Mutation Research, 31 (1975) 153-168 © Elsevier Scientific Publishing Company, A m s t e r d a m - - P r i n t e d in The Netherlands
163
V I N Y L C H L O R I D E E X P O S U R E AND HUMAN CHROMOSOME ABERRATIONS*
A L A N DUCATMAN, K U R T H I R S C H H O R N * * AND I R V I N G J. S E L I K O F F
Environmental Sciences Laboratory, Department of Community Medicine and the Division of Medical Genetics, Department of Pediatrics, Mount Sinai School of 3ledicine of the City University of New York, New York, N . Y . zoo29 (U.S.A.) (Received October 25th, 1974) (Revision received J a n u a r y I5th, 1975)
SUMMARY
Examination of lymphocyte cultures from I I vinyl chloride polymerization workers and IO controls revealed a significantly higher incidence of aberrations 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 the early I96O'S it has been clear that chromosome morphology examination could uncover evidence of genetic damage in man 1-3. 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 clastogenic (chromosome breaking), notably alkylating agents and cytostatic drugs, and DNA base analogues 6. 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 herbicides10,17. 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 identified 4. 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 in exposed wor* Supported by U S P H S Center Grants ES 00928 and GM 19443, and Research Grant H D 02552. ** K u r t Hirschhorn, M. D. (to w h o m reprint requests should be addressed) is a Career Scientist of the H e a l t h Research Council of the City of New York, I-5i 3.
16 4
A. DUCATMANet al.
kers 1°. There is additional suggestion from both animal and human data of an increased risk for cancers of the central nervous system, the respiratory system, and of the blood forming tissuesl°,i3,15. 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 i i 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 polyvinyl 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 I I 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 p p m 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 examined
I 2 3 4 5 6 7 8 9 IO II
61 5° 47 44 4° 39 36 36 33 32 25
23 28 I9 II I3 I7 17 I2 II IO 4
5° 5° 5° 5° 5° 5° 5° 5° 5° 5° 5°
443 4°
165 15
55 ° 5°
Total Average
Control No. I 2 3 4 5 6 7 8 9 IO
Age
Years exposed
Cells examined
43 3° 29 29 28 28 27 20 19 18
o o o o o o o o o o
5° 5° 5° 5° 5° 5° 5° 5° 5° 5o
271 27
o o
500 5°
Of the IO healthy male controls, 4 were from within the same factory population, without known vinyl chloride exposure. Nevertheless, as long-term employment implied 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 4o. METHODS
The chromosome studies were performed on cultures of peripheral blood lymphocytes incubated at 37 ° for 65-68 h with phytohemagglutinin (Wellcome), Harvests were performed according to micromethod modifications of procedures first described
CHROMOSOME DAMAGE BY VINYL CHLORIDE
165
b y MOORHEAD et a l . 14. Media used in this s t u d y came from a single b a t c h (Gibco) in order to insure u n i f o r m p H a n d o th e r culture conditions. In each i n d i v i d u a l studied, 50 m e t a p h a s e s were c o u n t e d directly u n d e r t h e microscope at 16oo × . T h e A , B , D , E , F , G , a n d Y ch r o m o so m es were e v a l u a t e d sep a r a t e l y so far as possible. Suspected aberrations an d some n o r m a l m e t a p h a s e s were p h o t o g r a p h e d ; k a r y o t y p e s were p e r f o r m e d where helpful. F r o m the t o t a l of lO5O cells e x a m i n e d t h ere were 281 p h o t o g r a p h s t a k e n an d 14o k a r y o t y p e s m a d e from t h e photographs. Confirmed aberrations were classified according to t w o systems in order to discern if a p a t t e r n of breakage m i g h t exist. The s y s t e m of BUCKTON et a l . 3 an d COURT BROWN 5 has th r e e categories of aberrations: B, cells w i t h simple aberrations such as breaks an d gaps; C~, cells w i t h " u n s t a b l e " c h r o m o s o m e changes such as fragments, dicentrics, a n d rings; Cs, cells w i t h " s t a b l e " c h r o m o s o m e changes such as monosomies, trisomies, deletions, a n d exchanges. Th e distinction b et w een cells with stable an d unstable aberrations is related to their t e n d e n c y to either r e m a i n in or disappear from t h e circulation. T h e s y s t e m of HIRSCHHORN AND COHEN11 differs in t w o respects. I t considers all breakage, including t h e t o t a l n u m b e r of breaks in cells w i t h more t h a n one aberTABLE II VINYL CHLORIDEEXPOSUREAND CHROMOSOMEABERRATIONS(CLASSIFICATIONSYSTEMOF COURT BROWN5 AND BUCKTONet al. 3) Case N o . i
2 3 4 5 6 7 8 9 IO II Total Mean(4- S.D.) Comparison
Ba
Ca a
Cs a
7
2
3
8 6 3 5 3 5 8 4 8 5 62.o 5.64(:j21.9i)
2 o i I o 2 2 o 4 3 17.o 1.55(4-1.29)
3 3 3 3 o 2 6 4 5 5 37.o 3.36(4-1.63)
t = 1.84 t = 2.863 o.i > p > 0.05 p = o.oi
t = 0.56 0.6 > p > o.5
I 2
5 4
I o
3 7
3 4 5 6 7 8 9
4 5 2 5 5 5 4
o o I o o i o
I 3 6 3 3 2 o
5
O
Control N o .
IO
Total Mean(4- S.D.)
44.o 4.40(4 - 0.97)
3.o 0.30(4- 0.48)
I
29.0 2.90(4- 2.18)
a B, Breaks and gaps; Cu, "unstable" changes (fragments, dicentrics, rings); Cs, " s t a b l e " changes (monosomy, trisomy, deletions, exchanges).
166
A. DUCATMAN et al.
ration, and it gives weighted consideration to those aberrations which are the apparent 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 lO5O cells examined, as seen in the B column of Table II. Subjects have nonsignificant increases of such aberrations when compared to controls by a t-test for comparison of the means (o.I > P >o.o5), and marginally significant increases by an F-ratio for comparison of the variances (o.o5 > P > o . o i ) . The difference in cells with stable aberrations, or those that BUCKTONet al. and COURT BROWN found to persist in circulating lymphocytes3,L 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 = o . o i ; F , o . o i > P
>o.ooi,
Table I I I focuses on breaking events only. The total of simple breaks, including TABLE
III
CHROMOSOME BREAK EVENTS (IN 5 ° C E L L S / I N D I V I D U A L ) AND VINYL CHLORIDE EXPOSURE (SYSTEM
OF HIRSCHHORN AND COHEN) Case No.
Sa
Ca
Total break events
(s ¢-2c) I 2 3 4 5 6 7 8 9 io ii Total M e a n ( ± S.D.) Comparison
Control No. i
4 2 2 i o o 3 i 4 I 3 21 1.91(±I.44 ) t = 1.12 o. 3 > p > o.2
3 3 o 2 i o 2 2 o 4 5
io 8 2 5 2 o 7 5 4 9 13
22 2,oo(±1.67) t = 2.8o o.o2 > p
> o.oi
65 5 . 9 I ( J2 3.91) t = 2-75 o.o2 > i t , > o . o i
3
i
5
2
I
0
I
3 4 5 6
i o 2 2
o o 2 o
i o 6 2
7
0
0
0
8 9
I 2
I o
3 2
I
0
I
13
4
2I
I0
Total
M e a n ( 4 - S.D.)
1 . 3 o ( = o.95)
0 . 4 o ( ± o.7o )
2 . 1 o ( + 2.o2)
a S, S i n g l e h i t e v e n t s ( b r e a k s , d e l e t i o n s ) ; C, c o m p l e x e v e n t s (rings, d i c e n t r i c s , e x c h a n g e s ) .
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: t, 0.02 > P > o . o i ; and F, o.oi > P > o . o o i . The total of breaking events (S + 2C) shows a similarly significant increase in the subjects (t, 0.02 > P > o . o I ; F, o.oi > P > o . o o I ) . Not included in the charts is a combined total of all breaks, gaps, and deletions. There was a marginally significant difference in these with t, 0.05 > P > 0.02; F was not significant. 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 (4- 2.47 S.D.) and the average control had 2.00 (4- 1.63) in the 50 cells examined per individual. Significance was marginal at most, P < 0.05 by a t-test; F not significant. 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 loss ~. Also, the relatively high degree of breaks and gaps in controls as well as subiects 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 increase 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 chromosomes 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 important 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 carcinogenesis 13.
168
A. DUCATMAN 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 I BENDER, M. A., AND P. C. GOOCH, P e r s i s t e n t c h r o m o s o m e a b e r r a t i o n s in irradiated h u m a n subjects, Radiat. Res., 16 (1962) 44-532 BLOOM, A. D., AND J. H. TJIO, I n vivo effects of diagnostic X - i r r a d i a t i o n on h u m a n c h r o m o sorties, New Engl. J. Med., 270 (1964) 1341-1344. 3 BUCKTON, K. E., 13. A. JACOBS, W. M. COURT BROWN AND R. DOLL, A s t u d y of t h e c h r o m o s o m e d a m a g e p e r s i s t i n g after X - r a y t h e r a p y for a n k y l o s i n g spondylitis, Lancet, ii (1962) 676-682. 4 CREECH, J. L., AND M. N. JOHNSON, A n g i o s a r c o m a of liver in t h e m a n u f a c t u r e of v i n y l chloride, J. Occup. Med., 16 (1974) 15o-151. 5 COURT BROWN, W. M., H u m a n p o p u l a t i o n cytogenics, in A. NEUBERGER AND E. L. TATUM (Eds.), Frontiers ofBiolagy, Vol. V, N o r t h - H o l l a n d , A m s t e r d a m , 1967, pp. i 31. 6 EVANS, H. J., P o p u l a t i o n c y t o g e n e t i c s a n d e n v i r o n m e n t a l factors, in 13ATRICIA A. JACOBS, W. H. PRICE AND PAMELA LAW (Eds.), Human Population Cytogenetics, ~vVilliauls a n d "vVilkins, Baltimore, 197 o, pp. 191-216. 7 FORNI, A. M., A. COPPELLINI, E. I~ACIFICO AND E. C. VIGLIANI, C h r o m o s o m e c h a n g e s a n d t h e i r e v o l u t i o n in s u b j e c t s w i t h p a s t e x p o s u r e to benzene, Arch. Environ. Health, 23 (1971 ) 385-391 . 8 FORN1, A. M., E. PACIFICO AND A. LIMONTA, C h r o m o s o m e s t u d i e s in w o r k e r s e x p o s e d to b e n z e n e or t o l u e n e or both, Arch. Environ. Health, 22 (1971 ) 373-378 . 9 GERMAN, J. (Ed.), Chromosomes and Cancer, Wiley, N e w York, 1974. IO HEATH, C., AND J. V~rAGONER,Report of a Working Group on Vinyl Chloride, Lyon, 24-25 June, i974, I A R C No. 74/oo5, W o r l d H e a l t h Organization, pp. 18-19. I I HIRSCHHORN, t{., AND M. M. COHEN, D r u g - i n d u c e d c h r o m o s o m a l a b e r r a t i o n s , / / n n . N. Y. ~t cad. Sci., 151 (1968) 9 5 5 - 9 8 7 . I2 t-~OOP1NGAMER, R. AND A. ~,'V. BLOOMER, L y m p h o c y t e c h r o m o s o m e a n a l y s i s of pesticide e x p o s e d individuals, in 7th Int. Congr. Plant Protection, Paris, I97o, p. 772. 13 MALTONI, C., AND G. LEFEMINE, Carcinogenicity b i o a s s a y s of v i n y l chloride, I. R e s e a r c h p l a n a n d early results, Environ. Res., 7 (1974) 387-405 • 14 MOORHEAD, l3. S., 13. C. HOWELL, W. J. MELLMAN, D. M. BATTIPS AND D. A. HUNGERFORD, C h r o m o s o m e p r e p a r a t i o n s of l e u k o c y t e s c u l t u r e d f r o m h u m a n peripheral blood, Exp. Cell Res., 20 (196o) 613-616. 15 NICHOLSON, W. J., E. C. HAMMOND, H. SEIDMAN AND I. J. SELIKOFF, M o r t a l i t y experience of a c o h o r t of v i n y l chloride-polyvinyl chloride workers, Ann. N . Y . Acad. Sci., 255 (1975) 225230. 16 TOUGH, I. M., AND ~V. M. COURT BROWN, C h r o m o s o m e a b e r r a t i o n s a n d e x p o s u r e to a m b i e n t benzene, Lancet, i (1965) 684. 17 YODER, J., M. WATSON AND ~\7. W. BENSON, L y m p h o c y t e c h r o m o s o m e a n a l y s i s of agricultural w o r k e r s d u r i n g e x t e n s i v e o c c u p a t i o n a l exposure, Mutation Res., 21 (1973) 335 34 °.
163
V I N Y L C H L O R I D E E X P O S U R E AND HUMAN CHROMOSOME ABERRATIONS*
A L A N DUCATMAN, K U R T H I R S C H H O R N * * AND I R V I N G J. S E L I K O F F
Environmental Sciences Laboratory, Department of Community Medicine and the Division of Medical Genetics, Department of Pediatrics, Mount Sinai School of 3ledicine of the City University of New York, New York, N . Y . zoo29 (U.S.A.) (Received October 25th, 1974) (Revision received J a n u a r y I5th, 1975)
SUMMARY
Examination of lymphocyte cultures from I I vinyl chloride polymerization workers and IO controls revealed a significantly higher incidence of aberrations 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 the early I96O'S it has been clear that chromosome morphology examination could uncover evidence of genetic damage in man 1-3. 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 clastogenic (chromosome breaking), notably alkylating agents and cytostatic drugs, and DNA base analogues 6. 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 herbicides10,17. 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 identified 4. 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 in exposed wor* Supported by U S P H S Center Grants ES 00928 and GM 19443, and Research Grant H D 02552. ** K u r t Hirschhorn, M. D. (to w h o m reprint requests should be addressed) is a Career Scientist of the H e a l t h Research Council of the City of New York, I-5i 3.
16 4
A. DUCATMANet al.
kers 1°. There is additional suggestion from both animal and human data of an increased risk for cancers of the central nervous system, the respiratory system, and of the blood forming tissuesl°,i3,15. 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 i i 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 polyvinyl 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 I I 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 p p m 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 examined
I 2 3 4 5 6 7 8 9 IO II
61 5° 47 44 4° 39 36 36 33 32 25
23 28 I9 II I3 I7 17 I2 II IO 4
5° 5° 5° 5° 5° 5° 5° 5° 5° 5° 5°
443 4°
165 15
55 ° 5°
Total Average
Control No. I 2 3 4 5 6 7 8 9 IO
Age
Years exposed
Cells examined
43 3° 29 29 28 28 27 20 19 18
o o o o o o o o o o
5° 5° 5° 5° 5° 5° 5° 5° 5° 5o
271 27
o o
500 5°
Of the IO healthy male controls, 4 were from within the same factory population, without known vinyl chloride exposure. Nevertheless, as long-term employment implied 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 4o. METHODS
The chromosome studies were performed on cultures of peripheral blood lymphocytes incubated at 37 ° for 65-68 h with phytohemagglutinin (Wellcome), Harvests were performed according to micromethod modifications of procedures first described
CHROMOSOME DAMAGE BY VINYL CHLORIDE
165
b y MOORHEAD et a l . 14. Media used in this s t u d y came from a single b a t c h (Gibco) in order to insure u n i f o r m p H a n d o th e r culture conditions. In each i n d i v i d u a l studied, 50 m e t a p h a s e s were c o u n t e d directly u n d e r t h e microscope at 16oo × . T h e A , B , D , E , F , G , a n d Y ch r o m o so m es were e v a l u a t e d sep a r a t e l y so far as possible. Suspected aberrations an d some n o r m a l m e t a p h a s e s were p h o t o g r a p h e d ; k a r y o t y p e s were p e r f o r m e d where helpful. F r o m the t o t a l of lO5O cells e x a m i n e d t h ere were 281 p h o t o g r a p h s t a k e n an d 14o k a r y o t y p e s m a d e from t h e photographs. Confirmed aberrations were classified according to t w o systems in order to discern if a p a t t e r n of breakage m i g h t exist. The s y s t e m of BUCKTON et a l . 3 an d COURT BROWN 5 has th r e e categories of aberrations: B, cells w i t h simple aberrations such as breaks an d gaps; C~, cells w i t h " u n s t a b l e " c h r o m o s o m e changes such as fragments, dicentrics, a n d rings; Cs, cells w i t h " s t a b l e " c h r o m o s o m e changes such as monosomies, trisomies, deletions, a n d exchanges. Th e distinction b et w een cells with stable an d unstable aberrations is related to their t e n d e n c y to either r e m a i n in or disappear from t h e circulation. T h e s y s t e m of HIRSCHHORN AND COHEN11 differs in t w o respects. I t considers all breakage, including t h e t o t a l n u m b e r of breaks in cells w i t h more t h a n one aberTABLE II VINYL CHLORIDEEXPOSUREAND CHROMOSOMEABERRATIONS(CLASSIFICATIONSYSTEMOF COURT BROWN5 AND BUCKTONet al. 3) Case N o . i
2 3 4 5 6 7 8 9 IO II Total Mean(4- S.D.) Comparison
Ba
Ca a
Cs a
7
2
3
8 6 3 5 3 5 8 4 8 5 62.o 5.64(:j21.9i)
2 o i I o 2 2 o 4 3 17.o 1.55(4-1.29)
3 3 3 3 o 2 6 4 5 5 37.o 3.36(4-1.63)
t = 1.84 t = 2.863 o.i > p > 0.05 p = o.oi
t = 0.56 0.6 > p > o.5
I 2
5 4
I o
3 7
3 4 5 6 7 8 9
4 5 2 5 5 5 4
o o I o o i o
I 3 6 3 3 2 o
5
O
Control N o .
IO
Total Mean(4- S.D.)
44.o 4.40(4 - 0.97)
3.o 0.30(4- 0.48)
I
29.0 2.90(4- 2.18)
a B, Breaks and gaps; Cu, "unstable" changes (fragments, dicentrics, rings); Cs, " s t a b l e " changes (monosomy, trisomy, deletions, exchanges).
166
A. DUCATMAN et al.
ration, and it gives weighted consideration to those aberrations which are the apparent 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 lO5O cells examined, as seen in the B column of Table II. Subjects have nonsignificant increases of such aberrations when compared to controls by a t-test for comparison of the means (o.I > P >o.o5), and marginally significant increases by an F-ratio for comparison of the variances (o.o5 > P > o . o i ) . The difference in cells with stable aberrations, or those that BUCKTONet al. and COURT BROWN found to persist in circulating lymphocytes3,L 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 = o . o i ; F , o . o i > P
>o.ooi,
Table I I I focuses on breaking events only. The total of simple breaks, including TABLE
III
CHROMOSOME BREAK EVENTS (IN 5 ° C E L L S / I N D I V I D U A L ) AND VINYL CHLORIDE EXPOSURE (SYSTEM
OF HIRSCHHORN AND COHEN) Case No.
Sa
Ca
Total break events
(s ¢-2c) I 2 3 4 5 6 7 8 9 io ii Total M e a n ( ± S.D.) Comparison
Control No. i
4 2 2 i o o 3 i 4 I 3 21 1.91(±I.44 ) t = 1.12 o. 3 > p > o.2
3 3 o 2 i o 2 2 o 4 5
io 8 2 5 2 o 7 5 4 9 13
22 2,oo(±1.67) t = 2.8o o.o2 > p
> o.oi
65 5 . 9 I ( J2 3.91) t = 2-75 o.o2 > i t , > o . o i
3
i
5
2
I
0
I
3 4 5 6
i o 2 2
o o 2 o
i o 6 2
7
0
0
0
8 9
I 2
I o
3 2
I
0
I
13
4
2I
I0
Total
M e a n ( 4 - S.D.)
1 . 3 o ( = o.95)
0 . 4 o ( ± o.7o )
2 . 1 o ( + 2.o2)
a S, S i n g l e h i t e v e n t s ( b r e a k s , d e l e t i o n s ) ; C, c o m p l e x e v e n t s (rings, d i c e n t r i c s , e x c h a n g e s ) .
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: t, 0.02 > P > o . o i ; and F, o.oi > P > o . o o i . The total of breaking events (S + 2C) shows a similarly significant increase in the subjects (t, 0.02 > P > o . o I ; F, o.oi > P > o . o o I ) . Not included in the charts is a combined total of all breaks, gaps, and deletions. There was a marginally significant difference in these with t, 0.05 > P > 0.02; F was not significant. 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 (4- 2.47 S.D.) and the average control had 2.00 (4- 1.63) in the 50 cells examined per individual. Significance was marginal at most, P < 0.05 by a t-test; F not significant. 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 loss ~. Also, the relatively high degree of breaks and gaps in controls as well as subiects 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 increase 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 chromosomes 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 important 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 carcinogenesis 13.
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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 I BENDER, M. A., AND P. C. GOOCH, P e r s i s t e n t c h r o m o s o m e a b e r r a t i o n s in irradiated h u m a n subjects, Radiat. Res., 16 (1962) 44-532 BLOOM, A. D., AND J. H. TJIO, I n vivo effects of diagnostic X - i r r a d i a t i o n on h u m a n c h r o m o sorties, New Engl. J. Med., 270 (1964) 1341-1344. 3 BUCKTON, K. E., 13. A. JACOBS, W. M. COURT BROWN AND R. DOLL, A s t u d y of t h e c h r o m o s o m e d a m a g e p e r s i s t i n g after X - r a y t h e r a p y for a n k y l o s i n g spondylitis, Lancet, ii (1962) 676-682. 4 CREECH, J. L., AND M. N. JOHNSON, A n g i o s a r c o m a of liver in t h e m a n u f a c t u r e of v i n y l chloride, J. Occup. Med., 16 (1974) 15o-151. 5 COURT BROWN, W. M., H u m a n p o p u l a t i o n cytogenics, in A. NEUBERGER AND E. L. TATUM (Eds.), Frontiers ofBiolagy, Vol. V, N o r t h - H o l l a n d , A m s t e r d a m , 1967, pp. i 31. 6 EVANS, H. J., P o p u l a t i o n c y t o g e n e t i c s a n d e n v i r o n m e n t a l factors, in 13ATRICIA A. JACOBS, W. H. PRICE AND PAMELA LAW (Eds.), Human Population Cytogenetics, ~vVilliauls a n d "vVilkins, Baltimore, 197 o, pp. 191-216. 7 FORNI, A. M., A. COPPELLINI, E. I~ACIFICO AND E. C. VIGLIANI, C h r o m o s o m e c h a n g e s a n d t h e i r e v o l u t i o n in s u b j e c t s w i t h p a s t e x p o s u r e to benzene, Arch. Environ. Health, 23 (1971 ) 385-391 . 8 FORN1, A. M., E. PACIFICO AND A. LIMONTA, C h r o m o s o m e s t u d i e s in w o r k e r s e x p o s e d to b e n z e n e or t o l u e n e or both, Arch. Environ. Health, 22 (1971 ) 373-378 . 9 GERMAN, J. (Ed.), Chromosomes and Cancer, Wiley, N e w York, 1974. IO HEATH, C., AND J. V~rAGONER,Report of a Working Group on Vinyl Chloride, Lyon, 24-25 June, i974, I A R C No. 74/oo5, W o r l d H e a l t h Organization, pp. 18-19. I I HIRSCHHORN, t{., AND M. M. COHEN, D r u g - i n d u c e d c h r o m o s o m a l a b e r r a t i o n s , / / n n . N. Y. ~t cad. Sci., 151 (1968) 9 5 5 - 9 8 7 . I2 t-~OOP1NGAMER, R. AND A. ~,'V. BLOOMER, L y m p h o c y t e c h r o m o s o m e a n a l y s i s of pesticide e x p o s e d individuals, in 7th Int. Congr. Plant Protection, Paris, I97o, p. 772. 13 MALTONI, C., AND G. LEFEMINE, Carcinogenicity b i o a s s a y s of v i n y l chloride, I. R e s e a r c h p l a n a n d early results, Environ. Res., 7 (1974) 387-405 • 14 MOORHEAD, l3. S., 13. C. HOWELL, W. J. MELLMAN, D. M. BATTIPS AND D. A. HUNGERFORD, C h r o m o s o m e p r e p a r a t i o n s of l e u k o c y t e s c u l t u r e d f r o m h u m a n peripheral blood, Exp. Cell Res., 20 (196o) 613-616. 15 NICHOLSON, W. J., E. C. HAMMOND, H. SEIDMAN AND I. J. SELIKOFF, M o r t a l i t y experience of a c o h o r t of v i n y l chloride-polyvinyl chloride workers, Ann. N . Y . Acad. Sci., 255 (1975) 225230. 16 TOUGH, I. M., AND ~V. M. COURT BROWN, C h r o m o s o m e a b e r r a t i o n s a n d e x p o s u r e to a m b i e n t benzene, Lancet, i (1965) 684. 17 YODER, J., M. WATSON AND ~\7. W. BENSON, L y m p h o c y t e c h r o m o s o m e a n a l y s i s of agricultural w o r k e r s d u r i n g e x t e n s i v e o c c u p a t i o n a l exposure, Mutation Res., 21 (1973) 335 34 °.
