- Email: [email protected]
Frequency of sister-chromatid exchange (SCE) in bone-marrow cells of severely malnourished animals during early life
Mutation Research, 175 (1986) 29-31 Elsevier
29
MRLett. 0888
Frequency of sister-chromatid exchange (SCE) in bone-marrow cells of severely malnourished animals during early life* Miguel Betancourt, Patricia Balvanera and Rocio Ortiz Departamento de Ciencias de la Salud, Universidad Autdnoma Metropolitana-Iztapalapa, Apartado Postal 55-535, C.P. 09340 D.F. M~xico (M~xico)
(Accepted 7 April 1986)
Summary
The frequency of sister-chromatid exchange (SCE) was examined in bone-marrow cells of 211-day-old Wistar rats malnourished during lactation and well-nourished controls of the same age. Malnutrition was obtained by increasing the litter size to 15 pups per mother. SCE were scored in 25 consecutive seconddivision metaphases in the femoral bone marrow cells from each animal. The average s c E in the malnourished animals was significantly higher than in the control group (p < 0.01). The distribution of SCE per mitosis was also significantly higher in the malnourished animals (p < 0.001). These results indicate that malnutrition per se during early life can increase SCE in the bone marrow of experimental animals.
Sister-chromatid exchange (SCE) methodology has been used for its accuracy in the study of genetic damage produced by different factors. Mutchinick et al. (1979) using this technique failed to find differences in the frequency of SCE in cultured lymphocytes from children suffering from protein energy malnutrition (PEM). In a similar study, however, Murthy et al. (1980) found an increase of SCE in lymphocytes from children with kwashiorkor. Experimentally Murthy and Srikantia (1981) found significant differences in the frequency of SCE in adult mice suffering from malnutrition caused by different diets when compared with controls. Cellular damage produced by PEM is not the same at different ages, being more pronounced during the lactation period than in adulthood
*Supported in part by grant-PCALXNA-000982 CONACYT (Mdxico).
(Winick and Noble, 1966). The aim of ~his study was to determine the frequency of SCE, as a measure of genetic damage, caused by severe PEM during the early life of experimental animals.
Material and methods
PEM was produced by the method of Winick and Noble (1966). Newborn outbred Wistar rats were placed in litters of 15 pups per mother during the first 21 days of life. Control animals !were kept during the same period in normal size litters of six animals per mother. The mothers were fed a commercial rat diet ad libitum. On the 21st day of life the weaned animals wbre injected intraperitoneally with a dose of 1 mg/g body weight of bromodeoxyuridine (BrdU) previously adsorbed onto activated charcoal following the method of Morales (1980). The animals were injected 20 h after the BrdU application with a
0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V, (Biomedical Division)
30 d o s e o f 1 m g / 1 0 0 g b o d y weight o f colchicine a n d sacrificed 1 h later. B o n e m a r r o w cells o b t a i n e d f r o m a f e m u r o f each a n i m a l were rinsed with w a r m e d M c C o y 5 A m e d i u m . T h e cell s u s p e n s i o n was t r e a t e d with 0.075 M KCI s o l u t i o n a n d fixed with m e t h a n o l - a c e t i c acid ( 3 : 1 ) , T h e a i r - d r i e d slides were s t a i n e d with G i e m s a b y the P e r r y - W o l f f m e t h o d (1974). S C E were s c o r e d in 25 consecutive s e c o n d division m e t a p h a s e s f r o m each a n i m a l .
DISTRIBUTION OF THE NUMBER OF SCE PER MITOSIS IN 2 GROUPS OF RATS SCE per mitosis 0 and 1 2 and 3 4 and 5 6 and 7 8 and 9 10 and 11
Malnourished rats
Well nourished rats
Number
°7o
Number
O7o
68 112 59 29 16 11
23 38 20 10 5 4
118 109 44 18 4 7
39 37 15 6 I 2
Significant difference between both groups of animals X2 = 14.75; p<0.001.
Results Results a r e p r e s e n t e d in T a b l e 1. T h e m e a n S C E f r e q u e n c y in the 12 m a l n o u r i s h e d rats was 3.4_+ 0.54, with a r a n g e o f 2 . 4 4 - 4 . 0 4 . T h e m e a n S C E in the c o n t r o l g r o u p was 2.5_+0.64, with a r a n g e o f 1.56-3.28. A p a i r e d "t' S t u d e n t test b e t w e e n these values gave a h i g h l y significant d i f f e r e n c e in f a v o r o f the m a l n o u r i s h e d g r o u p ( t = 3.69; p < 0 . 0 1 ) . T h 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 p e r cell was also a n a l y z e d ( T a b l e 2). T h e p e r c e n t a g e o f m i t o s i s s h o w i n g o n e o r n o S C E was h i g h e r in the well-
TABLE 1 INDIVIDUAL SCE FREQUENCY IN MALNOURISHED RATS DURING THE LACTATION PERIOD The BrdU was injected 21 h before sampling. Animal No.
TABLE 2
Malnourished rats
Well nourished rats
S.D.
~
S.D.
1 2 3 4
2.76 3.36 3.80 3.72
1.56 2.30 3.47 3.42
1.60 3.08 1.56 1.68
1.68 1.99 1.50 1.40
5 6 7 8
3.72 2.64 3.20 4.04
2.17 2.23 2.17 3.41
3.20 2.08 2.88 2.84
2.38 1.57 2.80 2.17
9 10 11 12
3.40 4.00 3.72 2.44
2.98 1.83 2.17 2.35
2.60 3.28 2.24 3.00
2.69 2.30 1.54 2.92
X = 3.40S.E. ±0.54 t = 3.69; p<0.01.
X = 2.50S.E. ±0.64
n o u r i s h e d a n i m a l s (39°7o) t h a n in the e x p e r i m e n t a l g r o u p (23070). T h e p e r c e n t a g e o f mitosis with two o r m o r e S C E was a l w a y s higher in the m a l n o u r i s h ed rats. These d a t a were a n a l y z e d with a Xz test o f p r o p o r t i o n s that g a v e a highly significant d i f f e r e n c e in f a v o r o f the m a l n o u r i s h e d g r o u p (X2 = 14.75; d f 1; p < 0 . 0 0 1 ) . Cell p r o l i f e r a t i o n was also a n a l y z e d in b o n e m a r r o w f r o m 6 m a l n o u r i s h e d a n i m a l s t r e a t e d for 21 h with B r d U . 100 r a n d o m l y selected cells f r o m each rat were s c o r e d a n d the n u m b e r o f cells in 1st, 2nd a n d 3rd d i v i s i o n were r e c o r d e d . 216 mitoses (36o70) were f o u n d in first division, 384 (64o70) in s e c o n d a n d n o n e in t h i r d division. O n the o t h e r h a n d in well n o u r i s h e d a n i m a l s , 143 mitoses (28.3o7o) were f o u n d in first division, 454 (75.6°/o) in s e c o n d a n d 3 mitoses a p p e a r e d in t h i r d division. Significant differences were s h o w n between b o t h g r o u p s o f a n i m a l s (X2= 8.56; p < 0 . 0 1 ) .
Discussion T h e results o f this e x p e r i m e n t clearly i n d i c a t e that animals suffering from experimental PEM d u r i n g early life h a v e an increase in the f r e q u e n c y o f S C E . O u r findings a r e in a g r e e m e n t with t h o s e o f M u r t h y a n d S r i k a n t i a (1981), which show t h a t d e p r i v a t i o n o f e n e r g y a n d p r o t e i n r e q u i r e m e n t s in a n i m a l s d u r i n g a d u l t h o o d p r o d u c e s an increase o f S C E in mice.
31
Since the malnourished animals in this experiment were free from infections, which are frequent in children with PEM, our results indicate that an increase in SCE is due to the malnutrition per se. Murthy and Bharkaram (1981) have demonstrated that children suffering from kwashiorkor are not deficient in DNA repair, the increase in SCE found in this study could therefore be due to a defect in DNA replication, rather than in DNA repair. In vitro studies with cells from malnourished children (Murthy et al., 1982), as well as in vivo studies with bone-marrow cells from animals (Ortiz and Betancourt, 1984) have demonstrated that PEM causes a prolongation of the cellular proliferation cycle, this is confirmed again, now at 21 h of BrdU intake, where the percentage of mitoses in the first division was higher in those cells from malnourished animals than in the control group. It is possible therefore, that a correlation exists between damage produced by SCE and prolongation of the cell cycle. It could be that differential uptake of BrdU accounts for the differences in SCE between malnourished and control groups. We have discarded the possibility of greater incorporation of BrdU into DNA in the malnourished rats because the minimum dose of BrdU was used to obtain differential staining in both groups. When we used less than 1 mg/g body weight, none of the ceils showed differential staining of their chromosomes. If malnourished cells would have incorporated more BrdU, differential staining would have been detected with lower doses of BrdU. Experimental support for the aforementioned could be obtained by correlating the SCE increase with BrdU concentration. A more accurate analysis of BrdU uptake could be done using monoclonal
antibodies against BrdU incorporated into DNA (Gratzner, 1982).
Acknowledgement We thank Mrs. Maryana Meaney for typing the manuscript.
References Gratzner, H.G. (1982) Monoclonal antibody to 5-bromo- and 5-iodo-deoxyuridine: A new reagent for detection of DNA replication, Science, 218, 474-475. Morales, P. (1980) Analysis in vivo of sister chr0matid exchange in bone marrow and salivary gland cellsl Mutation Res., 74, 61-69. Murthy, P.B., and P. Bhaskaram (1981) Unscheduled DNA synthesis in lymphocytes from malnourished children, Nutr. Rep. Int., 23, 321-325. Murthy, P.B., and S.G. Srikantia (1981) SCE frequency in malnourished mice, Metabolism, 30, 1-2. Murthy, P.B., P. Bhaskaram and S.G. Srikantia (~980) Sister chromatid exchanges in protein-energy malnutrilion, Hum. Genet., 55, 405-406. Murthy, P.B., M.A. Rahman and P.G. Tulpude (~982) Lymphocyte proliferation kinetics in malnourished children measured by differential chromatid staining, Br. J. Nutr., 47, 445-450. Mutchinick, O., R. Lisker, L. Ruz, F. SalamaOca and S. Armendares (1979) Frequency of sister chromati~t exchanges in severe protein calorie malnutrition, Ann. Genet., 22, 129-132. Ortiz, R., and M. Betancourt (1984) Cell proliferaiion in bone marrow cells of severely malnourished animali, J. Nutr., 114, 472-476. Perry, P., and S. Wolff (1974) New Giemsa method for differentiai staining of sister chromatids, Nature (Ldndon), 251, 156-158. Winick, M., and A. Noble (1966) Cellular respons d in rats during malnutrition at various ages, J. Nutr., 89, 500-306. /
Communicated by R.J. Preston
29
MRLett. 0888
Frequency of sister-chromatid exchange (SCE) in bone-marrow cells of severely malnourished animals during early life* Miguel Betancourt, Patricia Balvanera and Rocio Ortiz Departamento de Ciencias de la Salud, Universidad Autdnoma Metropolitana-Iztapalapa, Apartado Postal 55-535, C.P. 09340 D.F. M~xico (M~xico)
(Accepted 7 April 1986)
Summary
The frequency of sister-chromatid exchange (SCE) was examined in bone-marrow cells of 211-day-old Wistar rats malnourished during lactation and well-nourished controls of the same age. Malnutrition was obtained by increasing the litter size to 15 pups per mother. SCE were scored in 25 consecutive seconddivision metaphases in the femoral bone marrow cells from each animal. The average s c E in the malnourished animals was significantly higher than in the control group (p < 0.01). The distribution of SCE per mitosis was also significantly higher in the malnourished animals (p < 0.001). These results indicate that malnutrition per se during early life can increase SCE in the bone marrow of experimental animals.
Sister-chromatid exchange (SCE) methodology has been used for its accuracy in the study of genetic damage produced by different factors. Mutchinick et al. (1979) using this technique failed to find differences in the frequency of SCE in cultured lymphocytes from children suffering from protein energy malnutrition (PEM). In a similar study, however, Murthy et al. (1980) found an increase of SCE in lymphocytes from children with kwashiorkor. Experimentally Murthy and Srikantia (1981) found significant differences in the frequency of SCE in adult mice suffering from malnutrition caused by different diets when compared with controls. Cellular damage produced by PEM is not the same at different ages, being more pronounced during the lactation period than in adulthood
*Supported in part by grant-PCALXNA-000982 CONACYT (Mdxico).
(Winick and Noble, 1966). The aim of ~his study was to determine the frequency of SCE, as a measure of genetic damage, caused by severe PEM during the early life of experimental animals.
Material and methods
PEM was produced by the method of Winick and Noble (1966). Newborn outbred Wistar rats were placed in litters of 15 pups per mother during the first 21 days of life. Control animals !were kept during the same period in normal size litters of six animals per mother. The mothers were fed a commercial rat diet ad libitum. On the 21st day of life the weaned animals wbre injected intraperitoneally with a dose of 1 mg/g body weight of bromodeoxyuridine (BrdU) previously adsorbed onto activated charcoal following the method of Morales (1980). The animals were injected 20 h after the BrdU application with a
0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V, (Biomedical Division)
30 d o s e o f 1 m g / 1 0 0 g b o d y weight o f colchicine a n d sacrificed 1 h later. B o n e m a r r o w cells o b t a i n e d f r o m a f e m u r o f each a n i m a l were rinsed with w a r m e d M c C o y 5 A m e d i u m . T h e cell s u s p e n s i o n was t r e a t e d with 0.075 M KCI s o l u t i o n a n d fixed with m e t h a n o l - a c e t i c acid ( 3 : 1 ) , T h e a i r - d r i e d slides were s t a i n e d with G i e m s a b y the P e r r y - W o l f f m e t h o d (1974). S C E were s c o r e d in 25 consecutive s e c o n d division m e t a p h a s e s f r o m each a n i m a l .
DISTRIBUTION OF THE NUMBER OF SCE PER MITOSIS IN 2 GROUPS OF RATS SCE per mitosis 0 and 1 2 and 3 4 and 5 6 and 7 8 and 9 10 and 11
Malnourished rats
Well nourished rats
Number
°7o
Number
O7o
68 112 59 29 16 11
23 38 20 10 5 4
118 109 44 18 4 7
39 37 15 6 I 2
Significant difference between both groups of animals X2 = 14.75; p<0.001.
Results Results a r e p r e s e n t e d in T a b l e 1. T h e m e a n S C E f r e q u e n c y in the 12 m a l n o u r i s h e d rats was 3.4_+ 0.54, with a r a n g e o f 2 . 4 4 - 4 . 0 4 . T h e m e a n S C E in the c o n t r o l g r o u p was 2.5_+0.64, with a r a n g e o f 1.56-3.28. A p a i r e d "t' S t u d e n t test b e t w e e n these values gave a h i g h l y significant d i f f e r e n c e in f a v o r o f the m a l n o u r i s h e d g r o u p ( t = 3.69; p < 0 . 0 1 ) . T h 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 p e r cell was also a n a l y z e d ( T a b l e 2). T h e p e r c e n t a g e o f m i t o s i s s h o w i n g o n e o r n o S C E was h i g h e r in the well-
TABLE 1 INDIVIDUAL SCE FREQUENCY IN MALNOURISHED RATS DURING THE LACTATION PERIOD The BrdU was injected 21 h before sampling. Animal No.
TABLE 2
Malnourished rats
Well nourished rats
S.D.
~
S.D.
1 2 3 4
2.76 3.36 3.80 3.72
1.56 2.30 3.47 3.42
1.60 3.08 1.56 1.68
1.68 1.99 1.50 1.40
5 6 7 8
3.72 2.64 3.20 4.04
2.17 2.23 2.17 3.41
3.20 2.08 2.88 2.84
2.38 1.57 2.80 2.17
9 10 11 12
3.40 4.00 3.72 2.44
2.98 1.83 2.17 2.35
2.60 3.28 2.24 3.00
2.69 2.30 1.54 2.92
X = 3.40S.E. ±0.54 t = 3.69; p<0.01.
X = 2.50S.E. ±0.64
n o u r i s h e d a n i m a l s (39°7o) t h a n in the e x p e r i m e n t a l g r o u p (23070). T h e p e r c e n t a g e o f mitosis with two o r m o r e S C E was a l w a y s higher in the m a l n o u r i s h ed rats. These d a t a were a n a l y z e d with a Xz test o f p r o p o r t i o n s that g a v e a highly significant d i f f e r e n c e in f a v o r o f the m a l n o u r i s h e d g r o u p (X2 = 14.75; d f 1; p < 0 . 0 0 1 ) . Cell p r o l i f e r a t i o n was also a n a l y z e d in b o n e m a r r o w f r o m 6 m a l n o u r i s h e d a n i m a l s t r e a t e d for 21 h with B r d U . 100 r a n d o m l y selected cells f r o m each rat were s c o r e d a n d the n u m b e r o f cells in 1st, 2nd a n d 3rd d i v i s i o n were r e c o r d e d . 216 mitoses (36o70) were f o u n d in first division, 384 (64o70) in s e c o n d a n d n o n e in t h i r d division. O n the o t h e r h a n d in well n o u r i s h e d a n i m a l s , 143 mitoses (28.3o7o) were f o u n d in first division, 454 (75.6°/o) in s e c o n d a n d 3 mitoses a p p e a r e d in t h i r d division. Significant differences were s h o w n between b o t h g r o u p s o f a n i m a l s (X2= 8.56; p < 0 . 0 1 ) .
Discussion T h e results o f this e x p e r i m e n t clearly i n d i c a t e that animals suffering from experimental PEM d u r i n g early life h a v e an increase in the f r e q u e n c y o f S C E . O u r findings a r e in a g r e e m e n t with t h o s e o f M u r t h y a n d S r i k a n t i a (1981), which show t h a t d e p r i v a t i o n o f e n e r g y a n d p r o t e i n r e q u i r e m e n t s in a n i m a l s d u r i n g a d u l t h o o d p r o d u c e s an increase o f S C E in mice.
31
Since the malnourished animals in this experiment were free from infections, which are frequent in children with PEM, our results indicate that an increase in SCE is due to the malnutrition per se. Murthy and Bharkaram (1981) have demonstrated that children suffering from kwashiorkor are not deficient in DNA repair, the increase in SCE found in this study could therefore be due to a defect in DNA replication, rather than in DNA repair. In vitro studies with cells from malnourished children (Murthy et al., 1982), as well as in vivo studies with bone-marrow cells from animals (Ortiz and Betancourt, 1984) have demonstrated that PEM causes a prolongation of the cellular proliferation cycle, this is confirmed again, now at 21 h of BrdU intake, where the percentage of mitoses in the first division was higher in those cells from malnourished animals than in the control group. It is possible therefore, that a correlation exists between damage produced by SCE and prolongation of the cell cycle. It could be that differential uptake of BrdU accounts for the differences in SCE between malnourished and control groups. We have discarded the possibility of greater incorporation of BrdU into DNA in the malnourished rats because the minimum dose of BrdU was used to obtain differential staining in both groups. When we used less than 1 mg/g body weight, none of the ceils showed differential staining of their chromosomes. If malnourished cells would have incorporated more BrdU, differential staining would have been detected with lower doses of BrdU. Experimental support for the aforementioned could be obtained by correlating the SCE increase with BrdU concentration. A more accurate analysis of BrdU uptake could be done using monoclonal
antibodies against BrdU incorporated into DNA (Gratzner, 1982).
Acknowledgement We thank Mrs. Maryana Meaney for typing the manuscript.
References Gratzner, H.G. (1982) Monoclonal antibody to 5-bromo- and 5-iodo-deoxyuridine: A new reagent for detection of DNA replication, Science, 218, 474-475. Morales, P. (1980) Analysis in vivo of sister chr0matid exchange in bone marrow and salivary gland cellsl Mutation Res., 74, 61-69. Murthy, P.B., and P. Bhaskaram (1981) Unscheduled DNA synthesis in lymphocytes from malnourished children, Nutr. Rep. Int., 23, 321-325. Murthy, P.B., and S.G. Srikantia (1981) SCE frequency in malnourished mice, Metabolism, 30, 1-2. Murthy, P.B., P. Bhaskaram and S.G. Srikantia (~980) Sister chromatid exchanges in protein-energy malnutrilion, Hum. Genet., 55, 405-406. Murthy, P.B., M.A. Rahman and P.G. Tulpude (~982) Lymphocyte proliferation kinetics in malnourished children measured by differential chromatid staining, Br. J. Nutr., 47, 445-450. Mutchinick, O., R. Lisker, L. Ruz, F. SalamaOca and S. Armendares (1979) Frequency of sister chromati~t exchanges in severe protein calorie malnutrition, Ann. Genet., 22, 129-132. Ortiz, R., and M. Betancourt (1984) Cell proliferaiion in bone marrow cells of severely malnourished animali, J. Nutr., 114, 472-476. Perry, P., and S. Wolff (1974) New Giemsa method for differentiai staining of sister chromatids, Nature (Ldndon), 251, 156-158. Winick, M., and A. Noble (1966) Cellular respons d in rats during malnutrition at various ages, J. Nutr., 89, 500-306. /
Communicated by R.J. Preston