111 Mutation Research, 58 (1978) 111--114 © Elsevier/North-Holland Biomedical Press
Short Communication CYTOGENETIC TEST OF CAPTAN IN MOUSE BONE MARROW *
STEPHEN M. FRY and GYULA FICSOR **
Department of Biomedical Sciences, Western Michigan University, Kalamazoo, MI 49008 (U.S.A.) (Received 23 January 1978) (Revision received 6 March 1978) (Accepted 23 March 1978)
In troduction Captan is an extensively used fungicide known to produce base-pair subsitution in Escherichia colt [1], Salmonella typhimurium [4], and Neurospora crassa [8] and chromatid and chromosome breaks in cell culture [1]. Different studies using rats and mice gave conflicting results with respect to d o m i n a n t lethal mutations [2,3,7]. A recent review on captan by Bridges [1] pointed out the need for additional information on the possible cytogenetic effects of captan in vivo. This study reports results of bone-marrow cytogenetic tests with captan in mice. The clastogenic alkylating agent, TEM, was used as positive control. Animals and chemicals Random bred Upjohn strain Swiss albino male mice weighing 25--40 g were used. Captan 50 WP containing 50% technical captan lot No., 01007-10, CAS No., 133-06-2 was obtained from the Stauffer Chemical Co. and TEM from Pfaltz and Bauer Inc. Cytogenetic analysis of bone-marrow metaphases Appropriate quantities of captan or TEM (Table 1) were injected i.p. in 1 ml 0.9% NaC1. Controls were similarly injected with 1 ml 0.9% NaC1. Treated and control animals were killed by cervical dislocation 6, 12, 30 and 54 h following injection. 5 h prior to killing, the animals were injected i.p. with 1 mg/ kg colchicine in 1 ml 0.9% NaC1. With a 23 G needle, 1.5 ml of 2.2% sodium citrate were injected through each end of the excised femurs. The bone marrow
* Research by S.M.F. was performed in partial fulfillment Honors College. ** Send reprint requests to this author. Abbreviations: TEM, triethylenemelamine.
of requirements
of graduation
from The
12
30
54
Control Captan 250 T E M O.5 TEM 1.0
Control Captan 250 T E M O.5 T E M 1.0
Control Captan 250 TEM 0.5 T E M 1.0
a p ~ 0.05; significant. b p ( 0.01; highly significant.
6
Control Captan 250 TEM 0.5
Time
(h)
(mg/kg)
OF CONTROL,
Drug dose
EVALUATION
TABLE 1
4 2 3 1
4 3 3 1
3 3 3 2
4 3 3
Number of animals
1 l 44 b 5 a
2 3 21 b 1 3 0 1 0
400 300 303 100
404 101 300 100 3 1 0 1
2 5 40 b 60 b
300 300 259 195
391 300 307 0 2 6 30b
Chromatid breaks 4 0 39 b
Gaps
Number of cells
MOUSE BONE-MARROW
2 0 9
Number o f cels
CAPTAN AND TEM TREATED
1 0 6 0
2 1 6 0
3 0 9 13 b
1 0 13 b
Chromosome breaks
METAPHASES
0 1 0 0
0 1 34 b 9 b
0 0 8 a 39 b
0 1 7 a
Rearrangemeats
SAMPLED
0 0 1 1
0 0 4 5 b
0 0 0 24 b
0 0 1
Rings
AT DIFFERENT
0 0 0 0
0 0 50 b 72 b
0 0 0 42 b
0 0 1
Multiple breaks
1.73 1.98 2.67 2.00
1.25 1.67 35.6 b 77.0 b
1.67 2.33 19.31 b 58.97 b
1.79 0.33 17.92 b
cells (%)
Abnormal
TIME INTERVALS
b~
113 cells were suspended and collected in a Clay Adams Dynac Centrifuge with a 24 × 15 ml angle head at 1000 rpm for 5 min. Most of the supernatant was discarded, leaving about 0.25 ml of fluid in the test tube. The cells were thoroughly suspended and 3 ml of 0.56% KC1 hypotonic solution at 37°C were added and the suspension incubated in a water bath at the same temperature. In the last 5 min of the 30 rain hypotoni¢ treatment the cells were collected as before. 3 ml fresh fixative (3 parts absolute methanol : 1 part glacial acetic acid) were slowly added with constant agitation to prevent cell clumping. 5 aspirations with a pasteur pipette aided mixing. The fixative was changed 3 times. During the last resuspension enough fixative was added to give a turbid cell suspension. Grease-free microscope slides previously placed in a freezer were allowed to collect condensation. 3--4 drops of cell suspension were placed on each slide and immediately passed through a flame, allowing the methanol to burn completely. Once completely dry, the slides were flooded for 4--5 min with fresh Giemsa stain prepared by mixing 5 ml Giemsa stock solution, 6 ml acetone, and 100 ml tap water. Giemsa stock solution contained 3.6 g Giemsa powder (Fisher, U.S.A.) dissolved in 250 ml glycerol and 250 ml methanol. 100 cells per animal were scored from randomized slides when possible. A gap was an achromatic lesion less than the width of a chromatid. Gaps and isochromatid gaps were not scored separately. Fragments were scored as a single chromatid break. Cells with more than 10 breaks were scored as multiple breaks.
Statistical analysis The significance of differences in the frequencies of chromosome aberrations with captan and TEM in comparison to the control was determined using the m e t h o d described by Kastenbaum and Bowman [6].
Toxicity Within 12 h after injection of captan, toxicity was noted. Signs included edema, decreased body temperature, sluggishness, and bluish skin color. In the 6, 12, 30 and 54 h captan treatments, lethality increased over control animals by 0, 25, 20 and 68%, respectively. TEM-treated animals showed no signs of toxicity.
Cytogenetic observations In agreement with earlier observations [5], the highest frequency of TEMinduced chromosome aberrations was detected 30 h after treatment. 54 h after treatment, chromosome damage was not observed with either 0.5 or 1 mg/kg TEM, indicating that TEM-induced aberrations caused cell lethality and/or are not transmitted (Table 1). At no time did the incidence of chromosome aberrations in animals treated with captan deviate significantly from that of controls (Table 1). However, 3 metacentric chromosomes were observed (one in each of the 6, 30 and 54 h t r e a t m e n t groups) in 1001 metaphases from captan-treated animals, whereas none appeared in 1495 control spreads. Since mouse chromosomes are telocentric, the metacentric chromosomes in the captan-treated animals may have originated from Robertsonian fusion. Because of these unique chromosome rearrangements, it cannot be stated with certainty that 250 mg captan/kg i.p. does n o t break chromosomes in vivo.
114
Acknowledgements The authors are grateful to the Stauffer Company for the gift of captan of known production batch. We also appreciate the support to S.F. from the Russel H. Seibert Fund and reading of the manuscript by Drs. L. Beuving, M. McCarville and Ms. L. Ginsberg. References 1 Bridges, B.A., T h e m u t a g e n i c i t y of c a p t a n a n d r e l a t e d fungicides, M u t a t i o n Res., 32 ( 1 9 7 5 ) 3 - - 3 4 . 2 Cullins, T . F . X . , D o m i n a n t l e t h a l assay, I. C a p t a n , F o o d C o s m e t . T o x i c o l . , 10 ( 1 9 7 2 ) 3 5 3 7 6 1 . 3 E p s t e i n , S.E., E. A r n o l d , J. A n d r e a , W. Bass a n d Y. Bishop, D e t e c t i o n of c h e m i c a l m u t a g e n s by the d o m i n a n t l e t h a l assay in the m o u s e , T o x i c o l . A p p l . P h a r m a c o l . , 23 ( 1 9 7 2 ) 2 8 8 - - 3 2 5 . 4 Ficsor, G., S. Bordas, S.M. W a d e , E. M u t h i a n i , G.F. W e r t z and D.M. Z i m m e r , M a m m a l i a n host- and f l u i d - m e d i a t e d m u t a g e n i c i t y assays of c a p t a n a n d s t r e p t o z o t o c i n in S a l m o n e l l a t y p h i m u r i u m , Mutation Res., 48 ( 1 9 7 7 ) 1 - - 1 6 . 5 G r e e n , S., F.M. S a u r o a n d M.S. L e g a t o r , C y t o g e n e t i e e f f e c t s of h y e a n t h o n e in the rat, M u t a t i o n Res., 17 ( 1 9 7 3 ) 2 3 9 - - 2 4 4 . 6 K a s t e n b a u m , M.A., a n d K.O. B o w m a n , T a b l e s for d e t e r m i n i n g the statistical significance of m u t a t i o n f r e q u e n c i e s , M u t a t i o n R e s . , 9 ( 1 9 7 0 ) 527 549. 7 K e n n e d y , G . L . , D.W. A r n o l d a n d M.L. K e p l i n g e r , M u t a g e n i c i t y studies w i t h c a p t a n , e a p t a f o l , f o l p e t a n d t h a l i d o m i d e , F o o d C o s m e t . T o x i c o l . , 13 ( 1 9 7 5 ) 5 5 - - 6 1 . 8 Malling, H . V . , and F.J. de Serres, C a p t a n , a p o t e n t f u n g i c i d e w i t h m u t a g e n i e a c t i v i t y , 1st A n n . M e e t i n g EMS, EMS N e w s l e t t e r , 3 ( 1 9 7 0 ) 41.
Short Communication CYTOGENETIC TEST OF CAPTAN IN MOUSE BONE MARROW *
STEPHEN M. FRY and GYULA FICSOR **
Department of Biomedical Sciences, Western Michigan University, Kalamazoo, MI 49008 (U.S.A.) (Received 23 January 1978) (Revision received 6 March 1978) (Accepted 23 March 1978)
In troduction Captan is an extensively used fungicide known to produce base-pair subsitution in Escherichia colt [1], Salmonella typhimurium [4], and Neurospora crassa [8] and chromatid and chromosome breaks in cell culture [1]. Different studies using rats and mice gave conflicting results with respect to d o m i n a n t lethal mutations [2,3,7]. A recent review on captan by Bridges [1] pointed out the need for additional information on the possible cytogenetic effects of captan in vivo. This study reports results of bone-marrow cytogenetic tests with captan in mice. The clastogenic alkylating agent, TEM, was used as positive control. Animals and chemicals Random bred Upjohn strain Swiss albino male mice weighing 25--40 g were used. Captan 50 WP containing 50% technical captan lot No., 01007-10, CAS No., 133-06-2 was obtained from the Stauffer Chemical Co. and TEM from Pfaltz and Bauer Inc. Cytogenetic analysis of bone-marrow metaphases Appropriate quantities of captan or TEM (Table 1) were injected i.p. in 1 ml 0.9% NaC1. Controls were similarly injected with 1 ml 0.9% NaC1. Treated and control animals were killed by cervical dislocation 6, 12, 30 and 54 h following injection. 5 h prior to killing, the animals were injected i.p. with 1 mg/ kg colchicine in 1 ml 0.9% NaC1. With a 23 G needle, 1.5 ml of 2.2% sodium citrate were injected through each end of the excised femurs. The bone marrow
* Research by S.M.F. was performed in partial fulfillment Honors College. ** Send reprint requests to this author. Abbreviations: TEM, triethylenemelamine.
of requirements
of graduation
from The
12
30
54
Control Captan 250 T E M O.5 TEM 1.0
Control Captan 250 T E M O.5 T E M 1.0
Control Captan 250 TEM 0.5 T E M 1.0
a p ~ 0.05; significant. b p ( 0.01; highly significant.
6
Control Captan 250 TEM 0.5
Time
(h)
(mg/kg)
OF CONTROL,
Drug dose
EVALUATION
TABLE 1
4 2 3 1
4 3 3 1
3 3 3 2
4 3 3
Number of animals
1 l 44 b 5 a
2 3 21 b 1 3 0 1 0
400 300 303 100
404 101 300 100 3 1 0 1
2 5 40 b 60 b
300 300 259 195
391 300 307 0 2 6 30b
Chromatid breaks 4 0 39 b
Gaps
Number of cells
MOUSE BONE-MARROW
2 0 9
Number o f cels
CAPTAN AND TEM TREATED
1 0 6 0
2 1 6 0
3 0 9 13 b
1 0 13 b
Chromosome breaks
METAPHASES
0 1 0 0
0 1 34 b 9 b
0 0 8 a 39 b
0 1 7 a
Rearrangemeats
SAMPLED
0 0 1 1
0 0 4 5 b
0 0 0 24 b
0 0 1
Rings
AT DIFFERENT
0 0 0 0
0 0 50 b 72 b
0 0 0 42 b
0 0 1
Multiple breaks
1.73 1.98 2.67 2.00
1.25 1.67 35.6 b 77.0 b
1.67 2.33 19.31 b 58.97 b
1.79 0.33 17.92 b
cells (%)
Abnormal
TIME INTERVALS
b~
113 cells were suspended and collected in a Clay Adams Dynac Centrifuge with a 24 × 15 ml angle head at 1000 rpm for 5 min. Most of the supernatant was discarded, leaving about 0.25 ml of fluid in the test tube. The cells were thoroughly suspended and 3 ml of 0.56% KC1 hypotonic solution at 37°C were added and the suspension incubated in a water bath at the same temperature. In the last 5 min of the 30 rain hypotoni¢ treatment the cells were collected as before. 3 ml fresh fixative (3 parts absolute methanol : 1 part glacial acetic acid) were slowly added with constant agitation to prevent cell clumping. 5 aspirations with a pasteur pipette aided mixing. The fixative was changed 3 times. During the last resuspension enough fixative was added to give a turbid cell suspension. Grease-free microscope slides previously placed in a freezer were allowed to collect condensation. 3--4 drops of cell suspension were placed on each slide and immediately passed through a flame, allowing the methanol to burn completely. Once completely dry, the slides were flooded for 4--5 min with fresh Giemsa stain prepared by mixing 5 ml Giemsa stock solution, 6 ml acetone, and 100 ml tap water. Giemsa stock solution contained 3.6 g Giemsa powder (Fisher, U.S.A.) dissolved in 250 ml glycerol and 250 ml methanol. 100 cells per animal were scored from randomized slides when possible. A gap was an achromatic lesion less than the width of a chromatid. Gaps and isochromatid gaps were not scored separately. Fragments were scored as a single chromatid break. Cells with more than 10 breaks were scored as multiple breaks.
Statistical analysis The significance of differences in the frequencies of chromosome aberrations with captan and TEM in comparison to the control was determined using the m e t h o d described by Kastenbaum and Bowman [6].
Toxicity Within 12 h after injection of captan, toxicity was noted. Signs included edema, decreased body temperature, sluggishness, and bluish skin color. In the 6, 12, 30 and 54 h captan treatments, lethality increased over control animals by 0, 25, 20 and 68%, respectively. TEM-treated animals showed no signs of toxicity.
Cytogenetic observations In agreement with earlier observations [5], the highest frequency of TEMinduced chromosome aberrations was detected 30 h after treatment. 54 h after treatment, chromosome damage was not observed with either 0.5 or 1 mg/kg TEM, indicating that TEM-induced aberrations caused cell lethality and/or are not transmitted (Table 1). At no time did the incidence of chromosome aberrations in animals treated with captan deviate significantly from that of controls (Table 1). However, 3 metacentric chromosomes were observed (one in each of the 6, 30 and 54 h t r e a t m e n t groups) in 1001 metaphases from captan-treated animals, whereas none appeared in 1495 control spreads. Since mouse chromosomes are telocentric, the metacentric chromosomes in the captan-treated animals may have originated from Robertsonian fusion. Because of these unique chromosome rearrangements, it cannot be stated with certainty that 250 mg captan/kg i.p. does n o t break chromosomes in vivo.
114
Acknowledgements The authors are grateful to the Stauffer Company for the gift of captan of known production batch. We also appreciate the support to S.F. from the Russel H. Seibert Fund and reading of the manuscript by Drs. L. Beuving, M. McCarville and Ms. L. Ginsberg. References 1 Bridges, B.A., T h e m u t a g e n i c i t y of c a p t a n a n d r e l a t e d fungicides, M u t a t i o n Res., 32 ( 1 9 7 5 ) 3 - - 3 4 . 2 Cullins, T . F . X . , D o m i n a n t l e t h a l assay, I. C a p t a n , F o o d C o s m e t . T o x i c o l . , 10 ( 1 9 7 2 ) 3 5 3 7 6 1 . 3 E p s t e i n , S.E., E. A r n o l d , J. A n d r e a , W. Bass a n d Y. Bishop, D e t e c t i o n of c h e m i c a l m u t a g e n s by the d o m i n a n t l e t h a l assay in the m o u s e , T o x i c o l . A p p l . P h a r m a c o l . , 23 ( 1 9 7 2 ) 2 8 8 - - 3 2 5 . 4 Ficsor, G., S. Bordas, S.M. W a d e , E. M u t h i a n i , G.F. W e r t z and D.M. Z i m m e r , M a m m a l i a n host- and f l u i d - m e d i a t e d m u t a g e n i c i t y assays of c a p t a n a n d s t r e p t o z o t o c i n in S a l m o n e l l a t y p h i m u r i u m , Mutation Res., 48 ( 1 9 7 7 ) 1 - - 1 6 . 5 G r e e n , S., F.M. S a u r o a n d M.S. L e g a t o r , C y t o g e n e t i e e f f e c t s of h y e a n t h o n e in the rat, M u t a t i o n Res., 17 ( 1 9 7 3 ) 2 3 9 - - 2 4 4 . 6 K a s t e n b a u m , M.A., a n d K.O. B o w m a n , T a b l e s for d e t e r m i n i n g the statistical significance of m u t a t i o n f r e q u e n c i e s , M u t a t i o n R e s . , 9 ( 1 9 7 0 ) 527 549. 7 K e n n e d y , G . L . , D.W. A r n o l d a n d M.L. K e p l i n g e r , M u t a g e n i c i t y studies w i t h c a p t a n , e a p t a f o l , f o l p e t a n d t h a l i d o m i d e , F o o d C o s m e t . T o x i c o l . , 13 ( 1 9 7 5 ) 5 5 - - 6 1 . 8 Malling, H . V . , and F.J. de Serres, C a p t a n , a p o t e n t f u n g i c i d e w i t h m u t a g e n i e a c t i v i t y , 1st A n n . M e e t i n g EMS, EMS N e w s l e t t e r , 3 ( 1 9 7 0 ) 41.
