) 419--411. 14 Seeker Walker, L.M., The chromosomes of bone-marrow cells of bematologieally normal men and women. Bdt_ J. HaemstoL, 21 (1971) 465--*61. 15 Solomon. .. snd M. Bobrow, Sister chromatid exchanges --a sensitive assay of agents damaging human chromosomes. Mutation Res., 30 (1975) 273--278. 16 Stttka, D.G., and S. Wolff, Sister chromatid exchange as an assay for genetic damage induced by mutagen^arcinogens. 1. In vivo test for compounds rebuking metabolic activation. Mutation 41 (1976) 333--342- 17 Szenteii, I.. E. Hornyik. G. Ungviry, A. Czeixel. Z, SognZr and M. Tlmar. High rate of chromosomal aberrations m PVC workers. Mutation Res., 37 (1976) 313--316. 18 Waksvik, H., A. Brbgger and J. Steoe, Psomien/UVA treatment and chromosomes. 1. Aberrations and sister chromatid exchange in human lymphocytes in vitro and synergism with caffeine. Hum. Genet., 38 (1977) 195--207. 19 Yoder, J., M. Watson and W.W. Benson, Lymphocyte chromosome tnalysis of Agricultural workers during extensive occupational exposure to pesticides. Mutation Res., 21 (1973) 335--340. -V. :: r ::V:V:;:::;:::::::: ,v.\v;.v.v.v.v.v.v.v: V.V.V.V.V.V//.V.V.V.V.V/.V.V/.*. vv/-".--vv:" ' ' *.. v* ucc 023918