Mutation Research, 31 (1975) 225-233 © Elsevier Scientific Publishing Company, A m s t e r d a m - - P r i n t e d in The Netherlands
225
U M B R A L I M I : A MODEL FOR T H E STUDY OF CHROMOSOME ABERRATIONS IN FISHES
A. D. KLIGERMAN, S. E. BLOOM AND *V~r. M. HOWELL
Department of Poultry Science, Cornell University, lthaca, N.Y. ~4853 and *Department of Biology, Samford University, Birmingham, dla. 352o9 (U.S.A .) (Received February i8th, I975)
SUMMARY
Due to the lack of information available on the effects of various clastogenic agents on the chromosomes of fishes, an in vivo cytogenetics model system was developed. The central mudminnow, Umbra limi, was chosen for this study because of its ideal karyotype consisting of 22 large recta- and submetacentric chromosomes. Various organs of the fish were investigated to determine their suitability for chromosome preparations. The tissues of the intestines, stomach, kidneys, and gills were found to be the most suitable for clastogenic studies. Phase contrast observations were made on the chromosomes of control mudminnows and mudminnows exposed to 325 R of X-radiation. The control rate of spontaneous chromosome aberrations was found to be low (about 0.03%). In contrast, fish exposed to 325 R of X-rays had aberrations in approximately 30% of the metaphases per fish examined. An apparent increase in clumping and a decrease in the mitotic index were also noted. It was concluded that the chromosomes of Umbra limi displayed typical responses to low level radiation exposure and that this fish would be an ideal cytogenetics model for the study of induced chromosome aberrations in fishes.
INTRODUCTION
Several testing protocols have been developed in recent years which permit evaluation of the mutagenic (including clastogenic) properties of various chemical and physical agents4,7,9,1~,18. There is particular concern about possible mutagenic properties of substances that regularly enter the human body (e.g. drugs, food additives, sprays, chlorinated hydrocarbons). While it is essential that thorough mutagenic screening be performed in mammals, it is equally important to evaluate genetic damage that might occur in species man depends on for food and recreation. Numerous fish species (e.g. tuna, trout, salmon, carp, catfish, flounder, etc.) provide an important source of protein and other nutrients in the diet of man and certain animals raised for human consumption. It is essential to know, therefore,
220
A. I1. KLIGERMANet al.
what effects, if any, water-borne pollutants have on the genetic material of fishes. As a first step in this direction, we have investigated the possibility of using the central mudminnow, Umbra limi, to screen for the ehromosonle-breaking effects of chemical and physical agents. Various researchers, predominantly in the USSR, have investigated the effects of radiation of fish genomes ~*, but no totally successful system had been developed which permitted routine and precise analysis of the effects clastogenic agents might have on individual chromosomes. This was due in part to the choice of species and to difficulties encountered with the chromosomal methodologies used. Most fish species investigated had large numbers of small chromosomes that were difficult to resolve.
Fig. I. The central mudnlinnow, Untbra limi (actual size: 7.6 cm). ENDO AND [NGALLS6 suggested that the zebra fish, Brachydanio rerio, be used as an experimental model system for embryological, teratological, and cytological studies. However, this system is not well suited for cytological studies because of the fish's high chromosome number (2n = 50) and the difficulty in obtaining large numbers of well-spread metaphase figures for study. HOWELL1° proposed that the black ghost knifefish, Apteronotus albifrons, be used as a model aquatic vertebrate for studies on the effects of water pollution, drugs, and other environmental influences on its chromosome complement. Yet, although this fish has an excellent karyotype for such studies, it is a relatively expensive fish to obtain and keep in the laboratory. The central mudminnow, Umbra limi, seems to have none of these drawbacks (Fig. I). It has a low chromosome number (2n = 22) 1,~, large chromosomes, and large numbers of fish can be easily captured and held for study. The present study was carried out to determine (I) the best chromosome technique, (2) which organs were suitable for chromosome analysis and (3) if chromosome breakage could be easily assessed.
CYTOGENETICS MODEL* n
227
wvo
MATERIALS AND METHODS
Mudminnows were seined from mud-bottomed streams near Port Byron, N.Y. Fish were kept in an aerated, well-planted I5-gallon aquarium maintained at a temperature range from 14 ° to 15 ° and a pH range of 7 to 8. Fish to be used in chromosome studies were injected I P with 0. 5 ml of o.1% colchicine (Sigma) and returned to well-aerated water. 6 h after injection the fish were sacrificed and the gills, pancreas, spleen, heart, stomach, intestines, kidney, scale, liver, and brain were removed and placed in an 0.4% KC1 hypotonic solution for 15-2o rain.
b7.
_o .Q D
Z
i
L
1
2
3
4
5
6
7
8
I
9
I
10
Organs
Fig. 2. A n a l y s i s of t h e s u i t a b i l i t y of v a r i o u s o r g a n s of t h e m u d m i n n o w for c y t o g e n e t i c s s t u d i e s s h o w i n g s u p e r i o r i t y of intestines, s t o m a c h , kidneys, a n d gills. O r g a n code: I, i n t e s t i n e s ; 2, s t o m a c h ; 3, k i d n e y ; 4, gill; 5, scale; 6, spleen; 7, b r a i n ; 8, p a n c r e a s ; 9, h e a r t ; io, liver.
The hypotonic was removed and 3:1 ethanol:acetic acid was added. I h later the fixative was removed and fresh 3 :i ethanol:acetic acid was added. Squash preparations were made in 45% acetic acid S and examined using a Leitz Ortholux phase contrast microscope. Detailed examination of chromosomes was achieved at a magnification of 1125 × using an oil immersion objective. Squash preparations and phase optics were used since it was believed this would produce the least number of technically induced artifacts. For each fish examined a count was made of the total number of usable metaphases (at least 18 chromosomes clearly visible) found in each tissue and the results recorded (Fig. 2). Permanent preparations were made by freezing the
228
A.D. KLIGERMANet al.
10/.
II
lqg. 3. Intestinal metaph~se spread from Umbra limi showing diplc,id complen~ent (bright field). slide in liquid nitrogen, popping off the coverslip, a n d staining tile slide in IO°/{) Giemsa for 5 rain. Fish to be irradiated were placed two at a time in a IOOO ml beaker filled with 30o ml of water. The fish were X - i r r a d i a t e d with a Picker deep-therapy u n i t operating at 14o kV a n d 15 mA. Each set of two fish was exposed to 325 R of X - r a d i a t i o n a d m i n i s t e r e d at a rate of 65 R/rain. Following irradiation, the fish were injected I P with a p p r o x i m a t e l y 0.5 ml of an o.I°/~ colchicine solution a n d r e t u r n e d to well-
229
CYTOGENETICS MODEL ~n v~vo
aerated water for from 3.5 to 7.5 h. The fish were then sacrificed, and squash preparations were made (as described previously) from the kidneys, gills, stomach, and intestines, and these were examined using phase optics and oil immersion at a magnification of 1125 ×. Metaphases were scored for aberrations (breaks, gaps, translocations), and the number recorded for each fish examined. Photographs were taken with a Leitz Ortholux camera using high contrast copy film. Squash preparations were also made from control mudminnows not exposed to any known clastogen, and slides were examined for aberrations in a similar fashion to the method described above. RESULTS
Though the method of determining mitotic activity of the various organs was crude, Fig. 2 shows that four organs clearly stand out as giving superior numbers of usable metaphases: the intestines, stomach, kidney tissues and gills. The tissues of the alimentary canal consistently gave the best metaphase spreads (Fig. 3)- These results compare favorably with other methods previously used to determine the mitotic index in the mudminnow n. The gonads were not used in this study since it was found that mitoses were seasonal in nature, and when they did occur, they appeared in such great profusion that they were of little value for clastogenic studies. Examination of the data in Table I reveals that upon exposure to 325 R of X-radiation, breaks and gaps such as those shown in Figs. 4 a and 4 b were found in approximately 30% of the metaphases of each fish examined (X = 29.69 :~ 8.22). No translocations, inversions, or exchange figures were detected. When the X-ray induced aberration data are compared to the control data (Table II), two points are evident. First, the mudminnow has a low spontaneous chromosome breakage rate (about 0.03%), and secondly, 325 R of X-rays caused over a 3oo-fold increase in the detectable aberration rate in the somatic cells of the mudminnow. The induced aberration rate of o.ooli aberrations/metaphase/fish/R was in line with what other researchers have obtained with other animals exposed to X-rays3, 5. Typical radiation induced cytological effects such as the lowering of mitotic index and increased clumping of metaphase chromosomes were also observed. Thus, radiation induced chromosome damage could be detected in the cells of the mudminnow, and the amount and type of damage was similar to the damage TABLE I ANALYSIS OF CHROMOSOME A B E R R A T I O N S IN FISH E X P O S E D TO 3 2 5 R OF X - R A D I A T I O N
Fish code letter
N u m b e r of metaphases examined
N u m b e r of metaphases with at least one aberration a
Total number of aberrations
Percentage of metaphases with at least one aberration
Aberrations per metaphase
A B C D E F
34 ii 45 14 185 98
9 4 io 6 42 27
13 4 13 7 53 34
26.47 36-36 22.22 42.86 22.7 ° 27.55
0,3824 o.3636 o.2829 0.5000 0.2865 0-3469
Total6
387
98
124
29.69~8.22
o.3604±o.o795 b
X±S.D.
a A b e r r a t i o n ~ b reak, gap. b Mean n u m b e r of a b e r r a t i o n s per m e t a p h a s e per R p e r fish : o.ooi I -k 0.0002.
2]jO
A . D . KLIGERMAN el al.
°
i
B Fig. -t. X - R a y i n d u c e d c h r o m a t i d a b e r r a t i o n s in s t o m a c h cells of a m u d i n i n n o w (phase c o n t r a s t ) . A. C h r o m a t i d b r e a k (arrow). B. C h r o m a t i d gap (arrow),
found in cells of other vertebrates similarly exposed to X-radiation. Furthermore, these data illustrate that fishes can be useful tools in studying chromosome aberration. DISCUSSION
As mentioned previously, only a limited number of experimental studies have
CYTOGENETICS M O D E L ~ n v ~ v o
231
Fig. 5. Karyotype of Umbra timi showing eleven pairs of metacentric and submetacentric chromosomes (bright field). been carried out on the chromosomes of fishes. The fishes chosen for study had large chromosome numbers and/or small chromosomes. However, the mudminnow has an ideal karyotype for such studies. The diploid complement consists of 22 large metacentric and submetacentric chromosomes which facilitate clastogenic studies (Fig. 5). Furthermore, the mudminnow is a small hardy fish which is easy to obtain and keep in the laboratory.
232
A . D . KLIGERMAN el al.
"I'ABI3:11 ANALYSIS
OF CnROMOSOMF
ABERRATIONS
IN FISH NOT DELIBERATELY
EXPOSED
(CONTROL)
TO ANY
MUTAGENS
Irish No.
Number of metaphases examined
Number oj metaphases with at least ore" aberration a
Total number of aberrations
Percentage of metaphases with at least one aberration
Ab~rralions per metaphase
I
61
0
O
O
O.O0
2
49
o
o
o
o.oo
3
20
0
O
O
O.O0
4 5 6
40 I OO 1II
o 0 O
o O O
o O 0
o.oo O. O( ) O.O0
7
48
o
o
o
o.oo o.oo
8
27 I
o
o
o
9
21
O
O
O
O.OO
IO I I I 2
22 1 160 250
O o O
O o O
O o O
O .OO o.oo O.OO
13
299
l
4
0.334
1675
l
4
o.o134 o.oolo i o.oo37
Total ]3
~ ; q S.l).
O.O26~ o.o93
a Aberration ~ breaks, gaps. R a d i a t i o n s t u d i e s c a r r i e d o u t o n t h e m u d i n i n n o w t o d e t e r m i n e if, in f a c t , tile c h r o m o s o m e s of t h e n m d m i n n o w w e r e u s e f u l for c l a s t o g e n i c s t u d i e s p r o v e d s u c c e s s f u l . A d e q u a t e n u m b e r s of m e t a p h a s e s c o u l d b e o b t a i n e d f r o m v a r i o u s o r g a n s , a n d radiation damage was similar to that found in other vertebrates exposed to Xr a d i a t i o n . T o o u r k n o w l e d g e , t h i s is t h e first t i m e a c y t o g e n e t i c s s y s t e m h a s b e e n d e v e l o p e d w h i c h m a k e s u s e of a fish in w h i c h c h r o m o s o m e d a m a g e c a n b e a c c u r a t e l y d e t e c t e d . T h i s c o u l d p r o v e t o b e v a l u a b l e in c o r r e l a t i n g p h e n o t y p i c d i s t u r b a n c e s in fishes w i t h specific c h r o m o s o m e a b n o r m a l i t i e s ; s o m e t h i n g t h a t h a s b e e n l a c k i n g in l n u c h of t h e c l a s t o g e n e t i c a n d m u t a g e n i c w o r k d o n e w i t h fishes u p t o n o w . In a d d i t i o n it is felt t h a t t h e m u d m i n n o w c o u l d p r o v e t o b e a v a l u a b l e n l o d e l o r g a n i s m for s t u d y i n g t h e c l a s t o g e n i c effects of w a t e r - b o r n e c h e m i c a l s . P r e l i m i n a r y e x p e r i m e n t s c o n d u c t e d w i t h m u d m i n n o w s e x p o s e d t o v a r i o u s c o n c e n t r a t i o n s of m a l e i c h y d r a z i d e d i s s o l v e d in a q u a r i u m w a t e r i n d i c a t e d t h a t c h e m i c a l s c o u l d c a u s e a s m a l l i n c r e a s e in t h e o b s e r v e d r a t e of s o m a t i c c h r o m o : s o m e b r e a k a g e in fish so e x p o s e d . R E F I ~.RENC I;.S BEAMISH, B. J., M. J. ~IERRILb'ES AND E. J. CROSSMAN, Karyotype and DNA v a l u e s f o r members of the suborder Esocoidei, Chromoson'la, 34 1197 l) 436 4772 BLOOM, .q.E., G. POVAR AND D. B. PEAKALL, Chrnnlosome preparations from the avian allantoic sac, ,Stain Technol., 47 (1972) 123-1273 CORMIER, J. M., AND S. E. BLOO~a, An in vivo study of the el~fects of X-radiation on the chroniosomes of chick allantoic membranes, Mutation ICes., 20 (I973) 77-85 • 4 DE SERRES, F. J., AND W. SHERIDAN (Eds.), The evaluation of chemical mutagenicity data in relation to population risk, Environ. Health Per@., Experinmntal issue No. 6 (I973) i 232. 5 ELKIND, M. M., AND G. F. WHITMORE, The Radiobiology of Cultured Mammalian Cells, Gordon and Breach, New York, 1967, pp. 449 451. 6 ENDO, A., AND T. H. [NGALLS, Chromosomes of the zebra fish, J. Here&, 59 11968) 382 384 . I
7
FISHBEIN, L . , \ V . ( ; . F L A M M A N D M . L . F A L K , Chemical Mutagens: Environmental EjJ)cts on Biological Systems, Academic Press, New York, 197 o. FOLEY, J. o., The spermatogenesis of Umbra limi with special reference to the behavior of the spermatogonial chromosomes and the first maturation division, Biol. Bull. (Woods Hole, Mass.), 5o (I926) ~I7 I.t7.
CYTOGENETICS MODEL Zn V~VO
233
9 HOLLAENDER, A., Chemical Mutagens: Principles and Methods for their Detection, Vols. 1 a n d II, P l e n u m , N e w York, 1971. io HOWELL, W. M., S o m a t i c c h r o m o s o m e s of t h e Black G h o s t Knifefish, Apteronotus albifrons, Copeia, I (1972) I 9 I - I 9 3 . I I KLIGERMAN, A. D., S p o n t a n e o u s a n d i n d u c e d c h r o m o s o m e a b e r r a t i o n s in t h e m u d m i n n o w (Umbra limi): D e v e l o p m e n t of a n in vivo c y t o g e n e t i c s model, Master's Thesis, Cornell University, I t h a c a , N.Y., 1974. 12 LEGATOR, M. S., Chemical m u t a g e n e s i s c o m e s of age, J. Hered., 61 (197 o) 239-242. 13 LEGATOR, IV[. S., Deficiencies in our p r e s e n t protocol for chemical e v a l u a t i o n a n d possible remedies, Ann. N . Y . Acad. Sci., 179 (1971) 5o8-513 .
225
U M B R A L I M I : A MODEL FOR T H E STUDY OF CHROMOSOME ABERRATIONS IN FISHES
A. D. KLIGERMAN, S. E. BLOOM AND *V~r. M. HOWELL
Department of Poultry Science, Cornell University, lthaca, N.Y. ~4853 and *Department of Biology, Samford University, Birmingham, dla. 352o9 (U.S.A .) (Received February i8th, I975)
SUMMARY
Due to the lack of information available on the effects of various clastogenic agents on the chromosomes of fishes, an in vivo cytogenetics model system was developed. The central mudminnow, Umbra limi, was chosen for this study because of its ideal karyotype consisting of 22 large recta- and submetacentric chromosomes. Various organs of the fish were investigated to determine their suitability for chromosome preparations. The tissues of the intestines, stomach, kidneys, and gills were found to be the most suitable for clastogenic studies. Phase contrast observations were made on the chromosomes of control mudminnows and mudminnows exposed to 325 R of X-radiation. The control rate of spontaneous chromosome aberrations was found to be low (about 0.03%). In contrast, fish exposed to 325 R of X-rays had aberrations in approximately 30% of the metaphases per fish examined. An apparent increase in clumping and a decrease in the mitotic index were also noted. It was concluded that the chromosomes of Umbra limi displayed typical responses to low level radiation exposure and that this fish would be an ideal cytogenetics model for the study of induced chromosome aberrations in fishes.
INTRODUCTION
Several testing protocols have been developed in recent years which permit evaluation of the mutagenic (including clastogenic) properties of various chemical and physical agents4,7,9,1~,18. There is particular concern about possible mutagenic properties of substances that regularly enter the human body (e.g. drugs, food additives, sprays, chlorinated hydrocarbons). While it is essential that thorough mutagenic screening be performed in mammals, it is equally important to evaluate genetic damage that might occur in species man depends on for food and recreation. Numerous fish species (e.g. tuna, trout, salmon, carp, catfish, flounder, etc.) provide an important source of protein and other nutrients in the diet of man and certain animals raised for human consumption. It is essential to know, therefore,
220
A. I1. KLIGERMANet al.
what effects, if any, water-borne pollutants have on the genetic material of fishes. As a first step in this direction, we have investigated the possibility of using the central mudminnow, Umbra limi, to screen for the ehromosonle-breaking effects of chemical and physical agents. Various researchers, predominantly in the USSR, have investigated the effects of radiation of fish genomes ~*, but no totally successful system had been developed which permitted routine and precise analysis of the effects clastogenic agents might have on individual chromosomes. This was due in part to the choice of species and to difficulties encountered with the chromosomal methodologies used. Most fish species investigated had large numbers of small chromosomes that were difficult to resolve.
Fig. I. The central mudnlinnow, Untbra limi (actual size: 7.6 cm). ENDO AND [NGALLS6 suggested that the zebra fish, Brachydanio rerio, be used as an experimental model system for embryological, teratological, and cytological studies. However, this system is not well suited for cytological studies because of the fish's high chromosome number (2n = 50) and the difficulty in obtaining large numbers of well-spread metaphase figures for study. HOWELL1° proposed that the black ghost knifefish, Apteronotus albifrons, be used as a model aquatic vertebrate for studies on the effects of water pollution, drugs, and other environmental influences on its chromosome complement. Yet, although this fish has an excellent karyotype for such studies, it is a relatively expensive fish to obtain and keep in the laboratory. The central mudminnow, Umbra limi, seems to have none of these drawbacks (Fig. I). It has a low chromosome number (2n = 22) 1,~, large chromosomes, and large numbers of fish can be easily captured and held for study. The present study was carried out to determine (I) the best chromosome technique, (2) which organs were suitable for chromosome analysis and (3) if chromosome breakage could be easily assessed.
CYTOGENETICS MODEL* n
227
wvo
MATERIALS AND METHODS
Mudminnows were seined from mud-bottomed streams near Port Byron, N.Y. Fish were kept in an aerated, well-planted I5-gallon aquarium maintained at a temperature range from 14 ° to 15 ° and a pH range of 7 to 8. Fish to be used in chromosome studies were injected I P with 0. 5 ml of o.1% colchicine (Sigma) and returned to well-aerated water. 6 h after injection the fish were sacrificed and the gills, pancreas, spleen, heart, stomach, intestines, kidney, scale, liver, and brain were removed and placed in an 0.4% KC1 hypotonic solution for 15-2o rain.
b7.
_o .Q D
Z
i
L
1
2
3
4
5
6
7
8
I
9
I
10
Organs
Fig. 2. A n a l y s i s of t h e s u i t a b i l i t y of v a r i o u s o r g a n s of t h e m u d m i n n o w for c y t o g e n e t i c s s t u d i e s s h o w i n g s u p e r i o r i t y of intestines, s t o m a c h , kidneys, a n d gills. O r g a n code: I, i n t e s t i n e s ; 2, s t o m a c h ; 3, k i d n e y ; 4, gill; 5, scale; 6, spleen; 7, b r a i n ; 8, p a n c r e a s ; 9, h e a r t ; io, liver.
The hypotonic was removed and 3:1 ethanol:acetic acid was added. I h later the fixative was removed and fresh 3 :i ethanol:acetic acid was added. Squash preparations were made in 45% acetic acid S and examined using a Leitz Ortholux phase contrast microscope. Detailed examination of chromosomes was achieved at a magnification of 1125 × using an oil immersion objective. Squash preparations and phase optics were used since it was believed this would produce the least number of technically induced artifacts. For each fish examined a count was made of the total number of usable metaphases (at least 18 chromosomes clearly visible) found in each tissue and the results recorded (Fig. 2). Permanent preparations were made by freezing the
228
A.D. KLIGERMANet al.
10/.
II
lqg. 3. Intestinal metaph~se spread from Umbra limi showing diplc,id complen~ent (bright field). slide in liquid nitrogen, popping off the coverslip, a n d staining tile slide in IO°/{) Giemsa for 5 rain. Fish to be irradiated were placed two at a time in a IOOO ml beaker filled with 30o ml of water. The fish were X - i r r a d i a t e d with a Picker deep-therapy u n i t operating at 14o kV a n d 15 mA. Each set of two fish was exposed to 325 R of X - r a d i a t i o n a d m i n i s t e r e d at a rate of 65 R/rain. Following irradiation, the fish were injected I P with a p p r o x i m a t e l y 0.5 ml of an o.I°/~ colchicine solution a n d r e t u r n e d to well-
229
CYTOGENETICS MODEL ~n v~vo
aerated water for from 3.5 to 7.5 h. The fish were then sacrificed, and squash preparations were made (as described previously) from the kidneys, gills, stomach, and intestines, and these were examined using phase optics and oil immersion at a magnification of 1125 ×. Metaphases were scored for aberrations (breaks, gaps, translocations), and the number recorded for each fish examined. Photographs were taken with a Leitz Ortholux camera using high contrast copy film. Squash preparations were also made from control mudminnows not exposed to any known clastogen, and slides were examined for aberrations in a similar fashion to the method described above. RESULTS
Though the method of determining mitotic activity of the various organs was crude, Fig. 2 shows that four organs clearly stand out as giving superior numbers of usable metaphases: the intestines, stomach, kidney tissues and gills. The tissues of the alimentary canal consistently gave the best metaphase spreads (Fig. 3)- These results compare favorably with other methods previously used to determine the mitotic index in the mudminnow n. The gonads were not used in this study since it was found that mitoses were seasonal in nature, and when they did occur, they appeared in such great profusion that they were of little value for clastogenic studies. Examination of the data in Table I reveals that upon exposure to 325 R of X-radiation, breaks and gaps such as those shown in Figs. 4 a and 4 b were found in approximately 30% of the metaphases of each fish examined (X = 29.69 :~ 8.22). No translocations, inversions, or exchange figures were detected. When the X-ray induced aberration data are compared to the control data (Table II), two points are evident. First, the mudminnow has a low spontaneous chromosome breakage rate (about 0.03%), and secondly, 325 R of X-rays caused over a 3oo-fold increase in the detectable aberration rate in the somatic cells of the mudminnow. The induced aberration rate of o.ooli aberrations/metaphase/fish/R was in line with what other researchers have obtained with other animals exposed to X-rays3, 5. Typical radiation induced cytological effects such as the lowering of mitotic index and increased clumping of metaphase chromosomes were also observed. Thus, radiation induced chromosome damage could be detected in the cells of the mudminnow, and the amount and type of damage was similar to the damage TABLE I ANALYSIS OF CHROMOSOME A B E R R A T I O N S IN FISH E X P O S E D TO 3 2 5 R OF X - R A D I A T I O N
Fish code letter
N u m b e r of metaphases examined
N u m b e r of metaphases with at least one aberration a
Total number of aberrations
Percentage of metaphases with at least one aberration
Aberrations per metaphase
A B C D E F
34 ii 45 14 185 98
9 4 io 6 42 27
13 4 13 7 53 34
26.47 36-36 22.22 42.86 22.7 ° 27.55
0,3824 o.3636 o.2829 0.5000 0.2865 0-3469
Total6
387
98
124
29.69~8.22
o.3604±o.o795 b
X±S.D.
a A b e r r a t i o n ~ b reak, gap. b Mean n u m b e r of a b e r r a t i o n s per m e t a p h a s e per R p e r fish : o.ooi I -k 0.0002.
2]jO
A . D . KLIGERMAN el al.
°
i
B Fig. -t. X - R a y i n d u c e d c h r o m a t i d a b e r r a t i o n s in s t o m a c h cells of a m u d i n i n n o w (phase c o n t r a s t ) . A. C h r o m a t i d b r e a k (arrow). B. C h r o m a t i d gap (arrow),
found in cells of other vertebrates similarly exposed to X-radiation. Furthermore, these data illustrate that fishes can be useful tools in studying chromosome aberration. DISCUSSION
As mentioned previously, only a limited number of experimental studies have
CYTOGENETICS M O D E L ~ n v ~ v o
231
Fig. 5. Karyotype of Umbra timi showing eleven pairs of metacentric and submetacentric chromosomes (bright field). been carried out on the chromosomes of fishes. The fishes chosen for study had large chromosome numbers and/or small chromosomes. However, the mudminnow has an ideal karyotype for such studies. The diploid complement consists of 22 large metacentric and submetacentric chromosomes which facilitate clastogenic studies (Fig. 5). Furthermore, the mudminnow is a small hardy fish which is easy to obtain and keep in the laboratory.
232
A . D . KLIGERMAN el al.
"I'ABI3:11 ANALYSIS
OF CnROMOSOMF
ABERRATIONS
IN FISH NOT DELIBERATELY
EXPOSED
(CONTROL)
TO ANY
MUTAGENS
Irish No.
Number of metaphases examined
Number oj metaphases with at least ore" aberration a
Total number of aberrations
Percentage of metaphases with at least one aberration
Ab~rralions per metaphase
I
61
0
O
O
O.O0
2
49
o
o
o
o.oo
3
20
0
O
O
O.O0
4 5 6
40 I OO 1II
o 0 O
o O O
o O 0
o.oo O. O( ) O.O0
7
48
o
o
o
o.oo o.oo
8
27 I
o
o
o
9
21
O
O
O
O.OO
IO I I I 2
22 1 160 250
O o O
O o O
O o O
O .OO o.oo O.OO
13
299
l
4
0.334
1675
l
4
o.o134 o.oolo i o.oo37
Total ]3
~ ; q S.l).
O.O26~ o.o93
a Aberration ~ breaks, gaps. R a d i a t i o n s t u d i e s c a r r i e d o u t o n t h e m u d i n i n n o w t o d e t e r m i n e if, in f a c t , tile c h r o m o s o m e s of t h e n m d m i n n o w w e r e u s e f u l for c l a s t o g e n i c s t u d i e s p r o v e d s u c c e s s f u l . A d e q u a t e n u m b e r s of m e t a p h a s e s c o u l d b e o b t a i n e d f r o m v a r i o u s o r g a n s , a n d radiation damage was similar to that found in other vertebrates exposed to Xr a d i a t i o n . T o o u r k n o w l e d g e , t h i s is t h e first t i m e a c y t o g e n e t i c s s y s t e m h a s b e e n d e v e l o p e d w h i c h m a k e s u s e of a fish in w h i c h c h r o m o s o m e d a m a g e c a n b e a c c u r a t e l y d e t e c t e d . T h i s c o u l d p r o v e t o b e v a l u a b l e in c o r r e l a t i n g p h e n o t y p i c d i s t u r b a n c e s in fishes w i t h specific c h r o m o s o m e a b n o r m a l i t i e s ; s o m e t h i n g t h a t h a s b e e n l a c k i n g in l n u c h of t h e c l a s t o g e n e t i c a n d m u t a g e n i c w o r k d o n e w i t h fishes u p t o n o w . In a d d i t i o n it is felt t h a t t h e m u d m i n n o w c o u l d p r o v e t o b e a v a l u a b l e n l o d e l o r g a n i s m for s t u d y i n g t h e c l a s t o g e n i c effects of w a t e r - b o r n e c h e m i c a l s . P r e l i m i n a r y e x p e r i m e n t s c o n d u c t e d w i t h m u d m i n n o w s e x p o s e d t o v a r i o u s c o n c e n t r a t i o n s of m a l e i c h y d r a z i d e d i s s o l v e d in a q u a r i u m w a t e r i n d i c a t e d t h a t c h e m i c a l s c o u l d c a u s e a s m a l l i n c r e a s e in t h e o b s e r v e d r a t e of s o m a t i c c h r o m o : s o m e b r e a k a g e in fish so e x p o s e d . R E F I ~.RENC I;.S BEAMISH, B. J., M. J. ~IERRILb'ES AND E. J. CROSSMAN, Karyotype and DNA v a l u e s f o r members of the suborder Esocoidei, Chromoson'la, 34 1197 l) 436 4772 BLOOM, .q.E., G. POVAR AND D. B. PEAKALL, Chrnnlosome preparations from the avian allantoic sac, ,Stain Technol., 47 (1972) 123-1273 CORMIER, J. M., AND S. E. BLOO~a, An in vivo study of the el~fects of X-radiation on the chroniosomes of chick allantoic membranes, Mutation ICes., 20 (I973) 77-85 • 4 DE SERRES, F. J., AND W. SHERIDAN (Eds.), The evaluation of chemical mutagenicity data in relation to population risk, Environ. Health Per@., Experinmntal issue No. 6 (I973) i 232. 5 ELKIND, M. M., AND G. F. WHITMORE, The Radiobiology of Cultured Mammalian Cells, Gordon and Breach, New York, 1967, pp. 449 451. 6 ENDO, A., AND T. H. [NGALLS, Chromosomes of the zebra fish, J. Here&, 59 11968) 382 384 . I
7
FISHBEIN, L . , \ V . ( ; . F L A M M A N D M . L . F A L K , Chemical Mutagens: Environmental EjJ)cts on Biological Systems, Academic Press, New York, 197 o. FOLEY, J. o., The spermatogenesis of Umbra limi with special reference to the behavior of the spermatogonial chromosomes and the first maturation division, Biol. Bull. (Woods Hole, Mass.), 5o (I926) ~I7 I.t7.
CYTOGENETICS MODEL Zn V~VO
233
9 HOLLAENDER, A., Chemical Mutagens: Principles and Methods for their Detection, Vols. 1 a n d II, P l e n u m , N e w York, 1971. io HOWELL, W. M., S o m a t i c c h r o m o s o m e s of t h e Black G h o s t Knifefish, Apteronotus albifrons, Copeia, I (1972) I 9 I - I 9 3 . I I KLIGERMAN, A. D., S p o n t a n e o u s a n d i n d u c e d c h r o m o s o m e a b e r r a t i o n s in t h e m u d m i n n o w (Umbra limi): D e v e l o p m e n t of a n in vivo c y t o g e n e t i c s model, Master's Thesis, Cornell University, I t h a c a , N.Y., 1974. 12 LEGATOR, M. S., Chemical m u t a g e n e s i s c o m e s of age, J. Hered., 61 (197 o) 239-242. 13 LEGATOR, IV[. S., Deficiencies in our p r e s e n t protocol for chemical e v a l u a t i o n a n d possible remedies, Ann. N . Y . Acad. Sci., 179 (1971) 5o8-513 .
