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R&S 106681 BIO-MEDICAL RESEARCH DOCUMENT DESCRIPTION FORM 63 68 69 76 Duplicate in all cards:--> nnnnnt; year as-1961- File number [Right justify [Numeric only] Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded by one blank space 20 21 DVCA-TAi'l/y, A- HtSSt. K 40 41 Ssz/n'JAS . / J --S 77 78 l/<^ Sub-Index Code 60 61 62 11 12 13 Title of Report;, end with space-hyphen-hyphen-space. Follow with Index Terms, separated from each other with comma-space. Avoid other punctuation; do not abbreviate. ,t ,, 61 62 21 22 23 24 Source (Journal, Vol., Number, Pages,Date) 12 3/ , / f '-/A'f/ fp f~) 61 62 31 32 Brief Summary 12 10 SUMMARY: 61 62 61 62 63 64 000008? -....... Cka> et?L_ Mulatiaa Research, 31 (1975) 163-168 (') Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands 163 VINYL CHLORIDE EXPOSURE AND HUMAN CHROMOSOME Xberrations** ALAN DUCATMAN. KURT HIRSCHHORN* * and TRYING J. SELIKOFF Environmental Sciences Laboratory, Department of Coumumitv Medicine and the Division of Medical f Genetics, Department of Pediatrics, Mount Sinai Scioto/ of Medicine oj the City University oj New \ York, New York, N.Y. coozg (U\S,A.) ! (Kccoived October 25th, 1974) i (Ucvision received January 15th, 1975) R&S 106682 SUMMARY Examination of lymphocyte cultures from n vinyl chloride polymerization workers and xo controls revealed a significantly higher incidence of aberrations in the exposed population. Most of the excess damage was of the "unstable" varietv 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 the early 1960's it has been ciear that chromosome morphology examination could uncover evidence of genetic damage in man1-'. The earliest of these studies discovered the now well-known link between a carcinogenesis and chromosome changes (for recent review, see ref. 9). These were il soon followed by the discovery that a number of chemicals are clastogenic (chromosome breaking), notably alkylating agents and cytostatic drugs, and DNA base ^ analogues6. 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 had revealed the probable clastogenicity of some pesticides and herbicides16-17. Industrial exposure to benzene has also been implicated by a number of investigators in both leukemogenesis and clastogenesis (refs. 7, 8, 16). Within the last year, a new industrial carcinogen has been identified1. Vinyl chloride, the monomer used for production of the common polvmer polwinv! chloride, has been held responsible for at least 22 angiosarcomas of the liver in exposed wor- * Supported by USl'HS Center Grants ES ooo-S and GM 19443. and Research Grant HD 04334. * Kurt Hirschhom. M D. (to wnom reprint requests should bo addressed) is a Career Scientist of the Health Research Council of the City of Mere York, 1-3:3. 164 A. DBCATMAN Ct al. kers10. 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"-13-11. 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 unreacted 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 II 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 OF VINYL CHLORIDE EXPOSURE OF SELECTED CASES AND CONTROLS Case No. Age Years exposed Cells Control Age examined No. Years Cells exposed examined I 6l 23 5 1 43 O 5 2 5 2S 5 2 30 O 5 3 47 19 5 3 29 O 5 4 44 II 5 4 29 O 5 5 40 !3 50 5 28 O 50 6 39 17 50 6 28 0 50 7 36 17 5 7 27 0 5 S 36 12 5 8 20 0 50' 9 33 II 5 9 19 0 50 IO 32 10 5 IO 18 0 50 II 25 4 5 3J *> 0) Total 443 165 550 Average 4 15 50 271 0 500 27 0 5- O Of the 10 healthj' male controls, 4 were from within the same factory population', O) C7> without known vinyl chloride exposure. Nevertheless, as long-term employment im 00 plied the possibility of some vinyl chloride exposure, albeit at low' levels, we also CO 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 phytohemagglutinin (Wellcome). Harvests were performed according to micromethod modifications of procedures first'described T CHR01I050ST by Moorhead e order to insure 1 In each ii microscope at 1 parately so far a photographed; 1 cells examined t; photographs. Confirmed discern if a patt Court Brown5 ' such as breaks . fragments, dicen monosomies, tris stable and unsta disappear from t The svsten all breakage, incl TABLE II '-NYL CHLORIDE E> Brown5 and Buck Case No. E m 2 ^Ws 36 43 55 63 f5 88 94 IO 8 IX 5 Total 62. Mean(-S.D.) 5- Comparison t0. Control No. 1 2 3 4 5 6 7 8 9 IO Total Meant S.D.) 5 4 4 5 2 5 5 5 4 5 44-0 4-4 1 B. Breaks and gaps (monosomy, trisomy. CATMAN et al. luman data of an inratory system, and of n clastogenicity and this new carcinogen >ok a blind study of ers and none.xposed ed repeated exposure 'olymerization plant, as, fumes from poly operations. Reactor tion of vinyl chloride posures. Duration of 4-28 years with an levels in the factory, \es, based on reports CONTROLS mined factory population, rm employment imlow levels, we also e average age of the ripheral blood lym/elleome). Harvests lures first described CHROMOSOME DAMAGE BY VINYL CHLORIDE l65 by Moorhead et al.". Media used in this study came from a single batch (Gibeo) iij order to insure uniform pH and other culture conditions. In each individual studied, 50 metaphases were counted directly under the microscope at 1600 x. 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.* and Court Brown* has three categories of aberrations: B, cells with simple aberrations such as breaks and gaps; C,,, cells with "unstable" chromosome changes such as fragments, dicentrics, and rings; Cs, 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 either remain in or disappear from the circulation. The system of Hirschhorn and Cohenl1 differs in two respects. It considers all breakage, including the total number of breaks in cells with more than one aber- TABLE II vinyl chloride exposure and chromosome aberrations (classification system of Court Brown1 and Buckton et at.1) Case No. B I 2 3 4 5 6 7 S 9 10 IZ Total Mean(S.D.) Comparison 7 3 6 3 5 3 5 8 4 8 5 62.0 5.64(^1.91) 2 2 0 I X 0 2 2 0 4 3 17.0 r-55(zt !-29) t = 1.84 t = 2.S63 0.1 > p > 0.05 p = 0.01 3 3 3 3 3 0 2 6 4 5 5 37.0 3-36(t.63) t = 0.56 0.6 > p > 0.5 Control No. I 2 3 4 5 6 7 8 9 10 Total Mean(4-S.D.) 5 4 4 5 2 5 5 5 4 5 44.0 4-4( o-97) I O O O I O O I 0 0 30 o-3( 0.4S) 3 7 1 3 6 3 3 2 O T 29.0 2.90(4; 2.18) 1 B, Breaks and saps: C,,, "unstable'' changes (fragments, dicentrics, rings); C, "stable" changes (monosomy, trisomy, deletions, exchanges). f 166 A. ducatma.n cl ,d. ration, and it gives weighted consideration to those aberrations which are the ap parent result of two "hits". Therefore, complex breakage (C) such as rings, dicentrics, and exchanges is counted twice in the total, whereas simple breaks and deletions (S) are counted once. RESULTS Gaps and breaks were the predominant aberrations among the 1050 cells examined, as seen in the B column of Table II. Subjects have nonsignificant increases 33 of such aberrations when compared to controls by a <-test for comparison of the fio means (0,1 >P>0.05), and marginally significant increases by an F-ratio for com CO 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 O O) lymphocytes'* *, is also nonsignificant. In our study, stable aberrations were usually o> cells with random chromosome loss. However, cells with unstable aberrations were 00 <J1 observed significantly more frequently in the cultures from exposed workers: t, P = 0.01; F, 0.01 >P>0.001. Table III focuses on breaking events only. The total of simple breaks, including TABLE-III CHROMOSOME BREAK EVENTS (IN 50 CELLS/INDIVIDUAL) AND VINYL CHLORIDE EXPOSURE (SYSTEM of Hirschhorn and Cohen) Case No. S* c* Total break events (S+iC) I 2 3 4 5 6 7 8 9 IO II Total Mean(S.D.) Comparison 4 2 2 I O O 3 I 4 I 3 21 i-9i(i-44) t = 1.12 0.3 > p > 0.2 3 3 O 2 I O 2 2 O 4 5 22 2.00(i; I.67) 10 8 2 5 * O 7 5 4 9 J3 65 5-9l(3-9h t = 2.So t = 2.75 > > > >0.02 p 0.01 0.02 p 0.01 Control No. I 2 3 4 5 6 7 S 9 10 Total Mean(~S.D.) 3 r 1 0 2 2 0 1 2 1 13 1.30(^0.95) I O O O 2 O O I O O 4 0.40(^0.70) 5 1 1 0 6 2 0 3 2 I 21 2.10( 2.02) * 3. Single hit events (breaks, deletions): C. complex; events (rings, dicentrics, exchanges). CHROMOSOME DAM. those from multiai is shown in the S cc in those exposed, breaking events ( (t, 0.02 >P >0.01 total of all breaks, in these with t, o.c Exaggerated because of their hi 3-91 (2.47 S.D.) per individual. Sig ificant. DISCUSSION There are o and it would clea: experimental evidt than chromosome 1 as well as subject Within these limit vinyl chloride is c variety as dleeg^l most evidenl Hirschhorn and damage, shows tha crease in all breaki In this small with either the de former may never 1 is only a crude gue exhibited increased showed damage sin Stating that mosomes leaves us all predictive of en\ vinyl chloride expc with induced neop. studies, we will kn portant role to plat The second q chloride ? We feel s unwarranted. The t findings in any sing However, they mat exposure to unreact experimental findin / DUCATMAN ei til. iuSKe the aprings, dicentrics, md deletions (S) ' the 1050 cells aificant increases mparison of the F-ratio for comvith stable aberlist in circulating ons were usually aberrations were icposed workers: br-aks, including t_. POSURE (SYSTEM exchanges). CHROMOSOME DAMAGE BY VINYL CHLORIDE 167 those from multiaberrant cells, is increased but not significantly in the subjects. 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 F, 0.01 >P >0.001. The total of 1 breaking events (S + 2C) shows a similarly significant increase in the subjects I (/, 0.02 >P >0.01; F, 0.01 >F >0.001). Not included in the charts is a combined i total of all breaks, gaps, and deletions. There was a marginally significant difference : in these with l, 0.05 >P>0.02; F was notsignificant. Exaggerated secondary constrictions of the No. 9 chromosome were easily noted * because of their high degree of visibility. The average subject was observed to have 3.91 (2.47 S.D.) and the average control had 2.00 ( 1.63) in the 50 cells examined per individual. Significance was marginalat most, P < 0.05 by a /-test; F not signj ificant. i DISCUSSION I 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 loss1. 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 has 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 certainly damages chro mosomes leaves us with two questions. First, can chromosome damage studies be at all predictive of environmentally induced carcinogenesis ? Ionizing radiation and now i 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 j 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 be 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 carcinogenesis13. R&S 106687 rf1 168 A. DUCATJIAX et al. 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 suspect chemicals is now stronger than ever. REFERENCES 1 Bender, M. A., and P, C. Gooch, Persistent chromosome aberrations in irradiated human subjects, Radiat. Res., 16 (1962) 44-53. 2 Bloom, A. D., and J. H. Tjio, In vivo effects of diagnostic X-irradiation on human chromo somes, A'eu' Engl. J. Med., 270 (1964) 1341-1344. 3 Buckton. K. E.. P. A. J acobs, W. 31. Court Brown and R. Doll, A study of the chromosome damage persisting after X-ray therapy for ankylosing spondylitis, Lancet, ii (1962) 676-6S2. 4 Creech, J. L., and 31. X. Johnson, Angiosarcoma of liver in the manufacture of vinyl chloride, J. Occup. Med., 16 (1974) 150-151. 5 Court Brown, W. M,, Human population cvtogenics, in A. NeUberger and E, L. Tatum (Eds.), Frontiers of Biology, Vol. V, Xorth-Holland, Amsterdam, 1967, pp. 1-31. 6 Evans, H. J., Population cytogenetics and environmental factors, in Patricia A. Jacobs, \V. H. Price and Pamela Law (Eds.), Human Population Cytogenetics, Williams and Wilkins, Baltimore, 1970, pp. 191-216. 7 Forni. A. 31., A. Coppellini, E. Pacifico and E. C. Vigliani. Chromosome changes and their evolution in subjects with past e.Nposure to benzene. Arch. Environ. Health, 23 (t97i) 385-391. 8 Forni, A. M.. E. Pacifico and A. Limonta, Chromosome studies in workers exposed to benzene or toluene or both. Arch. Environ. Health. 22 (1971) 373-378. 9 German, J. (Ed.), Chromosomes and Cancer. Wiley. New York, 1974. to Heath, C., and J. Wagoner, Report of a Working Group on Vinyl Chloride, Lyon, 24-25 June, 1974, IARC No. 74/005, World Health Organization, pp. 18-19. ti Hirschhorn, K., and 31. 31. Cohen, Drug-induced chromosomal aberrations, Ann. A'. Y, Acad. Sci,, 151 (1968) 955-987. 12 Hoopingamer, R. and A. W. Bloomer, Lymphocyte chromosome analysis of pesticide exposed individuals, in 7th Int. Cangr. Plant Protection, Paris. 1970, p. 772. 13 Maltoni, C., and G. Lefemine, Carcinogenicity bioassays of vinyl chloride, I. Research plan and early results, Environ. Res., 7 (1974) 387-405. 14 3Ioorhead, P. S.. P. C. Nowell, W. i. 3Iellman, D, M. Battips and D. A. Hungerford, Chromosome preparations of leukocytes cultured from human peripheral blood. Exp. Cell Res., 20 (i960) 613-616, 15 NtcHOLSO.x, W. J., E. C. Hammond, H. Seidman and I. J. Selikoff, 3fortaIity experience of a cohort of vinyl chloride-polyvinyl chloride workers, Ann. A'.Y. Acad. Sci,, 255 (1975) 225230. 16 Tough, I. 31., and W. 3f. Court Brown, Chromosome aberrations and exposure to ambient benzene, Lancet, i (1965) 684. 17 Yoder, J., 31. Watson and W. W. Benson, Lymphocyte chromosome analysis of agricultural workers during extensive occupational exposure. Mutation Res.. 21 (1973) 335-340.