Document M4MyrJ8K3X2n2M5QdXgGL7vBM

Mutation Rest arch, 31 (1975) i) Klsevier Scientific ihibiishhi^ Company, Amsterdam Printed m The Netherlands 163 VINYL CHLORIDE EXPOSURE AND HUMAN CHROMOSOME ABERRATIONS* ALAN DUCATMAN, KUKT HIliSCHHOUN** and IRVING J. SE LI KOFI' Environmental Sciences Laboratory, Department of Community Medicine and the Dtvision of Medical dinettes, Department of Pediatrics, Mount Sinai School of Medicine of the City University of New j Yorhf New York, N.Y. 10029 (U.S.A.) (Received October 25th, 1974) (Revision received January 15th, *975) IOTICE: Hits material may protected by copyright law (Title 17 U.S. Cod ) SUMMARY Examination of lymphocyte cultures from n vinyl chloride polymerization workers and 10 controls revealed a significantly higher incidence of alienations in the exposed population. Most of the excess damage was of the "unstable" variety and involved the grossest kinds of changes such as fragments or rearrangements. When these complex changes were regarded as the product of two breaks, the incidence of all breaking events was also significantly increased in the workers. The results indicate the presence of chromosome damage in vinyl chloride exposed workers. INTRODUCTION Since the pioneering radiation studies of tile early 1960's it has been clear that chromosome morphology examination could uncover evidence of genetic damage in man1-11. The earliest of these studies discovered the now well-known link between carcinogenesis and chromosome changes {for recent review, see ref. 9). These were soon followed by the discovery that a number of chemicals are clastogeuic (chromo some breaking), notably alkylating agents and cytostatic drugs, and DNA base analogues*. In recent years, occupational exposure to various chemicals has been recognized as another potential source of genetic change in man. Chromosome examination following human exposures hud revealed the probable clustogenicity of 'Dine pesticides and herbicides1"'1'. Industrial exposure to benzene has also been implicated by a number of investigators in both ieukemogenesis and clastogenesis [ids. 7, ft, 16). Within the last year, a new industrial carcinogen has been identified1. Vinyl chloride, the monomer used for production of the common polymer polyvinyl chloride, has been held responsible ior at least 22 angiosarcomas of the liver in exposed wor- ' Supported by US1MIS Center Grants KS 0092N aiul GM 111443, and Research Grant HO 02552. ** Km t Ihrschhorn, M. J) (to whom reprint requests should be addicssed) is a Career Scientist 0/ ^^llealth Research C ouncil of the City nf New York, I -51 3 SL 041813 164 A. IJUCATMAN el ul. kerb10. There is additional suggestion from both animal and human data of an in creased risk for cancers of the central nervous system, the respiratory system, and of the blood forming tissues1"' 'L,6. With an association between clastogenicity and carcinogenesis established for other agents, it appeared that this new carcinogen might also cause chromosome damage. We therefore undertook a blind study of lymphocyte chromosomes from vinyl chloride-exposed workers and nonexposed controls. EXPOSURE TO VINYL CHLORINE The 11 subjects studied were male workers who had received repeated exposure to vinyl chloride in an upstate New York polyvinyl chloride polymerization plant. Major exposures came from leaks of un reacted vinyl chloride gas, fumes from poly vinyl chloride slurry, and from polymerization reactor cleaning operations. Reactor cleaning involved skin contact to polyvinyl chloride and inhalation of vinyl chloride gas residues; this operation accounted for the most intense exposures. Duration of recurrent occupational exposure in the n men ranged from 4-28 years with an average of 15 years (Table I). There is no record of ambient gas levels in the factory, but it is assumed that these must have exceeded 500 ppm at times, based on reports of odor detection, dizziness, and headaches. TABLE I AGE AND DEGREE OE VINYL CHLORIDE EXPOSURE OP SELECTED CASES AND CONTROLS Case No. Age Years exposed Celts Control examined No Age Years Cells exposed exarm. I 6l 23 50 1 43 2 50 28 50 t 3 3 47 19 50 3 4 44 11 5 4 29 5 40 !3 5 5 28 6 39 17 50 6 28 7 36 !7 50 7 27 S 36 12 50 8 20 9 33 11 50 9 19 IO 32 10 50 10 8 I I 25 4 50 Total 443 65 550 Average 40 !5 50 271 27 0 5 0 5*> 0 50 0 5 0 5" 0 50 0 50 0 5 0 50 0 50 0 500 0 50 Of the 10 healthy male controls, 4 were from within the same factory population, without known vinyl chloride exposure. Nevertheless, as long-term employment im plied the possibility of some vinyl chloride exposure, albeit at low levels, we also selected 6 older controls from outside the factory environment. The average age of the controls was 27, of the subjects 40. METHODS The chromosome studies were performed on cultures of peripheral blood lym phocytes incubated at 37 for 65-68 h with phytohemagglutimn (Wellcome). Harvests were performe(Recording to micronicthod modifications of procedures first described CHROMOSOME DAMAGE BY VINYL CHLORIDE 165 by Moorhead cl al.M. Media used in this study came from a single batch (Gibco) in order to insure uniform pH and other culture conditions. In each individual studied, 50 metaphases were counted directly under the microscope at 1600X. The A,B,D,E,F,G, and Y chromosomes were evaluated se parately so far as possible. Suspected aberrations and some normal metaphases were photographed; karyotypes were performed where helpful. From the total of 1050 cells examined there were 281 photographs taken and 140 karyotypes made from the photographs. | Confirmed aberrations were classified according to two systems in order to discern if a pattern of breakage might exist. The system of Buckton et al.3 and Court Brown6 has three categories of aberrations: B, cells with simple aberrations such as breaks and gaps; Cu, cells with ``unstable'' chromosome changes such as fragments, dicentrics, and rings; C, cells with "stable" chromosome changes such as monosomies, trisomies, deletions, and exchanges. The distinction between cells with stable and unstable aberrations is related to their tendency to eitlier remain in or disapjiear from the circulation. The system of Hikschhorn and Cohen11 differs in two respects. It considers all breakage, including the total number of breaks in cells with more than one aber- TAHLli U VINYL CHLORIDE EXPOSURE AND CHROMOSOME ABERRATIONS {CLASSIFICATION SYSTEM OF COURT JiKOWN* AND llUCKTON ft at.3) t'asr No. 1 i 3 t 5 h 7 8 <> lo JE Total Comparison 7 8 6 3 5 3 5 8 4 8 5 62 0 5-t>4( i:1-91! 2 2 O 1 1 O 2 2 O 4 3 17.0 i-55("-29) t = 1.84 t = 2863 0.1 > p > 0.05 p -- O.OI 6V 3 3 3 3 3 O 2 6 4 5 5 37 0 3-3&(t 63) 1 = 0 56 0.6 > p > 0.5 Control No. 1 2 3 4 5 6 7 8 9 10 Total Meaii( { S.D.) 5 4 4 5 2 5 5 5 4 5 44.0 +-4fi 0 97) l 0 0 0 1 0 0 E 0 0 30 -3(-i 0 48) 3 7 1 3 6 3 3 2 O I 29-0 2 go( j- 2.18) * B, Breaks ami gaps, Cu, "unstable" changes (fragments, dicentrics, rings); Ca, "stabje!!changes osomv. trisomy, deletions, exchanges}. SL 041814 lb() A. DUCATMAN el III. ration, and it gives weighted consideration to those aberrations which are tire ap parent result of two "hits". Therefore, complex breakage (f ) such as rings, dicentrics, and exchanges is counted twice in the total, whereas simple breaks and deletions {$) are counted once. RESULTS Gaps and breaks were the predominant aberrations among the 1050 cell-, examined, as seen in the B column of Table II. Subjects have nonsignificant increases of such aberrations when compared to controls by a f-test for comparison of the means (0.1 >P>o.o5), and marginally significant increases by an P-ratio for com parison of the variances {0.05 > P > 0.01). The difference in cells with stable aber rations, or those that Buckton el al. and Court Brown found to persist in circulating lymphocytes-6, is also nonsignificant. In our study, stable aberrations were usually cells with random chromosome loss. However, cells with unstable aberrations were observed significantly more frequently in the cultures from exposed workers: t, P = 0.01; F, 0.01 >P >0.001. Table fil focuses on breaking events only. The total of simple breaks, including TAHI.K 111 CHROMOSOME UREAK EVENTS (IN 50 CELLS fl N DIVIDUAL) AND VINYL CHLORIDE EXPOSURE (SYSTEM of Hirschhorn and Cohen) Case No. 1 2 3 4 5 6 7 8 9 10 11 Total Moan(J_ S.D.) Comparison 5* C Total break events (S { 2C) 4 2 2 I O 0 3 1 4 I 3 21 l-9l(i-44) 3 3 0 2 1 0 1 2 O 4 5 22 J.oo(:t 1,67) Io 8 2 a 2 o 7 5 4 9 <>5 5-9J( I y<n) t -- 1.12 t -- 2.80 t - 1.75 0 3 > p > 0.2 0.02 > p 0.01 0.02 > p > 0.01 Control No. 1 2 3 4 5 6 7 8 9 10 Tutul Mean l 13 l>) 1.3 1 -3{ i 0.95) o 4u( ] 0.70} 21 2 io( [ 2 02) * S, Single hit events (breaks, deletions); C, complex events (rings, dicentrics, exchanges). CHROMOSOME IIAMAOE BY VINYL CHLORIDE 167 those from multiaberrant cells, is increased but not significantly in the subjeets. This is shown in the .S' column. Complex breaking events in C are significantly more frequent in those exposed, with: l, 0.02 > P > 0.01; and P, 0.01 >P>o.ooi. The total of breaking events {S + 2C) shows a similarly significant increase in the subjects (/, 0.02 >P>o.oi; F, 0.01 >P >0.001). Not included in the charts is a combined total of all breaks, gaps, and deletions. There was a marginally significant difference 111 these with t, 0.05 >P >0.02; F was not significant. Exaggerated secondary constrictions of the No. 9 chromosome were easily noted liecause of their high degree of visibility. The average subject was observed to have j.qi ( + 2.47 S.D.) and the average control had 2.00 (ty 1.63) in the 50 cells examined jier individual. Significance was marginal at most, P<o.o5 by a f-test; F not sign ificant. I discussion There are obvious perils in drawing strong conclusions from small samples, and it would clearly be preferable to have an age-matched control group despite experimental evidence that age is generally unrelated to chromosome changes other than chromosome toss6. Also, the relatively high degree of breaks and gaps in controls as well as subjects is somewhat disconcerting, although subjects do have more. Within these limitations, it is clearly indicated that chronic high level exposure to vinyl chloride is clastogenic. Much of the increased damage was of the unstable variety as defined by Buckton et al.3. The difference between cases and controls is most evident for unstable aberrations in general and fragments in particular. Hirschhorn and Cohen's system, which is a better overall index of chromosome damage, shows that long-term exposure is associated with an evidently significant in crease in all breaking events. In this small sample it lias not been possible to correlate the degree of damage with either the degree of exposure or with vinyl chloride disease symptoms. The former may never be possible, as the best estimate of total exposure for any individual is only a crude guess. In general, the majority of subjects selected from this factory exhibited increased breakage rates, and those with the shortest duration of exposure showed damage similar to those with the longest duration. Stating that chronic vinyl chloride exposure almost certainty damages chro mosomes leaves us with two questions. First, can chromosome damage studies be at all predictive of environmentally induced carcinogenesis P Ionizing radiation and now vinyl chloride exposures have been studied for genetic properties after association with induced neoplasms. If we can reverse the order of events, perhaps in animal studies, we will know with more certainty if chromosome examination has an im portant role to play in predicting environmental carcinogenesis. The second question is: what kind of genetic studies should lie done with vinyl chloride ? We feel strongly that chromosome study of individual concerned fathers is unwarranted. The degree of damage discovered here is unlikely to yield meaningful findings in any single individual. Women are not employed in polymerization work. However, they may work in polyvinyl chloride processing industries, in which some exposure to unreacted vinyl chloride may occur. This could be important in light of experimental findings of transplacental carcinogenesis1. SL 041815 l()8 A, DUCATMAN el ill. Carefully controlled examination of larger groups with vinyl chloride and polyvinyl chloride exposure are obviously needed, first to provide the larger data base necessary to confirm clastogenicity for vinyl chloride exposure, and also to evaluate dose-response relationships. Mutagenicity is being assessed in other test systems, including bacterial studies, insect and animal studies, along with in vitro chromosome studies. The case for studying other genetically susjiect chemicals is now stronger than ever. REFERENCES 1 Bender, M. A., and P. C. Gooch, Fersistent chromosome aberrations in irradiated human subjects, Kadtat. Res., 16 (1962) 44-53 2 Bloom, A. D.. and J. H. Tjio, In vivo effects of diagnostic X-irradiation on human ctiruiim somes, New Engl. J. Med., 270 (1964) 1341-1344. 3 Buckton, K. E., F. A. Jacobs, W. M. Court Brown and K. Doll, A study of the chrouwixmiu damage persisting after X-ray therapy for ankylosing spondylitis. Lancet, ii (1962} 676-682. 4 Creech, J. E., and M. N. Johnson, Angiosarcoma of liver hi the manufacture of vmil chforide, J. Occup. Med., 16(1974) 150-151. 5 Court Brown, W. M., Human population cytogemcs, in A. Neuberger and E. L. Tatcm (Eds.}, Frontiers of Biology, Vol, V, North-Holland, Amsterdam, 1967. pp. ip 6 Evans, H. J., Population cytogenetics and environmental factors, in Patricia A. JakiiiW. H. Price and Pamela Eaw (Eds.), Human Population Cytogenetics, Williams and WilkmBaltimore, 1970. PP- 191-216. 7 Eorni, A. M., A. Coppellini, E. Paciitco and E. C. Vigliani, Chromosome changes and then evolution in subjects with past exposure to benzene, Arch. Environ. Health, 23 (197O 3W5 l*'1 B Eorni, A. M., E. Pacifico and A. Limonta, Chromosome studies in workers exposed t benzene or toluene or both. Arch. Environ. Health, 22 (1971) 373"37- 9 German, J. (Ed ), Chromosomes and Cancer, Wiley, New York, 197410 Heath, C., and J. Wagoner, Report of a Working Group on Piny/ Chloride, Lyon, 24 23 Jim iyj4, 1AKC No. 74/005, World Health Organization, pp. 18-19. 11 Hirschhorn, K., and M. M. Cohen, Drug-induced chromosomal aberrations, Ann. A . Y. -loui Set., 151 (1968) 955"9^712 Hoopingamer, R. and A. W. Bloomer, Lymphocyte chromosome analysis of pi-stnnk exposed individuals, in 7th hit. Congr. Plant Protection, Parts, 1970, p. 772. 13 Maltoni, C., and G. Lefemine, Carcinogenicity bioassays of vinyl chloride, 1. lieseaith pki1 and early results, Environ. Res., 7 (1974) 387-405. 14 Moorhead, P. S., P. C. Nowell, W. J. Mellman, D. M Hattips and D. A. Hunglri Chromosome preparations of leukocytes cultured Irom human peripheral blood, Exp. Oil lh 20 (i960) 613-616. 15 Nicholson, W. J., 1C C. Hammond, H Seidman and I. j. Sklikofk, Mortality expci ii iut a cohort of vinyl chloride-polyvinyl chloride workers, Ann. N.Y. Acad. Set., 255 (1975I -** 230. 16 Tough, I. M., and W. M. Court Brown, Chromosome aberrations and exposure to -mil"*;' benzene. Lancet, i (1965) 684. 17 Y'oder, J., M. Watson and W. W. Benson, Lymphocyte chromosome analysis ol agnciilim* workers during extensive occupational exposure, Millation Res , 21 (i973) 335-34- \htlation Research, 31 (1975) 169-173 , i-.lsevier Scientilic Publishing Company, Amsterdam--Punted in The Netherlands lb<) AN EVALUATION OF THE MUTAGENIC POTENTIAL OF AN AEROSOL SPRAY ADHESIVE IN THE RAT HUBERT E. OSTEKBEKG, JAMES C. MURPHY. GEORGE W. BIEKBUWEK ami FRANCES MORELAND SAIJRO* litvtsion of Biological Science, Bureau of Biomedical Science, Consumer Product Safety ( owmimon. \\ mhtngton, D.C. 20207 and *Diviston of Toxicology, Pood and Drug Administration, Department <) Health, Education and Welfare, Washington, D.C. 20204 (U.S.A.) iltvceived January 8th, 1975) 'I v mercially available aerosol adhesive formulation was examined for its im enfiat in adult rat bone marrow cells. Groups of rats were placed in a h" .lation chamber and were exposed to 10, 15, or 20 g of the aerosol once 'Lit * took place 21 times during 33 days. Treated and control animals itc iiicine intraperitoneally 2 h prior to sacrifice. Slides of bone marrow elb Mu, \.unined for the presence of chromosome and chromatid aberrations. Although no statistically significant increases in grossly visible chromosomal damage "`re produced, significant reductions in the mitotic index were obtained which were indicative of a cytotoxic effect. Acute toxicity was manifested by the occurrence of ataxia, eye and nasal irritation, hyperexcitability, and clonic convulsions; these mn t symptoms were dose dependent. IMHUIUJCTION b* i()73. a preliminary report on an aerosol adhesive was forwarded to the 1 *uisunier Product Safety Commission by Dr. j. Rodman Seely, Professor of Pediairns and Molecular Biology, University of Oklahoma Medical Center. Tins report indicated a causal connection between exposure to the aerosol adhesive and chromo'*une damage that might lead to genetic birth defects. The chromosomal analysis of peripheral blood drawn from exposed human subjects indicated a statistically in* leased incidence of visible chromosome damage as evidenced by breaks and gaps. Nn other aberrations were reported. Upon review of these data by academic, iniiKrial, and governmental experts, the Consumer Product Safety Commission alerted iln- general public to this potential hazard. Although the association between the uniMil adhesive and chromosome damage had not been positively established, the 'irength of the association based upon the available information and the potential ll,r 'tt'riiius personal injury to exposed humans led the Commission to conclude that dn distribution of these products for household use presented an imminent hazard 04j8 ^