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Induction of sister chromatid exchanges in chinese hamster cells by chlorpropamide
215
Mutation Research, 56 (1977) 215--217 © Elsevier/North-Holland Biomedical Press
Short communication INDUCTION OF SISTER C H R O M A T I D EXCHANGES IN CHINESE HAMSTER CELLS BY C H L O R P R O P A M I D E
ROGER F. BROWN and YUE WU Departtnent of Biology, Southwest Texas State University, San Marcos, Texas 78666 (U.S.A.) (Received June 7 th, 1977 ) (Accepted July 22nd, 1977)
Watson et al. [7] reported that l y m p h o c y t e s from diabetic patients receiving treatment with chlorpropamide, an oral hypoglycemic agent, exhibited a higher number of chromosome aberrations compared with l y m p h o c y t e s from untreated, nondiabetic individuals. They suggested that chlorpropamide m a y be mutagenic, although the possibility that the diabetic state itself has cytogenetic effects was n o t ruled out. This communication reports that chlorpropamide is mutagenic in vitro as judged b y the drug's ability to increase the frequency of sister chromatid exchanges (SCEs) in Chinese hamster V79 cells. Results obtained with cells exposed to EMS, a known mutagen--carcinogen, are included for comparison. Chlorpropamide (lot no. 51398-17000) was obtained from Pfizer. Ethanol was used as solvent for chlorpropamide and EMS (Sigma) was dissolved in water. Cells were grown in Ham's F12 medium supplemented with 10% fetal calf serum (Gibco) in a humidified atmosphere of 5% CO2 at 37°C. Culture vessels were from Falcon. Exponentially growing cultures in 100 mm plastic dishes were exposed for 24 h to medium containing 0.5% ethanol (control), 0.5% ethanol plus chlorpropamide, or EMS. The cultures were then trypsinized, centrifuged, and onefourth of each pellet transferred to 250 ml plastic flasks containing medium with 5-bromodeoxyuridine (10 -s M). The flasks were incubated for 30 h in the dark with colcemid present at a concentration of 0.1 pg/ml during the final 2 h. Mitotic cells were collected by shaking, treated for 10 min with 0.075 M KC1, fixed in methanol--acetic acid (3 : 1), and applied to microslides. The slides were then stained with Hoechst's 33258 and Giemsa essentially as described b y Perry and Wolff [5]. Slides were coded and SCEs scored " b l i n d " in 45 metaphase cells at each dosage. A high n u m b e r of SCEs were evident in the EMS-treated cells. Similar results of mutagen treatment were reported by Perry and Evans [4]. An increased n u m b e r of SCEs relative to control were also seen in the chlorpropamidetreated cell.
216 TABLE I E F F E C T S O F C H L O R P R O P A M I D E A N D EMS ON 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 EXC H A N G E S IN C H I N E S E H A M S T E R C E L L S M e a n n u m b e r s o f e x c h a n g e s per c h r o m o s o m e in the c h l o r p r o p a m i d e - and E M S - t r e a t e d cells w e r e signific a n t l y higher t h a n c o n t r o l at e a c h d o s a g e t e s t e d , P <~ 0 . 0 0 1 . V a l u e s in p a r e n t h e s e s are 9 9 % c o n f i d e n c e limits. Treatment
Number of chromosomes counted
Proportion of chromosomes with indicated number of exchanges
Mean n u m b e r o f e x c h a n g e s per chromosome
0
1
2
3
/>4
Control
943
0.64
0.32
0.04
--
--
0.400 (0.356--0.448)
Chlorpropamide 250 ~g/ml 500 ~g/ml 750 ug/ml
906 964 941
0.55 0.49 0.44
0.39 0.42 0.44
0.06 0.08 0.10
-0.01 0.02
----
0.509 (0.457--0.561) 0.608 (0.553--0.663) 0.699 (0.637-0.761)
EMS 100 ug/ml 200 ~g/ml
893 927
0.44 0.20
0.43 0.43
0.11 0.23
0.02 0.10
-0.04
0.695 (0.632--0.758) 1.386 (1.294--1.478)
Results of the enumeration of SCEs are presented in Table I. Chromosomes with 0 , 1 , 2, etc. SCEs-vcere found to be distributed in a Poisson fashion. The mean number of SCEs per chromosome increased as a function of concentration of both the hypoglycemic agent and the known mutagen. Even at the lowest dosage tested, chlorpropamide treatment significantly increased the mean. An activating system, such as the S-9 liver extract used in the Ames test [1], was not included in this experiment. This suggests that the parent drug rather than a metabolite of chlorpropamide was responsible for the increased number of SCEs. It is perhaps noteworthy that the major portion of chlorpropamide is apparently not metabolically altered in vivo [2]. Induction of sister chromatid exchanges has been shown to be a sensitive technique for detection of mutagens--carcinogens that are effective per se [4] as well as those that require metabolic activation [3,6]. In view of these findings, we believe that the potential genetic hazard of chlorpropamide should be further evaluated. Acknowledgements Chinese hamster V 7 9 cells were generously provided by Dr. E.H.Y. Chu. Hoechst's 3 3 2 5 8 was a gift from Dr. H. Loewe of Farbwerke Hoechst. References 1 A m e s , B.N., J. M c C a n n and E. Y a m a s a k i , M e t h o d s for d e t e c t i n g c a r c i n o g e n s and m u t a g e n s w i t h t h e S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y t e s t , M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 2 J o h n s t o n , P.C., A . R . H e n n e s , T. Driskoll a n d K.M. West, M e t a b o l i c fate o f c h l o r p r o p a m i d e in m a n , A n n . N.Y. A c a d . Sci., 7 4 ( 1 9 5 9 ) 4 5 9 - - 4 7 2 . 3 N a t a r a j a n , A . T . , A.D. T a t e s , P.P.W. V a n Buul, M. Meijers and N. De V o g e l , C y t o g e n e t i c e f f e c t s o f m u t a g e n s / c a r c i n o g e n s after a c t i v a t i o n in a m i c r o s o m a l s y s t e m in vitro. I. I n d u c t i o n o f c h r o m o s o m e
217
4 5 6
7
aberrations and sister chromatid exchanges by diethylnitrosamine ( D E N ) and dimethylnitrosamine ( D M N ) in C H O cellsin the presence of rat-livermicrosomes, Mutati6n Res., 37 (1976) 83--90. Perry, P. and H.J. Evans, Cytological detection of mutagen--carcinogen exposure by sister chromatid exchange, Nature, 258 (1975) 121--125. Perry, P. and S. Wolff, New Giemsa m e t h o d for the differehtial staining of sister chromatids, Nature, 251 (1974) 156--158. Stetka, D.G. and S. Wolff, Sister chromatid exchange as an assay for genetic damage induced by mutagen-carcinogens. II. In vitro test for c o m p o u n d s requiring metabolic activation, Mut a t i on Res., 41 (1976) 343--350. Watson, W.A.F., J.C. Petrie, D.B. Galloway, I. Bullock and J.C. Gilbert, In vivo cytogenetic activity of sulphonyluxea drugs in man, Mutation Res., 38 (1976) 71--80.
Mutation Research, 56 (1977) 215--217 © Elsevier/North-Holland Biomedical Press
Short communication INDUCTION OF SISTER C H R O M A T I D EXCHANGES IN CHINESE HAMSTER CELLS BY C H L O R P R O P A M I D E
ROGER F. BROWN and YUE WU Departtnent of Biology, Southwest Texas State University, San Marcos, Texas 78666 (U.S.A.) (Received June 7 th, 1977 ) (Accepted July 22nd, 1977)
Watson et al. [7] reported that l y m p h o c y t e s from diabetic patients receiving treatment with chlorpropamide, an oral hypoglycemic agent, exhibited a higher number of chromosome aberrations compared with l y m p h o c y t e s from untreated, nondiabetic individuals. They suggested that chlorpropamide m a y be mutagenic, although the possibility that the diabetic state itself has cytogenetic effects was n o t ruled out. This communication reports that chlorpropamide is mutagenic in vitro as judged b y the drug's ability to increase the frequency of sister chromatid exchanges (SCEs) in Chinese hamster V79 cells. Results obtained with cells exposed to EMS, a known mutagen--carcinogen, are included for comparison. Chlorpropamide (lot no. 51398-17000) was obtained from Pfizer. Ethanol was used as solvent for chlorpropamide and EMS (Sigma) was dissolved in water. Cells were grown in Ham's F12 medium supplemented with 10% fetal calf serum (Gibco) in a humidified atmosphere of 5% CO2 at 37°C. Culture vessels were from Falcon. Exponentially growing cultures in 100 mm plastic dishes were exposed for 24 h to medium containing 0.5% ethanol (control), 0.5% ethanol plus chlorpropamide, or EMS. The cultures were then trypsinized, centrifuged, and onefourth of each pellet transferred to 250 ml plastic flasks containing medium with 5-bromodeoxyuridine (10 -s M). The flasks were incubated for 30 h in the dark with colcemid present at a concentration of 0.1 pg/ml during the final 2 h. Mitotic cells were collected by shaking, treated for 10 min with 0.075 M KC1, fixed in methanol--acetic acid (3 : 1), and applied to microslides. The slides were then stained with Hoechst's 33258 and Giemsa essentially as described b y Perry and Wolff [5]. Slides were coded and SCEs scored " b l i n d " in 45 metaphase cells at each dosage. A high n u m b e r of SCEs were evident in the EMS-treated cells. Similar results of mutagen treatment were reported by Perry and Evans [4]. An increased n u m b e r of SCEs relative to control were also seen in the chlorpropamidetreated cell.
216 TABLE I E F F E C T S O F C H L O R P R O P A M I D E A N D EMS ON 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 EXC H A N G E S IN C H I N E S E H A M S T E R C E L L S M e a n n u m b e r s o f e x c h a n g e s per c h r o m o s o m e in the c h l o r p r o p a m i d e - and E M S - t r e a t e d cells w e r e signific a n t l y higher t h a n c o n t r o l at e a c h d o s a g e t e s t e d , P <~ 0 . 0 0 1 . V a l u e s in p a r e n t h e s e s are 9 9 % c o n f i d e n c e limits. Treatment
Number of chromosomes counted
Proportion of chromosomes with indicated number of exchanges
Mean n u m b e r o f e x c h a n g e s per chromosome
0
1
2
3
/>4
Control
943
0.64
0.32
0.04
--
--
0.400 (0.356--0.448)
Chlorpropamide 250 ~g/ml 500 ~g/ml 750 ug/ml
906 964 941
0.55 0.49 0.44
0.39 0.42 0.44
0.06 0.08 0.10
-0.01 0.02
----
0.509 (0.457--0.561) 0.608 (0.553--0.663) 0.699 (0.637-0.761)
EMS 100 ug/ml 200 ~g/ml
893 927
0.44 0.20
0.43 0.43
0.11 0.23
0.02 0.10
-0.04
0.695 (0.632--0.758) 1.386 (1.294--1.478)
Results of the enumeration of SCEs are presented in Table I. Chromosomes with 0 , 1 , 2, etc. SCEs-vcere found to be distributed in a Poisson fashion. The mean number of SCEs per chromosome increased as a function of concentration of both the hypoglycemic agent and the known mutagen. Even at the lowest dosage tested, chlorpropamide treatment significantly increased the mean. An activating system, such as the S-9 liver extract used in the Ames test [1], was not included in this experiment. This suggests that the parent drug rather than a metabolite of chlorpropamide was responsible for the increased number of SCEs. It is perhaps noteworthy that the major portion of chlorpropamide is apparently not metabolically altered in vivo [2]. Induction of sister chromatid exchanges has been shown to be a sensitive technique for detection of mutagens--carcinogens that are effective per se [4] as well as those that require metabolic activation [3,6]. In view of these findings, we believe that the potential genetic hazard of chlorpropamide should be further evaluated. Acknowledgements Chinese hamster V 7 9 cells were generously provided by Dr. E.H.Y. Chu. Hoechst's 3 3 2 5 8 was a gift from Dr. H. Loewe of Farbwerke Hoechst. References 1 A m e s , B.N., J. M c C a n n and E. Y a m a s a k i , M e t h o d s for d e t e c t i n g c a r c i n o g e n s and m u t a g e n s w i t h t h e S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y t e s t , M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 2 J o h n s t o n , P.C., A . R . H e n n e s , T. Driskoll a n d K.M. West, M e t a b o l i c fate o f c h l o r p r o p a m i d e in m a n , A n n . N.Y. A c a d . Sci., 7 4 ( 1 9 5 9 ) 4 5 9 - - 4 7 2 . 3 N a t a r a j a n , A . T . , A.D. T a t e s , P.P.W. V a n Buul, M. Meijers and N. De V o g e l , C y t o g e n e t i c e f f e c t s o f m u t a g e n s / c a r c i n o g e n s after a c t i v a t i o n in a m i c r o s o m a l s y s t e m in vitro. I. I n d u c t i o n o f c h r o m o s o m e
217
4 5 6
7
aberrations and sister chromatid exchanges by diethylnitrosamine ( D E N ) and dimethylnitrosamine ( D M N ) in C H O cellsin the presence of rat-livermicrosomes, Mutati6n Res., 37 (1976) 83--90. Perry, P. and H.J. Evans, Cytological detection of mutagen--carcinogen exposure by sister chromatid exchange, Nature, 258 (1975) 121--125. Perry, P. and S. Wolff, New Giemsa m e t h o d for the differehtial staining of sister chromatids, Nature, 251 (1974) 156--158. Stetka, D.G. and S. Wolff, Sister chromatid exchange as an assay for genetic damage induced by mutagen-carcinogens. II. In vitro test for c o m p o u n d s requiring metabolic activation, Mut a t i on Res., 41 (1976) 343--350. Watson, W.A.F., J.C. Petrie, D.B. Galloway, I. Bullock and J.C. Gilbert, In vivo cytogenetic activity of sulphonyluxea drugs in man, Mutation Res., 38 (1976) 71--80.