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Comparative evaluation of the frequency of chromosomal aberrations and the SCE numbers in peripheral lymphocytes of workers occupationally exposed to vinyl chloride monomer
97
Mutation Research, 67 (1979) 97--100 © Elsevier/North-Holland Biomedical Press
Short Communication COMPARATIVE E V A L U A T I O N O F THE F R E Q U E N C Y O F CHROMOSOMAL A B E R R A T I O N S AND THE SCE NUMBERS IN P E R I P H E R A L LYMPHOCYTES O F W O R K E R S OCCUPATIONALLY EXPOSED TO VINYL C H L O R I D E MONOMER
M. KU(~EROV.~, Z. POLIVKOV.~ and J. B/kTORA *
Pediatric Department of Postgraduate Medical Institute, Institute of Hygiene and Epidemiology, Prague, Srobdrova 48 (Czechoslovakia) (Received 31 March 1978) (Revision received 15 November 1978) (Accepted 23 November 1978)
There are n o w available plenty of authorative data on the carcinogenic [1--3, 13] and mutagenic [1,2,10] activity of vinyl chloride monomer. Increased frequency of chromosomal aberrations detectable in peripheral l y m p h o c y t e s of chronically exposed workers has been reported by numerous authors [4--6, 11,12]. We have repeatedly analysed, using routine cytogenetic techniques [8], 3 blood samples from each of 9 workers occupationally exposed for 10--27 years to relatively high mean annual doses of vinyl chloride m o n o m e r (about 20--150 p p m of air). In the third blood sample series we also used the SCE technique [14] and compared the frequency of detected chromosomal aberrations with the n u m b e r of SCEs in each sample. The blood samples from 8 healthy persons were used as controls and were collected with the 3rd blood sampling from the vinyl chloride (VC) workers. The control persons were matched for age and sex and they were n o t exposed to known mutagens during 3 months before the time of blood collection. The results are summarized in three t~bles. Tables I and 2 show the instability and non-homogeneity in the frequency of chromosomal aberrations over a period of t w o years. Mostly chromatid and chromosome breaks were detected; chromatid and chromosome exchanges occurred only sporadically. Table 2 compares the frequency of chromosomal aberrations and the number of SCE per cell in 7 blood samples. There is an apparent increase in b o t h parameters in comparison with control levels. The mathematical evaluation of our results was calculated only for the 3rd collection. Conventional aberrations in VC workers were non-homogeneous.
.~istrict Hygienic Station, Prievidza.
98 TABLE 1 C H R O M O S O M A L A N A L Y S E S OF 9 W O R K E R S O C C U P A T I O N A L L Y EXPOSED TO V I N Y L CHLORIDE (TYPES OF ABERRATION)
Exposure (years)
Aberrant ceBs (%)
Number of chromatid breaks / 1 0 0 cells
Number of chromatid exchanges / 1 0 0 cells
Number of chromosome breaks / 1 0 0 cells
Number of chromosome exchanges / 1 0 0 cells
Number of despiralizations / 1 0 0 cells
E.H. 25--27
1 2 3
11 0 11
5 0 5
1 0 0
4 0 4
0 0 3
1 0 0
J.P.
1 2 3
4 2 2
4 0 1
0 0 0
0 1 0
0 1 1
0 0 0
K.K. 15--17
1 2 3
2 7 3
2 6 3
0 1 0
0 0 0
0 0 0
0 0 0
A.H. 14--16
1 2 3
1 1 6
1 1 2
0 0 0
0 0 4
0 0 0
0 0 0
M.M. 13--15
1 2 3
0 1 7
0 0 3
0 0 0
0 0 4
0 1 0
0 0 0
E~I. 12--14
1 2 3
3 6 II
1 4 2
1 0 1
0 2 8
1 0 0
0 0 0
J.K. 12--14
1 2 3
1 4 3
0 3 3
0 0 0
1 1 0
0 0 0
0 0 0
F.M. 11--13
1 2 3
1 2 1
1 1 1
0 0 0
0 1 0
0 0 0
0 0 0
A.T. 10--12
1 2 3
4 1 3
2 0 0
0 0 0
1 1 3
0 0 0
1 0 0
1.8
1
0
0.8
0.2
0
16--18
8 controls (mean)
The frequency of SCEs was significantly higher in cells of VC workers in comparison with controls. The level of significance was P ~ 0.001, according to the t test. The detailed control data are in Table 4. The medical histories, and the smoking and drinking habits of workers tested are presented in Table 3. Smoking and other habits of workers appeared to have no effect on the frequency of any chromosomal changes. Our positive results are contradictory to findings reported by Hansteen [7] w h o did not observe any increase either in the frequency of aberrations or in the SCE numbers. However, the subjects examined by this author had been exposed to very low doses of vinyl chloride (0.1 ppm) and only for the relatively short period of two years. This can be taken as a further proof that VCM-
99 TABLE
2
CHROMOSOMAL RIDE
ANALYSES
Exposure
OF
9 WORKERS
OCCUPATIONALLY
EXPOSED
years
ppm
1st collection a b e r r a n t cells (%) a
E.H. J.P. K.K. A.H. M.M. E.J. J.K. F.M.
25--27 16--18 15--17 14--16 13--15 12--14 12--14 11--13
20--150 20---80 15--70 20--150 20--90 20-150 20-90 20---90
11 4 2 1 0 3 1 1
0 2 7 1 1 6 4 2
11 c 2 3 6 7 c 11 c 3 1
A.T.
10--12
20--90
4
1
3
TO VINYL
2nd collection a b e r r a n t cells
3hi collection
(%)
aberrant (%) a
a
cells
CHLO-
SCE/cell b
17.50 9.56 15.02 -15.26 16.41 10.60 12.22
c c c c c
--
Mean
5 . 2 -+ 1 . 3
1 3 . 8 0 -+ 1 . 1 3
8 controls (mean)
1 . 8 -+ 0 . 4
9.41 + 0.40
a 100 cells were scored per sample. b 5 0 cells w e r e s c o r e d p e r s a m p l e . c Significantly increased in comparison
with control values.
induced chromosomal changes depend both on dose and length of exposure to the vinyl chloride monomer. Our results seem to lead to a conclusion t h a t the t w o cytogenetic m e t h o d s -routine and SCE -- are equally suitable for testing of high-dose vinyl chloride mutagenicity in vivo. The increase of SCEs in our results is more homogeneous. The sensitivities of the two methods t o d e t e c t mutagenic activity seem to be about the same.
TABLE
3
MEDICAL
HISTORY,
AND SMOKING
AND DRINKING
HABITS
OF WORKERS
TESTED
Diagnostic radiation
Smoking
Drinking
Drugs
E.H.
--
+
-+
J.p.
--
_+
++
Meprobamat Papaverin Chloramphenicol Synt ophyllin
K.K.
+
+ +_
A.H. M.M.
--
--
E.J. J.K. F.M.
----
+ -+÷
A.T.
_+ +_ _+ ++ +_
Solutan Syntophyllin Bellaspon Superpyrin Thiospasmin Ketazon
100
TABLE 4 CHROMOSOMAL ANALYSIS OF 8 CONTROLS Aberrant cells a (%)
SCE/cell b
Smoking
Drinking
M.K.
3
10.88
--
--
J.K. V.B. J.~. K.B. M.Ku. Z.P. R.~.
0 3 3 1 1 2 1
8.22 10.30 10.16 10.46 8.26 7.94 9.02
-++ ++ --+ --
-+ + ---+
Mean
1.8 + 0 . 4
9.41 +- 0 . 4 0
a 1 0 0 cells w e r e s c o r e d p e r s a m p l e . b 5 0 cells w e r e s c o r e d p e r s a m p l e . T h e c o n t r o l s w e r e n o t e x p o s e d t o X-rays a n d d r u g s a t t h e t i m e o f b l o o d c o l l e c t i o n .
References 1 B a r t s c h , H., C. Malaveille a n d R. M o n t e s a n o , H u m a n , r a t a n d m o u s e l i v e r - m e d i a t e d m u t a g e n i c i t y of v i n y l c h l o r i d e in S. t y p h i m u r i u m strains, I n t . J. C a n c e r , 15 ( 1 9 7 5 ) 4 2 9 - - 4 3 7 . 2 B a r t s e h , H., a n d R. M o n t e s a n o , M u t a g e n i c a n d c a r c i n o g e n i c e f f e c t s of v i n y l c h l o r i d e , M u t a t i o n Res., 32 (1975) 93--114. 3 C r e e c h , J . L . , a n d M.N. J o h n s o n , A n g i o s a r c o m a o f the liver in t h e m a n u f a c t u r e o f p o l y v i n y l c h l o r i d e , J. O c c u p . Med., 16 ( 1 9 7 4 ) 1 5 0 - - 1 5 1 . 4 D u c a t m a n , V., K. H i r s c h h o r n a n d I.J. Selikoff, V i n y l c h l o r i d e e x p o s u r e a n d h u m a n c h r o m o s o m e a b e r r a t i o n s , M u t a t i o n Res., 31 ( 1 9 7 5 ) 1 6 3 - - 1 6 9 . 5 F u n e s - C r a v i o t o , F., B. L a m b e r t , J. L i n d s t e n , L. E h r e n b e r g , A . T . N a t a r a j a n a n d S. O s t e r m a n - G o l k a r , C h r o m o s o m e a b e r r a t i o n s in w o r k e r s e x p o s e d to v i n y l c h l o r i d e , L a n c e t , 1 ( 1 9 7 5 ) 4 5 9 . 6 H a n s t e e n , I . L . , L. Hillestad a n d E. T h i i s - E v e n s e n , 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 t o v i n y l c h l o r i d e , M u t a t i o n Res., 38 ( 1 9 7 6 ) 1 1 2 . 7 H a n s t e e n , I.L., C h r o m o s o m e b r e a k a g e f r e q u e n c y a n d sister c h r o m a t i d e e x c h a n g e s in PVC w o r k e r s t w o years after exposure, A follow-up study, Abstract of 6th International C h r o m o s o m e Conference, Helsinki, A u g u s t 1 9 7 7 . 8 Hungerford, D.A., L e u k o c y t e s cultured f r o m small inocula of whole blood and preparation of rectap h a s e c h r o m o s o m e s b y t r e a t m e n t w i t h h y p o t o n i c KC1, S t a i n T e c h n o l . , 4 0 ( 1 9 6 5 ) 3 3 3 - - 3 3 8 . 9 Ku~erov~i, M., C y t o g e n e t i c analysis o f h u m a n c h r o m o s o m e s a n d its v a l u e f o r t h e e s t i m a t i o n o f g e n e t i c risk, M u t a t i o n Res., 41 ( 1 9 7 6 ) 1 2 3 - - 1 3 0 . 1 0 L o p r i e n o , N., R. Barale, S. B a r o n c e l l i , C. B a u e r , G. B r o n z e t t i , A. C a m m e U i n i , G. C e r c i g n a n i , C. Corsi, G. Gervasi, C. L e p o r i n i , R. Nieri, A.M. Rossi, G. S t r e t t i a n d G. T u r c h i , E v a l u a t i o n of t h e g e n e t i c e f f e c t s i n d u c e d b y v i n y l c h l o r i d e m o n o m e r ( V C M ) u n d e r m a m m a l i a n m e t a b o l i c a c t i v a t i o n : in v i t r o a n d in v i v o s t u d i e s , M u t a t i o n Res., 4 0 ( 1 9 7 6 ) 8 5 - - 9 6 . 11 P u r c h a s e , I . F . H . , C. R i c h a r d s o n a n d D. A n d e r s o n , C h r o m o s o m a l a n d d o m i n a n t l e t h a l e f f e c t s o f v i n y l c h l o r i d e , L a n c e t , II ( 1 9 7 5 ) 4 1 0 - - 4 1 1 . 12 P u r c h a s e , I . F . H . , C. R i c h a r d s o n a n d D. A n d e r s o n , C h r o m o s o m a l e f f e c t s in p e r i p h e r a l l y m p h o c y t e s , Proe. R o y . Soc. Med., 6 9 ( 1 9 7 6 ) 2 9 0 - - 2 9 1 . 13 V i o h , P.L., A. Bigotti a n d A. C a p u t o , O n c o g e n i c r e s p o n s e of r a t skin, l u n g s a n d b o n e s t o v i n y l c h l o r i d e , C a n c e r Res., 31 ( 1 9 7 1 ) 5 1 6 - - 5 2 2 . 1 4 W o l f f , S., a n d P. P e r r y , D i f f e r e n t i a l G i e m s a s t a i n i n g of sister c h r o m a t i d s a n d t h e s t u d y o f sister c h r o matid exchanges without autoradiography, C h r o m o s o m a , 48 (1974) 341--353.
Mutation Research, 67 (1979) 97--100 © Elsevier/North-Holland Biomedical Press
Short Communication COMPARATIVE E V A L U A T I O N O F THE F R E Q U E N C Y O F CHROMOSOMAL A B E R R A T I O N S AND THE SCE NUMBERS IN P E R I P H E R A L LYMPHOCYTES O F W O R K E R S OCCUPATIONALLY EXPOSED TO VINYL C H L O R I D E MONOMER
M. KU(~EROV.~, Z. POLIVKOV.~ and J. B/kTORA *
Pediatric Department of Postgraduate Medical Institute, Institute of Hygiene and Epidemiology, Prague, Srobdrova 48 (Czechoslovakia) (Received 31 March 1978) (Revision received 15 November 1978) (Accepted 23 November 1978)
There are n o w available plenty of authorative data on the carcinogenic [1--3, 13] and mutagenic [1,2,10] activity of vinyl chloride monomer. Increased frequency of chromosomal aberrations detectable in peripheral l y m p h o c y t e s of chronically exposed workers has been reported by numerous authors [4--6, 11,12]. We have repeatedly analysed, using routine cytogenetic techniques [8], 3 blood samples from each of 9 workers occupationally exposed for 10--27 years to relatively high mean annual doses of vinyl chloride m o n o m e r (about 20--150 p p m of air). In the third blood sample series we also used the SCE technique [14] and compared the frequency of detected chromosomal aberrations with the n u m b e r of SCEs in each sample. The blood samples from 8 healthy persons were used as controls and were collected with the 3rd blood sampling from the vinyl chloride (VC) workers. The control persons were matched for age and sex and they were n o t exposed to known mutagens during 3 months before the time of blood collection. The results are summarized in three t~bles. Tables I and 2 show the instability and non-homogeneity in the frequency of chromosomal aberrations over a period of t w o years. Mostly chromatid and chromosome breaks were detected; chromatid and chromosome exchanges occurred only sporadically. Table 2 compares the frequency of chromosomal aberrations and the number of SCE per cell in 7 blood samples. There is an apparent increase in b o t h parameters in comparison with control levels. The mathematical evaluation of our results was calculated only for the 3rd collection. Conventional aberrations in VC workers were non-homogeneous.
.~istrict Hygienic Station, Prievidza.
98 TABLE 1 C H R O M O S O M A L A N A L Y S E S OF 9 W O R K E R S O C C U P A T I O N A L L Y EXPOSED TO V I N Y L CHLORIDE (TYPES OF ABERRATION)
Exposure (years)
Aberrant ceBs (%)
Number of chromatid breaks / 1 0 0 cells
Number of chromatid exchanges / 1 0 0 cells
Number of chromosome breaks / 1 0 0 cells
Number of chromosome exchanges / 1 0 0 cells
Number of despiralizations / 1 0 0 cells
E.H. 25--27
1 2 3
11 0 11
5 0 5
1 0 0
4 0 4
0 0 3
1 0 0
J.P.
1 2 3
4 2 2
4 0 1
0 0 0
0 1 0
0 1 1
0 0 0
K.K. 15--17
1 2 3
2 7 3
2 6 3
0 1 0
0 0 0
0 0 0
0 0 0
A.H. 14--16
1 2 3
1 1 6
1 1 2
0 0 0
0 0 4
0 0 0
0 0 0
M.M. 13--15
1 2 3
0 1 7
0 0 3
0 0 0
0 0 4
0 1 0
0 0 0
E~I. 12--14
1 2 3
3 6 II
1 4 2
1 0 1
0 2 8
1 0 0
0 0 0
J.K. 12--14
1 2 3
1 4 3
0 3 3
0 0 0
1 1 0
0 0 0
0 0 0
F.M. 11--13
1 2 3
1 2 1
1 1 1
0 0 0
0 1 0
0 0 0
0 0 0
A.T. 10--12
1 2 3
4 1 3
2 0 0
0 0 0
1 1 3
0 0 0
1 0 0
1.8
1
0
0.8
0.2
0
16--18
8 controls (mean)
The frequency of SCEs was significantly higher in cells of VC workers in comparison with controls. The level of significance was P ~ 0.001, according to the t test. The detailed control data are in Table 4. The medical histories, and the smoking and drinking habits of workers tested are presented in Table 3. Smoking and other habits of workers appeared to have no effect on the frequency of any chromosomal changes. Our positive results are contradictory to findings reported by Hansteen [7] w h o did not observe any increase either in the frequency of aberrations or in the SCE numbers. However, the subjects examined by this author had been exposed to very low doses of vinyl chloride (0.1 ppm) and only for the relatively short period of two years. This can be taken as a further proof that VCM-
99 TABLE
2
CHROMOSOMAL RIDE
ANALYSES
Exposure
OF
9 WORKERS
OCCUPATIONALLY
EXPOSED
years
ppm
1st collection a b e r r a n t cells (%) a
E.H. J.P. K.K. A.H. M.M. E.J. J.K. F.M.
25--27 16--18 15--17 14--16 13--15 12--14 12--14 11--13
20--150 20---80 15--70 20--150 20--90 20-150 20-90 20---90
11 4 2 1 0 3 1 1
0 2 7 1 1 6 4 2
11 c 2 3 6 7 c 11 c 3 1
A.T.
10--12
20--90
4
1
3
TO VINYL
2nd collection a b e r r a n t cells
3hi collection
(%)
aberrant (%) a
a
cells
CHLO-
SCE/cell b
17.50 9.56 15.02 -15.26 16.41 10.60 12.22
c c c c c
--
Mean
5 . 2 -+ 1 . 3
1 3 . 8 0 -+ 1 . 1 3
8 controls (mean)
1 . 8 -+ 0 . 4
9.41 + 0.40
a 100 cells were scored per sample. b 5 0 cells w e r e s c o r e d p e r s a m p l e . c Significantly increased in comparison
with control values.
induced chromosomal changes depend both on dose and length of exposure to the vinyl chloride monomer. Our results seem to lead to a conclusion t h a t the t w o cytogenetic m e t h o d s -routine and SCE -- are equally suitable for testing of high-dose vinyl chloride mutagenicity in vivo. The increase of SCEs in our results is more homogeneous. The sensitivities of the two methods t o d e t e c t mutagenic activity seem to be about the same.
TABLE
3
MEDICAL
HISTORY,
AND SMOKING
AND DRINKING
HABITS
OF WORKERS
TESTED
Diagnostic radiation
Smoking
Drinking
Drugs
E.H.
--
+
-+
J.p.
--
_+
++
Meprobamat Papaverin Chloramphenicol Synt ophyllin
K.K.
+
+ +_
A.H. M.M.
--
--
E.J. J.K. F.M.
----
+ -+÷
A.T.
_+ +_ _+ ++ +_
Solutan Syntophyllin Bellaspon Superpyrin Thiospasmin Ketazon
100
TABLE 4 CHROMOSOMAL ANALYSIS OF 8 CONTROLS Aberrant cells a (%)
SCE/cell b
Smoking
Drinking
M.K.
3
10.88
--
--
J.K. V.B. J.~. K.B. M.Ku. Z.P. R.~.
0 3 3 1 1 2 1
8.22 10.30 10.16 10.46 8.26 7.94 9.02
-++ ++ --+ --
-+ + ---+
Mean
1.8 + 0 . 4
9.41 +- 0 . 4 0
a 1 0 0 cells w e r e s c o r e d p e r s a m p l e . b 5 0 cells w e r e s c o r e d p e r s a m p l e . T h e c o n t r o l s w e r e n o t e x p o s e d t o X-rays a n d d r u g s a t t h e t i m e o f b l o o d c o l l e c t i o n .
References 1 B a r t s c h , H., C. Malaveille a n d R. M o n t e s a n o , H u m a n , r a t a n d m o u s e l i v e r - m e d i a t e d m u t a g e n i c i t y of v i n y l c h l o r i d e in S. t y p h i m u r i u m strains, I n t . J. C a n c e r , 15 ( 1 9 7 5 ) 4 2 9 - - 4 3 7 . 2 B a r t s e h , H., a n d R. M o n t e s a n o , M u t a g e n i c a n d c a r c i n o g e n i c e f f e c t s of v i n y l c h l o r i d e , M u t a t i o n Res., 32 (1975) 93--114. 3 C r e e c h , J . L . , a n d M.N. J o h n s o n , A n g i o s a r c o m a o f the liver in t h e m a n u f a c t u r e o f p o l y v i n y l c h l o r i d e , J. O c c u p . Med., 16 ( 1 9 7 4 ) 1 5 0 - - 1 5 1 . 4 D u c a t m a n , V., K. H i r s c h h o r n a n d I.J. Selikoff, V i n y l c h l o r i d e e x p o s u r e a n d h u m a n c h r o m o s o m e a b e r r a t i o n s , M u t a t i o n Res., 31 ( 1 9 7 5 ) 1 6 3 - - 1 6 9 . 5 F u n e s - C r a v i o t o , F., B. L a m b e r t , J. L i n d s t e n , L. E h r e n b e r g , A . T . N a t a r a j a n a n d S. O s t e r m a n - G o l k a r , C h r o m o s o m e a b e r r a t i o n s in w o r k e r s e x p o s e d to v i n y l c h l o r i d e , L a n c e t , 1 ( 1 9 7 5 ) 4 5 9 . 6 H a n s t e e n , I . L . , L. Hillestad a n d E. T h i i s - E v e n s e n , 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 t o v i n y l c h l o r i d e , M u t a t i o n Res., 38 ( 1 9 7 6 ) 1 1 2 . 7 H a n s t e e n , I.L., C h r o m o s o m e b r e a k a g e f r e q u e n c y a n d sister c h r o m a t i d e e x c h a n g e s in PVC w o r k e r s t w o years after exposure, A follow-up study, Abstract of 6th International C h r o m o s o m e Conference, Helsinki, A u g u s t 1 9 7 7 . 8 Hungerford, D.A., L e u k o c y t e s cultured f r o m small inocula of whole blood and preparation of rectap h a s e c h r o m o s o m e s b y t r e a t m e n t w i t h h y p o t o n i c KC1, S t a i n T e c h n o l . , 4 0 ( 1 9 6 5 ) 3 3 3 - - 3 3 8 . 9 Ku~erov~i, M., C y t o g e n e t i c analysis o f h u m a n c h r o m o s o m e s a n d its v a l u e f o r t h e e s t i m a t i o n o f g e n e t i c risk, M u t a t i o n Res., 41 ( 1 9 7 6 ) 1 2 3 - - 1 3 0 . 1 0 L o p r i e n o , N., R. Barale, S. B a r o n c e l l i , C. B a u e r , G. B r o n z e t t i , A. C a m m e U i n i , G. C e r c i g n a n i , C. Corsi, G. Gervasi, C. L e p o r i n i , R. Nieri, A.M. Rossi, G. S t r e t t i a n d G. T u r c h i , E v a l u a t i o n of t h e g e n e t i c e f f e c t s i n d u c e d b y v i n y l c h l o r i d e m o n o m e r ( V C M ) u n d e r m a m m a l i a n m e t a b o l i c a c t i v a t i o n : in v i t r o a n d in v i v o s t u d i e s , M u t a t i o n Res., 4 0 ( 1 9 7 6 ) 8 5 - - 9 6 . 11 P u r c h a s e , I . F . H . , C. R i c h a r d s o n a n d D. A n d e r s o n , C h r o m o s o m a l a n d d o m i n a n t l e t h a l e f f e c t s o f v i n y l c h l o r i d e , L a n c e t , II ( 1 9 7 5 ) 4 1 0 - - 4 1 1 . 12 P u r c h a s e , I . F . H . , C. R i c h a r d s o n a n d D. A n d e r s o n , C h r o m o s o m a l e f f e c t s in p e r i p h e r a l l y m p h o c y t e s , Proe. R o y . Soc. Med., 6 9 ( 1 9 7 6 ) 2 9 0 - - 2 9 1 . 13 V i o h , P.L., A. Bigotti a n d A. C a p u t o , O n c o g e n i c r e s p o n s e of r a t skin, l u n g s a n d b o n e s t o v i n y l c h l o r i d e , C a n c e r Res., 31 ( 1 9 7 1 ) 5 1 6 - - 5 2 2 . 1 4 W o l f f , S., a n d P. P e r r y , D i f f e r e n t i a l G i e m s a s t a i n i n g of sister c h r o m a t i d s a n d t h e s t u d y o f sister c h r o matid exchanges without autoradiography, C h r o m o s o m a , 48 (1974) 341--353.