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Sister-chromatid exchanges in children treated with nalidixic acid
371
Mutation Research, 77 (1980) 371--375 © Elsevier/North-Holland Biomedical Press
Short Communication SISTER-CHROMATID EXCHANGES IN CHILDREN TREATED WITH NALIDIXIC ACID
JERZY KOWALCZYK Institute of Pediatrics, Staszica 11, 20-081 Lublin (Poland) (Received 18 July 1979) (Revision received 8 October 1979) (Accepted 17 October 1979)
The recently developed BrdU-labelling techniques have provided a sensitive and reproducible means of measuring chromosomal damage induced by chemical mutagens and carcinogens [1,11,13,14,16]. An increased rate of sister-chromatid exchanges (SCEs) induced by some drugs, mainly cytostatics, has been demonstrated in human lymphocytes [11,12]. In the present study the effect of the treatment with nalidixic acid (negram, 1-ethyl-l,4-dihydro-7-methyl-4-oxo-l,S-naphthiridine-3-carboxylic acid) on SCEs was investigated. The mode of action of this compound is not exactly known. It does not inhibit the DNA synthesis in a direct way and does not intercalate with DNA [3]. The most recent studies suggest that negram inhibits the activity of DNA-gyrase in Escherichia coli [15]. The mechanisms for DNA repair in E. coli also play a role in its action because recombination~leficient mutants of E. coli show increased sensitivity to negram and inability to repair the damage that accumulates during the inhibition of DNA synthesis by this compound [10]. In the present study, 12 children (3 boys and 9 girls), aged from 5 months to 12 years, with an infection of the urinary tract were investigated. These children were not treated with any drug for a few months before the diagnosis. The control group comprised 12 apparently healthy children aged from 3 to 19 years. Venous blood was collected from each sick donor before and after 10 days of the treatment with negram (Nevigramon, Chinoin) at 50 mg per kg body weight. Peripheral leukocytes were grown in total darkness at 37°C for 72 h in culture medium containing phytohaemagglutinin and 32.6 pM 5-bromodeoxyuridine (Koch-Light). Colcemid (Ciba) was added during the last 2 h of culture. Slides were prepared according to standard procedures and stained with acridine orange, at 0.125 mg/ml, dissolved in phosphate buffer at pH 6.0 [6]. The metaphases were examined by fluorescent microscopy and analyzed from microphotographs. Only cells showing a complete chromosome complement, good differential staining of chromatids and an absence of confusing chromosome overlaps were accepted for scoring. The number of SCEs in at
372
TABLE 1 M E A N V A L U E S O F SCEs I N C H I L D R E N W I T H U R I N A R Y T R A C T I N F E C T I O N S B E F O R E A N D AFT E R T R E A T M E N T W I T H N A L I D I X I C A C I D , I N C O M P A R I S O N W I T H V A L U E S IN H E A L T H Y SUBJECTS Number of
Av. n u m ber of
Cells
Chromosomes
SCEs/ chrom.
12
180
8280
0.269
2.3
1 2 . 4 -+ 2.5
before treat.
12
182
8372
0.285
2.9
13.1 -+ 3.0
after treat.
12
162
7452
0.572
3.1
26.3 -+ 7.6
Group
Number of children
Healthy
Av, n u m b e r o f CMEs/metaph.
Av. number o f SCEs/ m e t a p h . ± SD
CMEs, c e n t r o m e r i c e x c h a n g e s ; S D , s t a n d a r d d e v i a t i o n . Significant differences were found by t test when healthy children were compared with sick children b e f o r e t h e t r e a t m e n t (p <: 0 . 0 5 ) a n d c h i l d r e n b e f o r e t h e t r e a t m e n t w e r e c o m p a r e d w i t h c h i l d r e n after t r e a t m e n t (p ~ 0 . 0 0 1 ) .
least 13 metaphases was recorded from each culture. The results are presented in Tables 1 and 2. An average of 13.1 + 3.0 SCEs per metaphase was observed in 182 cells obtained from children with urinary tract infections. This result differs statistically from that obtained with healthy children {12.4 -+ 2.5, p < 0.05). Analysis of variance, used to test the intragroup variability, indicated that, in both groups, the differences between individuals were statistically significant (p < 0.01). This variability may reflect inter-individual differences in the efficiency of mechanisms involved in the production of exchanges. It appears to indicate that there is probably no relation between the slightly higher mean value of SCEs in children with a
TABLE 2 SCE F R E Q U E N C I E S IN I N D I V I D U A L C H I L D R E N W I T H U R I N A R Y T R A C T I N F E C T I O N S B E F O R E AND AFTER TREATMENT Patient
Sex
Before treatment
After treatment
SCE/cen
Range
SCE/cell
Range
7--13 8--12 10--14 7--14
21.7 22.5 23.7 21.3
18--24 17--26 21--27 19--24
1 2
M M
3 4
M F
9.7 10.3 11.7 12.6
5 6 7 8
F F F F
15.9 14.8 12.9 9.7
11--24 12--18 10--15 8--11
21.6 24.7 20.7 25.6
16--33 21--40 16--24 21--33
9 10 11 12
F F F F
14.5 14.8 14.3 15.1
12--21 12--19 12--18 13--18
43.5 35.3 23.4 35.9
36--53 20--43 21--26 30--46
M, m a l e ; F, f e m a l e .
373 of metaphases t5' ii
14
il
13
~
,
12 11
!~
9-
I ~'
~
8765"
1
~. 8
,,,, I
~
~
•'
~ ,
~,
, , , / ,~ . . . . . . . ~, , I I . ,', , .. ". =_ 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 SCE/metaph.
F i g . I . D i s t r i b u t i o n o f f r e q u e n c i e s o f s i s t e r - c h r o m a t i d e x c h a n g e s e x p r e s s e d in p e r c e n t a g e . F u l l llne, cells f r o m c h i l d r e n w i t h u r i n a r y t r a c t i n f e c t i o n s b e f o r e t r e a t m e n t . D a s h e d line, ceils f r o m c h i l d r e n a f t e r treatment with nalidixic acid.
urinary tract infection and pathological agents. The number of centromeric exchanges (CME) was similar in both groups. There was a more than twofold increase of the SCE frequency in lymphocytes of children after 10 days of treatment with nalidixic acid. The mean value of 26.3 + 7.6 SCEs/metaphase was significantly higher than in children before the treatment (p < 0.001). The increase in the SCE frequency after negram was observed in all patients studied. However, a wide range of SCE values (from 16 to 53) was found in 162 metaphases scored. Treatment with negram caused only a small increase in mean value of centromeric exchanges to 3.1/metaphase. The results may indicate that negram used in normal, therapeutic d o s e s damages DNA and the damage is repaired by special mechanisms resulting in SCEs. The distribution of cells with various numbers of SCEs resembled a bimodal curve in cultures from donors both before and after treatment (Fig. 1). Similar results were obtained by Lezana et al. [9] in normal human beings and in those with Down syndrome, and by Raposa [12] in haematological disorders, o n e may speculate that a differential uptake or a variable cellular sensitivity to the drug produces different rates of SCEs [9]. The frequency of SCEs in blood cultures may be the average of the rates exhibited by two or more lymphocyte sub-populations with different sensitivities to mutagens. The tendency to a bimodal distribution in the cells with different numbers of SCEs seems to support the above assumption. In both examinations, before and after the treatment, the distribution of SCEs was related to the lengths of each chromosomal group. Regression analyses of the number of SCEs and chromosome lengths for the two groups of
374 • ; iCE-,
11 10 9 8 7 6 5 4 3 2 1
.v'/N
;~11~1l i GFED
g ~1 ~ Ig ~ relative C B A length
Fig. 2. R e l a t i v e f r e q u e n c y d i s t r i b u t i o n o f S C E s as a f u n c t i o n o f c h r o m o s o m e l e n g t h . A - - G , c h r o m o s o m a l groups. - - , s a m p l e r e g r e s s i o n o f S C E s o n l e n g t h o f c h r o m o s o m a l g r o u p in c h i l d r e n b e f o r e a n d ...... , a f t e r t h e t r e a t m e n t , o, o b s e r v e d m e a n f r e q u e n c y o f S C E s f o r e a c h o f t h e i n d i c a t e d c h r o m o s o m e g r o u p s in c h i l d r e n b e f o r e a n d o a f t e r t h e t r e a t m e n t .
children are shown in Fig. 2. This finding matched well with some previous reports that numbers of SCEs are proportional to the lengths of chromosomes [2,4,5,7,8]. However, a chi-square analysis of the distribution of numbers of SCEs in individual chromosomal groups indicated a highly significant difference
F i g . 3. 43 S C E s w e r e o b s e r v e d in t h i s m e t a p h a s e o f a child w i t h a u r i n a r y t r a c t i n f e c t i o n a f t e r t r e a t m e n t with nalidixic acid.
375
(p < 0.001) between the expected and observed values in both examinations. In particular, an elevated number of SCEs was noted on chromosomes of the D group as well as a decrease of the C group before the treatment and a decrease of the C group chromosomes after the treatment. No explanation of this phenomenon is known. It may reflect differences in base composition along the length of these chromosomes [2]. The results of these investigations indicate that negram may cause damage to DNA and ought to be used with care, especially in pregnant w o m e n and small babies. Acknowledgements I most gratefully thank Professor Danuta Ko~ynkowa and Professor Antoni G~bala for valuable advice and criticism in the preparation of the manuscript. References 1 Beek, B., and G. Obe, The h u m a n l e u k o c y t e test system, VI. The use of sister c h r o m a t i d exchanges as possible indicators for mutagenic activities, Hum. Genet., 29 (1975) 127--134. 2 Crossen, P.E., M.E. Drets, F.E. Arrighi and D.A. Johnston, Analysis of the frequency and di s t ri but i on of sister chro matid exchanges in cultured h u m a n l y m p h o c y t e s , Hum. Genet., 35 (1977) 345--352. 3 Crumplin, G.C., and J.T. Smith, Nalidixic acid and bacterial c h r o m o s o m e replication, Nature (London), 260 (1976) 643--645. 4 Dutrillaux, B., A.M. Fosse, M. Prieur and J. Lejeune, Analyse des exchanges de chrornatides dans les cellules somatiques humalnes, Chromosoma, 48 (1974) 327--340. 5 Galloway, S.M., and H.J. Evans, Sister c h r o m a t i d exchange in h u m a n c h r o m o s o m e s from n o r m a l individuals and patients with ataxia-telangiectasia, Cytogenet. Cell Genet., 15 (1975) 17--29. 6 Kato, H., SPontaneous sister chromatid exchanges detec t e d by a BUdR-labelling m e t h o d , Nature (London), 251 (1974) 70--72. 7 Lambert, B., K. Hansson J. Lindsten, M. Sten and B. Werellus, B r o m o d e o x y u r l d t n e - i n d u c e d sistez chr omatid exchanges in h u m a n l y m p h o c y t e s , Hereditas, 83 (1976) 163--174. 8 Latt, S.A., Microfluorometric detection of deoxyribonucl e i c acid replication in h u m a n me t a pha s e chromosomes, Proc. Natl. Acad. Sci. (U.S.A.), 70 (1973) 3395--3399. 9 Lezana, E.A., N.O. Bianchi, M.S. Bianchi and J.E. Zabala-Suarez, Sister c hroma t i d exchanges in Down syndromes and normal h u m a n beings, Mutation Res., 45 (1977) 85--90. 10 McDaniel, L.S., L.H. Rogers and W.E. Hill, Survival of recombination-deficient m u t a n t s of Escherichia coli during incubation with nalidixic acid, J. Bacteriol., 134 (1978) 1195--1198. 11 Perry, P., and H.J. Evans, Cytological d e t e c t i o n of mutagen--carcinogen exposure by sister c hroma t i d exchange, Nature (London), 258 (1975) 121--125. 12 Raposa, T., Sister ehromatid exchange studies for m o n i t o r i n g DNA damage and repair capacity after cytostatics in vitro and in l y m p h o c y t e s of leukaemic pa t i e nt s under c yt os t a t i c t he ra py, Mut a t i on Res., 57 (1978) 241--251. 13 Savage, J.R.K., Chro mosomal aberrations as test for mu t a ge ni c i t y, Nature (London), 258 (1975) 103--104. 14 Solomon, E., and M. Bohrow, Sister c h r o m a t i d exchanges -- a sensitive assay of agents damaging h u m a n chromosomes, Mutation Res., 30 (1975) 273--278, 15 Smith, C.L., M. Kubo and F. I m a m o t o , Promoter-specific i n h i b i t i o n of t ra ns c ri pt i on by a nt i bi ot i c s which act on DNA gyrase, Nature (London), 275 (1978) 420--423. 16 Wolff, S., Sister c h r o m a t i d exchange, Annu. Rev. Genet., 11 (1977) 183--201.
Mutation Research, 77 (1980) 371--375 © Elsevier/North-Holland Biomedical Press
Short Communication SISTER-CHROMATID EXCHANGES IN CHILDREN TREATED WITH NALIDIXIC ACID
JERZY KOWALCZYK Institute of Pediatrics, Staszica 11, 20-081 Lublin (Poland) (Received 18 July 1979) (Revision received 8 October 1979) (Accepted 17 October 1979)
The recently developed BrdU-labelling techniques have provided a sensitive and reproducible means of measuring chromosomal damage induced by chemical mutagens and carcinogens [1,11,13,14,16]. An increased rate of sister-chromatid exchanges (SCEs) induced by some drugs, mainly cytostatics, has been demonstrated in human lymphocytes [11,12]. In the present study the effect of the treatment with nalidixic acid (negram, 1-ethyl-l,4-dihydro-7-methyl-4-oxo-l,S-naphthiridine-3-carboxylic acid) on SCEs was investigated. The mode of action of this compound is not exactly known. It does not inhibit the DNA synthesis in a direct way and does not intercalate with DNA [3]. The most recent studies suggest that negram inhibits the activity of DNA-gyrase in Escherichia coli [15]. The mechanisms for DNA repair in E. coli also play a role in its action because recombination~leficient mutants of E. coli show increased sensitivity to negram and inability to repair the damage that accumulates during the inhibition of DNA synthesis by this compound [10]. In the present study, 12 children (3 boys and 9 girls), aged from 5 months to 12 years, with an infection of the urinary tract were investigated. These children were not treated with any drug for a few months before the diagnosis. The control group comprised 12 apparently healthy children aged from 3 to 19 years. Venous blood was collected from each sick donor before and after 10 days of the treatment with negram (Nevigramon, Chinoin) at 50 mg per kg body weight. Peripheral leukocytes were grown in total darkness at 37°C for 72 h in culture medium containing phytohaemagglutinin and 32.6 pM 5-bromodeoxyuridine (Koch-Light). Colcemid (Ciba) was added during the last 2 h of culture. Slides were prepared according to standard procedures and stained with acridine orange, at 0.125 mg/ml, dissolved in phosphate buffer at pH 6.0 [6]. The metaphases were examined by fluorescent microscopy and analyzed from microphotographs. Only cells showing a complete chromosome complement, good differential staining of chromatids and an absence of confusing chromosome overlaps were accepted for scoring. The number of SCEs in at
372
TABLE 1 M E A N V A L U E S O F SCEs I N C H I L D R E N W I T H U R I N A R Y T R A C T I N F E C T I O N S B E F O R E A N D AFT E R T R E A T M E N T W I T H N A L I D I X I C A C I D , I N C O M P A R I S O N W I T H V A L U E S IN H E A L T H Y SUBJECTS Number of
Av. n u m ber of
Cells
Chromosomes
SCEs/ chrom.
12
180
8280
0.269
2.3
1 2 . 4 -+ 2.5
before treat.
12
182
8372
0.285
2.9
13.1 -+ 3.0
after treat.
12
162
7452
0.572
3.1
26.3 -+ 7.6
Group
Number of children
Healthy
Av, n u m b e r o f CMEs/metaph.
Av. number o f SCEs/ m e t a p h . ± SD
CMEs, c e n t r o m e r i c e x c h a n g e s ; S D , s t a n d a r d d e v i a t i o n . Significant differences were found by t test when healthy children were compared with sick children b e f o r e t h e t r e a t m e n t (p <: 0 . 0 5 ) a n d c h i l d r e n b e f o r e t h e t r e a t m e n t w e r e c o m p a r e d w i t h c h i l d r e n after t r e a t m e n t (p ~ 0 . 0 0 1 ) .
least 13 metaphases was recorded from each culture. The results are presented in Tables 1 and 2. An average of 13.1 + 3.0 SCEs per metaphase was observed in 182 cells obtained from children with urinary tract infections. This result differs statistically from that obtained with healthy children {12.4 -+ 2.5, p < 0.05). Analysis of variance, used to test the intragroup variability, indicated that, in both groups, the differences between individuals were statistically significant (p < 0.01). This variability may reflect inter-individual differences in the efficiency of mechanisms involved in the production of exchanges. It appears to indicate that there is probably no relation between the slightly higher mean value of SCEs in children with a
TABLE 2 SCE F R E Q U E N C I E S IN I N D I V I D U A L C H I L D R E N W I T H U R I N A R Y T R A C T I N F E C T I O N S B E F O R E AND AFTER TREATMENT Patient
Sex
Before treatment
After treatment
SCE/cen
Range
SCE/cell
Range
7--13 8--12 10--14 7--14
21.7 22.5 23.7 21.3
18--24 17--26 21--27 19--24
1 2
M M
3 4
M F
9.7 10.3 11.7 12.6
5 6 7 8
F F F F
15.9 14.8 12.9 9.7
11--24 12--18 10--15 8--11
21.6 24.7 20.7 25.6
16--33 21--40 16--24 21--33
9 10 11 12
F F F F
14.5 14.8 14.3 15.1
12--21 12--19 12--18 13--18
43.5 35.3 23.4 35.9
36--53 20--43 21--26 30--46
M, m a l e ; F, f e m a l e .
373 of metaphases t5' ii
14
il
13
~
,
12 11
!~
9-
I ~'
~
8765"
1
~. 8
,,,, I
~
~
•'
~ ,
~,
, , , / ,~ . . . . . . . ~, , I I . ,', , .. ". =_ 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 SCE/metaph.
F i g . I . D i s t r i b u t i o n o f f r e q u e n c i e s o f s i s t e r - c h r o m a t i d e x c h a n g e s e x p r e s s e d in p e r c e n t a g e . F u l l llne, cells f r o m c h i l d r e n w i t h u r i n a r y t r a c t i n f e c t i o n s b e f o r e t r e a t m e n t . D a s h e d line, ceils f r o m c h i l d r e n a f t e r treatment with nalidixic acid.
urinary tract infection and pathological agents. The number of centromeric exchanges (CME) was similar in both groups. There was a more than twofold increase of the SCE frequency in lymphocytes of children after 10 days of treatment with nalidixic acid. The mean value of 26.3 + 7.6 SCEs/metaphase was significantly higher than in children before the treatment (p < 0.001). The increase in the SCE frequency after negram was observed in all patients studied. However, a wide range of SCE values (from 16 to 53) was found in 162 metaphases scored. Treatment with negram caused only a small increase in mean value of centromeric exchanges to 3.1/metaphase. The results may indicate that negram used in normal, therapeutic d o s e s damages DNA and the damage is repaired by special mechanisms resulting in SCEs. The distribution of cells with various numbers of SCEs resembled a bimodal curve in cultures from donors both before and after treatment (Fig. 1). Similar results were obtained by Lezana et al. [9] in normal human beings and in those with Down syndrome, and by Raposa [12] in haematological disorders, o n e may speculate that a differential uptake or a variable cellular sensitivity to the drug produces different rates of SCEs [9]. The frequency of SCEs in blood cultures may be the average of the rates exhibited by two or more lymphocyte sub-populations with different sensitivities to mutagens. The tendency to a bimodal distribution in the cells with different numbers of SCEs seems to support the above assumption. In both examinations, before and after the treatment, the distribution of SCEs was related to the lengths of each chromosomal group. Regression analyses of the number of SCEs and chromosome lengths for the two groups of
374 • ; iCE-,
11 10 9 8 7 6 5 4 3 2 1
.v'/N
;~11~1l i GFED
g ~1 ~ Ig ~ relative C B A length
Fig. 2. R e l a t i v e f r e q u e n c y d i s t r i b u t i o n o f S C E s as a f u n c t i o n o f c h r o m o s o m e l e n g t h . A - - G , c h r o m o s o m a l groups. - - , s a m p l e r e g r e s s i o n o f S C E s o n l e n g t h o f c h r o m o s o m a l g r o u p in c h i l d r e n b e f o r e a n d ...... , a f t e r t h e t r e a t m e n t , o, o b s e r v e d m e a n f r e q u e n c y o f S C E s f o r e a c h o f t h e i n d i c a t e d c h r o m o s o m e g r o u p s in c h i l d r e n b e f o r e a n d o a f t e r t h e t r e a t m e n t .
children are shown in Fig. 2. This finding matched well with some previous reports that numbers of SCEs are proportional to the lengths of chromosomes [2,4,5,7,8]. However, a chi-square analysis of the distribution of numbers of SCEs in individual chromosomal groups indicated a highly significant difference
F i g . 3. 43 S C E s w e r e o b s e r v e d in t h i s m e t a p h a s e o f a child w i t h a u r i n a r y t r a c t i n f e c t i o n a f t e r t r e a t m e n t with nalidixic acid.
375
(p < 0.001) between the expected and observed values in both examinations. In particular, an elevated number of SCEs was noted on chromosomes of the D group as well as a decrease of the C group before the treatment and a decrease of the C group chromosomes after the treatment. No explanation of this phenomenon is known. It may reflect differences in base composition along the length of these chromosomes [2]. The results of these investigations indicate that negram may cause damage to DNA and ought to be used with care, especially in pregnant w o m e n and small babies. Acknowledgements I most gratefully thank Professor Danuta Ko~ynkowa and Professor Antoni G~bala for valuable advice and criticism in the preparation of the manuscript. References 1 Beek, B., and G. Obe, The h u m a n l e u k o c y t e test system, VI. The use of sister c h r o m a t i d exchanges as possible indicators for mutagenic activities, Hum. Genet., 29 (1975) 127--134. 2 Crossen, P.E., M.E. Drets, F.E. Arrighi and D.A. Johnston, Analysis of the frequency and di s t ri but i on of sister chro matid exchanges in cultured h u m a n l y m p h o c y t e s , Hum. Genet., 35 (1977) 345--352. 3 Crumplin, G.C., and J.T. Smith, Nalidixic acid and bacterial c h r o m o s o m e replication, Nature (London), 260 (1976) 643--645. 4 Dutrillaux, B., A.M. Fosse, M. Prieur and J. Lejeune, Analyse des exchanges de chrornatides dans les cellules somatiques humalnes, Chromosoma, 48 (1974) 327--340. 5 Galloway, S.M., and H.J. Evans, Sister c h r o m a t i d exchange in h u m a n c h r o m o s o m e s from n o r m a l individuals and patients with ataxia-telangiectasia, Cytogenet. Cell Genet., 15 (1975) 17--29. 6 Kato, H., SPontaneous sister chromatid exchanges detec t e d by a BUdR-labelling m e t h o d , Nature (London), 251 (1974) 70--72. 7 Lambert, B., K. Hansson J. Lindsten, M. Sten and B. Werellus, B r o m o d e o x y u r l d t n e - i n d u c e d sistez chr omatid exchanges in h u m a n l y m p h o c y t e s , Hereditas, 83 (1976) 163--174. 8 Latt, S.A., Microfluorometric detection of deoxyribonucl e i c acid replication in h u m a n me t a pha s e chromosomes, Proc. Natl. Acad. Sci. (U.S.A.), 70 (1973) 3395--3399. 9 Lezana, E.A., N.O. Bianchi, M.S. Bianchi and J.E. Zabala-Suarez, Sister c hroma t i d exchanges in Down syndromes and normal h u m a n beings, Mutation Res., 45 (1977) 85--90. 10 McDaniel, L.S., L.H. Rogers and W.E. Hill, Survival of recombination-deficient m u t a n t s of Escherichia coli during incubation with nalidixic acid, J. Bacteriol., 134 (1978) 1195--1198. 11 Perry, P., and H.J. Evans, Cytological d e t e c t i o n of mutagen--carcinogen exposure by sister c hroma t i d exchange, Nature (London), 258 (1975) 121--125. 12 Raposa, T., Sister ehromatid exchange studies for m o n i t o r i n g DNA damage and repair capacity after cytostatics in vitro and in l y m p h o c y t e s of leukaemic pa t i e nt s under c yt os t a t i c t he ra py, Mut a t i on Res., 57 (1978) 241--251. 13 Savage, J.R.K., Chro mosomal aberrations as test for mu t a ge ni c i t y, Nature (London), 258 (1975) 103--104. 14 Solomon, E., and M. Bohrow, Sister c h r o m a t i d exchanges -- a sensitive assay of agents damaging h u m a n chromosomes, Mutation Res., 30 (1975) 273--278, 15 Smith, C.L., M. Kubo and F. I m a m o t o , Promoter-specific i n h i b i t i o n of t ra ns c ri pt i on by a nt i bi ot i c s which act on DNA gyrase, Nature (London), 275 (1978) 420--423. 16 Wolff, S., Sister c h r o m a t i d exchange, Annu. Rev. Genet., 11 (1977) 183--201.