Document VJkp0YJeaOQyNKxr6Qer8QEwN

Mutation Research, 31 (1975) i(i,v-il)S i> Klsevier SciehtiJic Rublislun^ Company, Amsterdam Printed m I'hc Netherlands VINYL CHLORIDE EXPOSURE AND HUMAN CHROMOSOME ABERRATIONS* ALAN DIJCATMAN, KURT HIRSCHHORN** anl> IRVING j. SKUKOI-F Environmental Sciences Laboratory, Department of Community Medicine and the Division of Medical in tuttcs. Department of Pediatrics, Mount .Stnai School of Medicine of the City University of New York, New York, N.V. X0029 (U S.A.) __ (Received October 25th, 1974} ^ (Revision received January 15th, 1975) I fOTICl: This material may >>* protected by copyright la* I (Title 17 U.S. Code) SUMMARY 0 Examination of lymphocyte cultures from II vinyl chloride polymerization workers and 10 controls revealed a significantly higher incidence of alienations in the exjaised 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 file pioneering radiation studies of the early 1960's it has been clear that chromosome morphology examination could uncover evidence of genetic damage in man1"1. The earliest of these studies discovered the now well-known link between carcinogenesis and chromosome changes (for recent review, see ref. cj). These were soon followed by the discovery that a number of chemicals are clastogenic (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 had revealed the probable clastogenicity of some pesticides and herbicides1"*1'. Industrial exposure to benzene lias also been implicated by a number of investigators in botli leukemogenesis and clastogenesis (refs. 7, 8, 16). Within the last year, a new industrial carcinogen has been identified*. Vinyl chloride, the monomer used for production of the common polymer polyvinyl chloride, has been held responsible for at least 22 angiosarcomas of the liver m exposed wor- ( * Supported by USRMS Center Grants LS 00928 and GM 19443, and Research Grant HI) 025^2. ** ICtirt Hirschhoru, M. J) (to whom reprint requests should be addressed) is a Career Scientist of the Health Research Council of the City of New York, I-513. SI* 102050 T 16.4 A. DUCATMAN et til. chromosome damage by vinyl chloride kers10. There is additional suggestion funn 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 tissues10'13-15. Will) an association between clastogenicity and carcinogenesis established for other agents, it appeared that 111 is 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 bad received repeated exposure to vmyi 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 uf recurrent occupational exposure in the ri men ranged from 4-28 years with an average of 15 years (Table 1). 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 OK VINYL CHLORIDE EXPOSURE OK SELECTED CASES AND CONTROLS Case No. Age Years exposed Cells Control examined No. Age Years Cells exposed examined I 6x *3 50 I 43 2 5 28 50 2 3'J 3 47 19 5 3 29 4 44 11 50 4 29 5 40 13 50 5 28 6 39 *7 50 6 28 7 36 17 50 7 -*7 8 3& 12 50 8 20 9 33 11 5 9 19 10 31 10 50 IO 18 it *5 4 50 Total 443 165 55 Average 40 15 5 271 27 0 5 0 50 0 50 0 5< 0 50 0 0 50 0 50 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 370 for 65-68 li with phytohemagglutinin (Wellcome). Harvests were performed according to micromethod modifications of procedures first described by Moorhead et al.1*. 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 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 celts 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 Brown5 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 either remain in or disappear from the circulation. The system of Hirschhorn and Cohen11 differs in two respects. It considers all breakage, including the total number of breaks in cells with more than one aber- r utui n VINYL CHLORIDE EXPOSURE AND CHROMOSOME ABERRATIONS (CLASSIEICATION SYSTEM OR COURT Drown5 and Buckton el at 3) No. 1 2 \ 4 "i (> 7 8 9 10 11 Total /i* 7 S 6 3 5 3 5 8 4 8 5 62.0 5.64(4191) 2 2 O 1 I O 2 2 0 4 3 17 0 3 3 3 i 3 O 2 6 4 5 5 37 0 29) 3.36(41.63) Comparison t = 1.H4 / = 2.863 0,1 > p > 0.05 p -- 0.01 t = 0.56 0.6 > p > 0.5 Control No. 2 3 4 5 6 7 8 9 to Total Meaofy S D.) 5 4 4 5 2 5 5 5 4 5 44.0 4 4( 0 97) j O O O I O O I O O 3.0 0 30(4 0.48) 3 7 1 3 6 3 3 2 0 1 29 0 2 t)o( 4 2.18) 5 H, Breaks and Raps, C,,, "unstable" changes (fragments, dicentrics, rings), CB, "stable" changes (monosomy, trisomv. deletions, exchaneesl. SL 102051 5(>(l A. DUCATMAN H III. ration, and it gives weighted consideration to those aberrations which are the 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 (S) are counted once. RESULTS (iaps and breaks were the predominant aberrations among the 1050 celb examined, as seen tn flit; H column of Table II. Subjects have nonsignificant increases of such aberrations when compared to controls by a /-test tor comparison of the means (0.1 >P>o.o5), and marginally significant increases by an /'-ratio for com parison of the variances (0.05 > P > 001). The difference in cells with stable aber rations, or those that Hucktun et al. and Court Brown found to persist in circulating lymphocytes5'1, 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 /, P = 0.01; F, 0.01 > P > 0.001. Table III focuses on breaking events only. The total of simple breaks, including taiii.i-: hi 1 IIKOMOSOME HREAK EVENTS (IN 50 CELLS/[N 1)1 VUJUAL) AND VINYL CHLORIDE EXPOSURE (SYSTEM op Hirschhorn and Cohen) Case No. 1 2 3 4 5 6 7 8 9 [O 11 Tot.il MisuidST).) i'ompanson S* 0 Total break events fS ( ;C) 4 2 2 [ 0 0 3 f 4 1 3 21 i-9i(i 44) 3 3 0 2 t 0 2 2 0 4 5 22 2.0O(;t 1 -67) J0 8 2 5 2 O 7 5 4 9 13 f>3 5 9t( i 3-yO t = l 12 t - 2.80 / ,= 2.75 0 j > p > a 2 O 02 > p > 0.01 0.02 > p > O.OI Control No. 1 2* * 4 S (i 7 8 q 10 Total Mwm( | S.D.) 3 r i 0 i 2 0 j 1 1 13 <30(10.95) 1 O O O 2 O O ] O O 4 <M1 i 0 7`>) 5 0 6 2 0 3 2 1 2j ri( 1 2.02) to tr* M O (XJ O tcon 5 S, Single hit events (breaks, deletions); C, complex events (ruifjs, dicentrics, exchanges). chromosome damage by vinyl chloride 167 those front multiaberrant cells, is increased hut 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: /, 0.02 > P > o.oi; and F, o.oi >P>o.ooi. The total of breaking events (-S' -f- 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 f, 0.05 > P >0.02; F was not significant. Exaggerated secondary constrictions of the No. 9 chromosome were easily noted In-cause of their high degree of visibility. The average subject was observed to have jqi ( f 2.47 S.D.) and the average control had 2.00 (-( 1.63) in the 50 cells examined [>er individual. Significance was marginal at most, P<o.o5 by a /-test; F not sign ificant. IIISCUSSION There are obvious perils in drawing strong conclusions from small samples, ,md it would clearly be preferable to have an age-matched control group despite vxjiemuental evidence that age is generally unrelated to chromosome changes other than chromosome loss5. 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 must evident for unstable aberrations in general and fragments in particular. Hi itsch horn 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 witli 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 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 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, 111 which some exposure to unreacted vinyl chloride may occur. This could be important in light of experimental findings of transplacental carcinogenesis15. l(t A. DUCATMAh' el ill. Carefully controlled examination of larger groups with vinyl ctiloride 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 hunKin subjects, Raduit. Res., 16 (1962) 44-53- 2 Bloom, A. D., and J. H. Tjio, In vivo effects of diagnostic X-irradiation on human chromu somes, New Engl J. Med., 270(1964) 1341-1344. 3 Bulkton, K. E., P. A. Jacobs, W M. Court Brown and R. Doll, A study of the chromosunu damage persisting after X-ray therapy for ankylosing spondylitis. Lancet, li (1962) 676-6S2 4 Creech, J. L, and M. N. Johnson, Angiosarcoma of liver m the manufacture ol vnnl chloride, J. Occup. Med , 16 (1974} 150-151. 5 Court Brown, W M., Human population cytogenics, in A. Nkuberger and E. L. Tati si (Eds.). Frontiers oj Biology, Vol. V, North-Holland, Amsterdam, 1967, pp. 1-3*- 0 Evans, H. j., Population cytogenetics and environmental factors, in Patricia A Jacob* W. H. Price and Pamela Law (Eds.), Human Population Cytogenetics, Williams and WilkinBaltimore, 1970, pp. 191-216. 7 Form, A. M., A. Coppellini, E. Pacjkico and E C. Vigliam, Chromosome changes ami linn evolution in subjects with past exposure to benzene, Arch. F.nviron. Health, 23 (1971) 385 pu 8 Eorni, A. M., E. Pacieico and A Limonta, Chromosome stiubes 111 workers exposed l benzene or toluene or both, Arch. Lnvtron Health, 22 (1971) 373-J78. 9 tiKRMAN, J (Ed.), Chromosome* and Cancer, Wiley, New York, 197410 Heath, C , and J. Wagoner, Report of a Working Group on Vinyl Chloride, Lyon, 24-25 Jitn, i<4J4, I ARC No. 74/005, World Health Organization, pp. 18-19. 11 ItiRSCHhorn, K.( and M. M. Cohen, Drug-induced chromosomal aberrations, A nn. A' V Inu Sr*-, 151 (1968) 955-V8712 Hoopingamer, It. and A. W, Bloomer, Lymphocyte chromosome analysis of pesLimU exposed individuals, in 7th Jnt. Congr, Plant Protection, Parts, lyjo, p 772. 1 3 Maltoni, C., and G. Lefemine, Carcinogenicity bioassays ol vinyl chloride, 1. Rcscmth pl< and early results, Environ. Res., 7 (1974} 387~4514 Moorhead, P. S., P. C. Nowell, W. J. Mellman, 1>. M. Battips and D. A. Hunch-kmum Chromosome preparations of leukocytes cultured from human peripheral blood, Exp. hit R 20 (1960) 613-616 15 Nicholson, W. J., K. C. Hammond, H. Seidman and I. J. Seljkokf, Mortality exptrUna 1 a cohort of vinyl chloride-polyvinyl chloride workers, Ann. AW. Acad. Set , 255 (1976) 230, ib Tough, I. M , and W. M. Court Brown, Chromosome aberrations and exjwjsure to amlnti* benzene, Lancet, 1 (1965) 684. 17 Yoder, J., M. Watson and W. W. Henson, Lymphocyte chromosome analysis of agricultuu workers during extensive occupational exposure. Mutation Res., 21 (*973) 335-34 CO tr* H O bJ O t to OJ Mntatuw Research, 31 (1975) **9-173 , Klsevier Scientitic Publishing Company, Amsterdam--Printed in The Netherlands 169 AN EVALUATION OF THE MUTAGENIC POTENTIAL OF AN AEROSOL SPRAY ADHESIVE IN THE RAT HUBERT E. OSTEKBERG, JAMES C. Ml'REHV, GEORGE W. BIERBOWER *\i> ERANCES MORELAND SAURO* En liii'rt of Biological Science, Bureau of Biomedical Science, Consumer Product Safety Commission, II mMington, B C. 20207 and * Division of Toxicology, Food and Drug Administration. Department A Health. Education and Welfare, Washington, D.C. 20204 (U.S.A.) iHvccivet! January 8th, 1975) .I i menially available aerosol adhesive formulation was examined for its h" t ntial in adult rat bone marrow cells. Groups of rats were placed in a |l" dation chamber and were exposed to 10, 15, or 20 g of the aerosol once 'Lb s took place 21 times during 33 days. Treated and control animals "lI,` Heine intraperitoneally 2 li prior to sacrifice. Slides of bone marrow ell-, mu. \.imined for tlie presence of chromosome and chromatid aberrations. Although no statistically significant increases in grossly visible chromosomal damage "i 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 .mixta, eye and nasal irritation, hyperexcitability, and clonic convulsions; these 'ant symptoms were dose dependent.* 1 IMMODUCTION !* 1(J73. a preliminary report on an aerosol adhesive was forwarded to the 1 "iisumer Product Safety Commission by Dr. J. Rodman Seely, Professor of Pediairtcs and Molecular Biology, University of Oklahoma Medical Center. This report indicated a causal connection between exposure to the aerosol adhesive and chromo'*ime damage that might lead to genetic birth defects. The chromosomal analysts of peripheral blood drawn from exposed human subjects indicated a statistically in1 reused incidence of visible chromosome damage as evidenced by breaks and gaps. x<* (>tl,er aberrations were reported. Upon review of these data by academic, in dustrial, and governmental experts, the Consumer Product Safety Commission alerted I"' general public to this potential hazard. Although the association lietween the J'-rns<d adhesive and chromosome damage had not been positively established, the 'length of the association based upon the available information and the potential t,,r *TMus personal injury to exposed humans led the Commission to conclude that G distribution of these products for household use presented an imminent hazard