149
Mutation Research, 68 (1979) 149--152
© Elsevier/North-Holland Biomedical Press
SISTER-CHROMATID EXCHANGES IN O R A L CONTRACEPTIVE USERS
P. B A L A K R I S H N A M U R T H Y
and K. P R E M A
National Institute of Nutrition, Indian Council of Medical Research, Jamai Osmania (P.O.), Hyderabad-500 007, A.P. (India)
(Received 2 March 1979) (Revisionreceived22 May 1979) (Accepted 29 May 1979)
Summary The effect of the use o f an oral contraceptive on the frequency of sisterchromatid exchanges (SCEs) was investigated. Oral contraceptive users showed significantly higher mean SCE per cell compared with b o t h normal and pregnant women. This result obviously means an increased mutagenic environment in these cells -- due either to the pill itself or to a metabolite(s).
There have so far been only few studies on the mutagenic effect of oral contraceptive drugs. Earlier work conducted by Badr and Badr had demonstrated a high incidence of dominant lethal mutations in female mice given a large dose o f these drugs [1]. However, the results of the subsequent studies dealing with the frequency of chromosomal aberrations in w o m e n using the pill have been controversial [ 2,6,7 ]. Following the development of new cytological techniques [3,4,8,10--12], the cellular significance of sister-chromatid exchange (SCE) has been extensively studied. Results o f these and other investigations have shown that SCE is a sensitive indicator of subtle alterations in genetic material [e.g. 5,9]. Therefore we investigated the frequency of SCEs in w o m e n who used an oral contraceptive (OC) preparation. Materials and methods
A sample o f heparinized venous blood was obtained from 15 normal healthy non-pregnant women, 15 w o m e n in the third trimester o f pregnancy and 15 w o m e n taking an oral contraceptive preparation which contained 150 #g d-norgestrel and 50 or 30 #g ethenyl estradiol/day for periods ranging from 6 to 24 months (mean 16 months). The w o m e n were aged b e t w e e n 18 and 35 years (mean 23.6 years). Peripheral lymphocytes were cultured in Tc 199 me-
150
Fig. 1. A m e t a p h a s e s h o w i n g d i f f e r e n t i a l l y s t a i n e d c h r o m a t i d s a n d SCEs.
dium supplemented with 20% fetal calf serum, penicillin (100 u/ml), streptomycin (100 pg/ml) and PHA (Wellcome). Bromodeoxyuridine, at a final concentration of 10 #M, was added. All cultures were incubated in the dark for 72 h at 37°C. The cells were collected, exposed to colchicine (0.5 pg/ml) for 2 h, hypotonically shocked (0.075 M KC1) and fixed for chromosome preparations. Air-dried slides aged for a day were stained for SCEs by the HoechstGiemsa m e t h o d [8]. 25 differentially stained metaphases (Fig. 1) (M2 cells) were scored for each subject under a × 100 oil immersion objective. All slides were coded. Results Table 1 shows the mean frequencies o f SCE per cell. Oral contraceptive users had significantly higher mean SCEs per cell as compared with both non-pregnant controls and pregnant women. Mean values of SCEs in non-pregnant and pregnant women were similar. The increased mean SCE frequency seen in women on oral contraceptives was n o t due to a relatively large increase in a few subjects but it was a p h e n o m e n o n seen in most subjects (Table 2).
151 TABLI~ 1 MEAN
FREQUENCIES
OF SCEs SCEs p e r cell
Number of cells examined
T o t a l SCEs
15
375
2143
Pregnant women
15
375
2360
6.3±0.2
OC users
15
375
3641
9.7±0.6
Group
Number of women
N on-pregnant women (control)
Mean a
Range
5.8±0.3
0--17 0--15 b
1--21
a V a l u e s are m e a n ± S.E. b S i g n i f i c a n t at P ~ 0 . 0 0 1 . P b y S t u d e n t ' s " t " t e s t .
TABLE
2
INDIVIDUAL Serial No.
SCE FREQUENCY
IN W O M E N
ON THE ORAL
CONTRACEPTIVE
M e a n S C E ± S.D.
1 2 3
9.0±2.4 8.8±2.5 11.2±3.7
4 5 6
11.0±3.9 12.1±2.9 12.4±3.6
7 8 9
11.7±1.9 11.8±3.2 11.8±3.7
10 11 12
11.0±2.9 7.2±1.2 7.8±1.4
13 14 15
7.3±2.0 6.5±1.4 5.9±1.9
Total
9.7±0.6
a
a M e a n + S.E.
Discussion These results suggest that, in pill users, the mean SCE per cell is increased, the increase being about 75%. Another consistent finding was that, in these women, more than 45% of cells examined had SCEs ranging from 10 to 21 as against only 17% of cells in control subjects. Pregnant women were included in the study, to assess the role, if any, of an altered hormonal profile on SCE. The absence o f changes suggest that an altered hormonal profile per se had not contributed to altered SCE rates. The increased frequency of SCEs in the cells of OC users obviously means an increased mutagenic environment in these cells -either due to OC itself or to a metabolite(s). It is also possible that excipients in the pill may have had a role. This, however, was not tested, because it is believed that such substances are inert. The mechanism by which the use of the pill brings about these changes is not
152 known, nor is the biological significance. Until such time as the functional significance of the elevated SCE frequency is known, it would be wise to view it with caution.
Acknowledgements We are grateful to Dr. S.G. Srikantia, Director, National Institute of Nutrition, Hyderabad, for his active guidance and interest. We thank Prof. H. Sharatchandra of the Cell Biology and Microbiology Laboratory, Indian Institute o f Science, Bangalore, for his help during the preparation of the manuscript. Our special thanks are due to Dr. T.C. Raghuram, Research Officer, National Institute o f Nutrition, for his useful suggestions.
References 1 B a d r , F . M . , a n d R.S. B a d r , S t u d i e s o n t h e m u t a g e n i c e f f e c t o f c o n t r a c e p t i v e d r u g s , I. I n d u c t i o n o f d o m i n a n t l e t h a l m u t a t i o n s in f e m a l e m i c e , M u t a t i o n R e s . , 2 6 ( 1 9 7 4 ) 5 2 9 - - 5 3 4 . 2 B i s h u n , N., N. S m i t h , D. Williams a n d J. Mills, C y t o l o g i c a l e f f e c t s of t h e o r a l c o n t r a c e p t i v e , M u t a t i o n Res., 39 (1976) 97--110. 3 G o t o , K., T. A k e m a t s u , H. S h i m a z u a n d T. S u g i y a m a , S i m p l e d i f f e r e n t i a l s t a i n i n g o f sister c h r o m a t i d s after treatment with photosensitive dyes and exposure to light and the mechanism of staining, Chromosoma, 53 (1975) 223--230. 4 K a t o , H., S p o n t a n e o u s sister c h r o m a t i d e x c h a n g e s d e t e c t e d b y ~ a B U d R - l a b e l l i n g m e t h o d , N a t u r e (London), 251 (1974) 70--72. 5 L a t t , S . A . , Sister e h r o m a t i d e x c h a n g e s , i n d i c e s o f h u m a n c h r o m o s o m e d a m a g e a n d r e p a i r : D e t e c t i o n b y f l u o r e s c e n c e a n d i n d u c t i o n b y M i t o m y c i n - C , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 1 ( 1 9 7 4 ) 3 1 6 2 - 3166. 6 L a u r i s t e n , J . G . , T h e s i g n i f i c a n c e o f o r a l c o n t r a c e p t i v e s in c a u s i n g a n o m a l i e s in s p o n t a n e o u s a b o r t i o n s , Acta Obstet. Gynecol. Scand., 54 (1975) 261--264. 7 L i t t l e f i e l d , L . G . , W.E. L e v e r , F . L . Miller a n d K . O . G o h , C h r o m o s o m e b r e a k a g e s t u d i e s in l y m p h o c y t e s f r o m n o r m a l w o m e n , p r e g n a n t w o m e n , a n d w o m e n t a k i n g o r a l c o n t r a c e p t i v e s , A m . J. O b s t e t . Gynecol., 121 (1975) 976--980. 8 P e r r y , P., a n d S. W o l f f , N e w G i e m s a m e t h o d f o r t h e d i f f e r e n t i a l s t a i n i n g o f sister c h r o m a t i d s , N a t u r e (London), 251 (1974) 156--158. 9 P e r r y , P., a n d H . J . E v a n s , C y t o l o g i c a l d e t e c t i o n o f m u t a g e n - c a r c i n o g e n e x p o s u r e b y sister c h r o m a t i d exchange, Nature (London), 258 (1975) 121--125. 1 0 S c h n e i d e r , E . L . , R . R . Tice a n d D. K r a m , B r o m o d e o x y u r i d i n e - d i f f e r e n t i a l s t a i n i n g ' t e c h n i q u e : A n e w a p p r o a c h t o e x a m i n i n g sister c h r o m a t i d e x c h a n g e a n d cell r e p l i c a t i o n k i n e t i c s , i n : M e t h o d s in Cell B i o l o g y , A c a d e m i c Press, N e w Y o r k , 1 9 7 7 . 11 S o l o m o n , E., a n d M. B o b r o w , Sister c h r o m a t i d e x c h a n g e s , A sensitive a s s a y o f a g e n t s d a m a g i n g human chromosomes, Mutation Res., 30 (1975) 273--278. 1 2 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 o f sister e h r o m a t i d s a n d t h e s t u d y o f s i s t e r c h r o matid exchanges without autoradiography, Chromosoma, 48 (1974) 341--353.
Mutation Research, 68 (1979) 149--152
© Elsevier/North-Holland Biomedical Press
SISTER-CHROMATID EXCHANGES IN O R A L CONTRACEPTIVE USERS
P. B A L A K R I S H N A M U R T H Y
and K. P R E M A
National Institute of Nutrition, Indian Council of Medical Research, Jamai Osmania (P.O.), Hyderabad-500 007, A.P. (India)
(Received 2 March 1979) (Revisionreceived22 May 1979) (Accepted 29 May 1979)
Summary The effect of the use o f an oral contraceptive on the frequency of sisterchromatid exchanges (SCEs) was investigated. Oral contraceptive users showed significantly higher mean SCE per cell compared with b o t h normal and pregnant women. This result obviously means an increased mutagenic environment in these cells -- due either to the pill itself or to a metabolite(s).
There have so far been only few studies on the mutagenic effect of oral contraceptive drugs. Earlier work conducted by Badr and Badr had demonstrated a high incidence of dominant lethal mutations in female mice given a large dose o f these drugs [1]. However, the results of the subsequent studies dealing with the frequency of chromosomal aberrations in w o m e n using the pill have been controversial [ 2,6,7 ]. Following the development of new cytological techniques [3,4,8,10--12], the cellular significance of sister-chromatid exchange (SCE) has been extensively studied. Results o f these and other investigations have shown that SCE is a sensitive indicator of subtle alterations in genetic material [e.g. 5,9]. Therefore we investigated the frequency of SCEs in w o m e n who used an oral contraceptive (OC) preparation. Materials and methods
A sample o f heparinized venous blood was obtained from 15 normal healthy non-pregnant women, 15 w o m e n in the third trimester o f pregnancy and 15 w o m e n taking an oral contraceptive preparation which contained 150 #g d-norgestrel and 50 or 30 #g ethenyl estradiol/day for periods ranging from 6 to 24 months (mean 16 months). The w o m e n were aged b e t w e e n 18 and 35 years (mean 23.6 years). Peripheral lymphocytes were cultured in Tc 199 me-
150
Fig. 1. A m e t a p h a s e s h o w i n g d i f f e r e n t i a l l y s t a i n e d c h r o m a t i d s a n d SCEs.
dium supplemented with 20% fetal calf serum, penicillin (100 u/ml), streptomycin (100 pg/ml) and PHA (Wellcome). Bromodeoxyuridine, at a final concentration of 10 #M, was added. All cultures were incubated in the dark for 72 h at 37°C. The cells were collected, exposed to colchicine (0.5 pg/ml) for 2 h, hypotonically shocked (0.075 M KC1) and fixed for chromosome preparations. Air-dried slides aged for a day were stained for SCEs by the HoechstGiemsa m e t h o d [8]. 25 differentially stained metaphases (Fig. 1) (M2 cells) were scored for each subject under a × 100 oil immersion objective. All slides were coded. Results Table 1 shows the mean frequencies o f SCE per cell. Oral contraceptive users had significantly higher mean SCEs per cell as compared with both non-pregnant controls and pregnant women. Mean values of SCEs in non-pregnant and pregnant women were similar. The increased mean SCE frequency seen in women on oral contraceptives was n o t due to a relatively large increase in a few subjects but it was a p h e n o m e n o n seen in most subjects (Table 2).
151 TABLI~ 1 MEAN
FREQUENCIES
OF SCEs SCEs p e r cell
Number of cells examined
T o t a l SCEs
15
375
2143
Pregnant women
15
375
2360
6.3±0.2
OC users
15
375
3641
9.7±0.6
Group
Number of women
N on-pregnant women (control)
Mean a
Range
5.8±0.3
0--17 0--15 b
1--21
a V a l u e s are m e a n ± S.E. b S i g n i f i c a n t at P ~ 0 . 0 0 1 . P b y S t u d e n t ' s " t " t e s t .
TABLE
2
INDIVIDUAL Serial No.
SCE FREQUENCY
IN W O M E N
ON THE ORAL
CONTRACEPTIVE
M e a n S C E ± S.D.
1 2 3
9.0±2.4 8.8±2.5 11.2±3.7
4 5 6
11.0±3.9 12.1±2.9 12.4±3.6
7 8 9
11.7±1.9 11.8±3.2 11.8±3.7
10 11 12
11.0±2.9 7.2±1.2 7.8±1.4
13 14 15
7.3±2.0 6.5±1.4 5.9±1.9
Total
9.7±0.6
a
a M e a n + S.E.
Discussion These results suggest that, in pill users, the mean SCE per cell is increased, the increase being about 75%. Another consistent finding was that, in these women, more than 45% of cells examined had SCEs ranging from 10 to 21 as against only 17% of cells in control subjects. Pregnant women were included in the study, to assess the role, if any, of an altered hormonal profile on SCE. The absence o f changes suggest that an altered hormonal profile per se had not contributed to altered SCE rates. The increased frequency of SCEs in the cells of OC users obviously means an increased mutagenic environment in these cells -either due to OC itself or to a metabolite(s). It is also possible that excipients in the pill may have had a role. This, however, was not tested, because it is believed that such substances are inert. The mechanism by which the use of the pill brings about these changes is not
152 known, nor is the biological significance. Until such time as the functional significance of the elevated SCE frequency is known, it would be wise to view it with caution.
Acknowledgements We are grateful to Dr. S.G. Srikantia, Director, National Institute of Nutrition, Hyderabad, for his active guidance and interest. We thank Prof. H. Sharatchandra of the Cell Biology and Microbiology Laboratory, Indian Institute o f Science, Bangalore, for his help during the preparation of the manuscript. Our special thanks are due to Dr. T.C. Raghuram, Research Officer, National Institute o f Nutrition, for his useful suggestions.
References 1 B a d r , F . M . , a n d R.S. B a d r , S t u d i e s o n t h e m u t a g e n i c e f f e c t o f c o n t r a c e p t i v e d r u g s , I. I n d u c t i o n o f d o m i n a n t l e t h a l m u t a t i o n s in f e m a l e m i c e , M u t a t i o n R e s . , 2 6 ( 1 9 7 4 ) 5 2 9 - - 5 3 4 . 2 B i s h u n , N., N. S m i t h , D. Williams a n d J. Mills, C y t o l o g i c a l e f f e c t s of t h e o r a l c o n t r a c e p t i v e , M u t a t i o n Res., 39 (1976) 97--110. 3 G o t o , K., T. A k e m a t s u , H. S h i m a z u a n d T. S u g i y a m a , S i m p l e d i f f e r e n t i a l s t a i n i n g o f sister c h r o m a t i d s after treatment with photosensitive dyes and exposure to light and the mechanism of staining, Chromosoma, 53 (1975) 223--230. 4 K a t o , H., S p o n t a n e o u s sister c h r o m a t i d e x c h a n g e s d e t e c t e d b y ~ a B U d R - l a b e l l i n g m e t h o d , N a t u r e (London), 251 (1974) 70--72. 5 L a t t , S . A . , Sister e h r o m a t i d e x c h a n g e s , i n d i c e s o f h u m a n c h r o m o s o m e d a m a g e a n d r e p a i r : D e t e c t i o n b y f l u o r e s c e n c e a n d i n d u c t i o n b y M i t o m y c i n - C , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 1 ( 1 9 7 4 ) 3 1 6 2 - 3166. 6 L a u r i s t e n , J . G . , T h e s i g n i f i c a n c e o f o r a l c o n t r a c e p t i v e s in c a u s i n g a n o m a l i e s in s p o n t a n e o u s a b o r t i o n s , Acta Obstet. Gynecol. Scand., 54 (1975) 261--264. 7 L i t t l e f i e l d , L . G . , W.E. L e v e r , F . L . Miller a n d K . O . G o h , C h r o m o s o m e b r e a k a g e s t u d i e s in l y m p h o c y t e s f r o m n o r m a l w o m e n , p r e g n a n t w o m e n , a n d w o m e n t a k i n g o r a l c o n t r a c e p t i v e s , A m . J. O b s t e t . Gynecol., 121 (1975) 976--980. 8 P e r r y , P., a n d S. W o l f f , N e w G i e m s a m e t h o d f o r t h e d i f f e r e n t i a l s t a i n i n g o f sister c h r o m a t i d s , N a t u r e (London), 251 (1974) 156--158. 9 P e r r y , P., a n d H . J . E v a n s , C y t o l o g i c a l d e t e c t i o n o f m u t a g e n - c a r c i n o g e n e x p o s u r e b y sister c h r o m a t i d exchange, Nature (London), 258 (1975) 121--125. 1 0 S c h n e i d e r , E . L . , R . R . Tice a n d D. K r a m , B r o m o d e o x y u r i d i n e - d i f f e r e n t i a l s t a i n i n g ' t e c h n i q u e : A n e w a p p r o a c h t o e x a m i n i n g sister c h r o m a t i d e x c h a n g e a n d cell r e p l i c a t i o n k i n e t i c s , i n : M e t h o d s in Cell B i o l o g y , A c a d e m i c Press, N e w Y o r k , 1 9 7 7 . 11 S o l o m o n , E., a n d M. B o b r o w , Sister c h r o m a t i d e x c h a n g e s , A sensitive a s s a y o f a g e n t s d a m a g i n g human chromosomes, Mutation Res., 30 (1975) 273--278. 1 2 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 o f sister e h r o m a t i d s a n d t h e s t u d y o f s i s t e r c h r o matid exchanges without autoradiography, Chromosoma, 48 (1974) 341--353.
