Mutation Research, 280 (1992) 279-283 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00
279
MUTGEN 01817
Effects of amino acids on sister-chromatid exchanges Zili Zhang
a
and Jie Yang b
a Department of Biology, Nankai University, Tianjin 300071, China and ~ Gynecology-Obstetrics Department, Second Affiliated Hospital of Tianjin Medical College, Tianjin 300211, China (Received 17 December 1991) (Revision received 23 March 1992) (Accepted 4 May 1992)
Keywords." Sister-chromatid exchange; Amino acids; Human Bromodeoxyuridine
peripheral blood lymphocytes; Hordeum
vulgare; Lysine;
Summary
The effects of 10 amino acids on sister-chromatid exchange (SCE) frequency in human peripheral blood lymphocytes (PBL) and six amino acids on the SCE frquency in root tip cells of Hordeum vulgare were studied. Alanine (Ala), glycine (Gly), phenylalanine (Phe), valine (Val), histidine (His) and serine (Ser) induced a significant increase in SCE in PBL but threonine (Thr), isoleucine (lie), lysine (Lys) and arginine (Arg) did not. Ala, Gly, Thr, lie and Val induced a significant increase in SCE in root tip cells of Hordeum vulgare but Lys did not. The effect of Lys and bromodeoxyuridine (BrdU) on SCE levels in PBL and the interaction between them were also studied. The results show that Lys can inhibit the SCE induced by BrdU.
The existence of sister-chromatid exchanges (SCE) was first detected by J. Herbert Taylor and his colleagues in 1957 (Taylor et al., 1957). In the following years, scientific interest in the nature, significance and utility of this cytogenetically observed phenomenon expanded (Latt, 1974). SCE are now known to be associated with chromosome damage a n d / o r repair. Many researchers discovered that numerous mutagens and carcinogens can induce SCE and the concentration for inducing SCE is far lower than that needed to induce a significant increase in chromosomal aberrations. This has resulted in SCE being widely
Correspondence: Dr. Zili Zhang, Department of Biology, Nankai University, Tianjin 300071, China.
accepted as an indicator of mutagenic and carcinogenic potency (Tice et al., 1984; Carrano et al., 1978; Kato, 1975; Latt et al., 1975; Perry and Evans, 1975; Solomon and Bobrow, 1975). Few contributions have been published about the effects of chemical agents such as amino acids (AAs) which are often used and are thought to be non-genotoxic. Thus, we evaluated whether AAs can induce SCE in human peripheral blood lymphocytes (PBL) and root tip cells of Hordeum vulgate.
Materials and methods
PBL SCE test The whole-blood microculture technique was used. 0.4 ml of venous whole blood was inocu-
280 l a t e d to 5 ml of the c u l t u r e m e d i u m ( p H 7.0) c o n t a i n i n g 41.6 mg of R P M I 1640 ( J R Scientific Inc., W o o d l a n d , CA), 1 ml of fetal calf s e r u m ( p u r c h a s e d from T i a n j i n M e d i c a l College, China), 1.5 mg of P H A ( p r o d u c e d by G u a n g d o n g Biological P r o d u c t Institute, China), 10,000 IU each o f penicillin a n d s t r e p t o m y c i n , and 0.1 ml of 5 % N a H C O 3. T h e mixture was i n c u b a t e d for 24 h. B r d U , at 10 p , g / m l , was a d d e d at 24 h of culture. A t 48 h of culture, A A s (dissolved in saline solution) or saline solution (for c o n t r o l ) was a d d e d a n d the c u l t u r e s w e r e i n c u b a t e d for a n o t h e r 24 h. P B L were h a r v e s t e d at the e n d of the 24-h exposure p e r i o d (total c u l t u r e time = 72 h) with 0.05 / x g / m l colchicine b e i n g a d d e d 2 h b e f o r e harvesting. All c u l t u r e s w e r e i n c u b a t e d at 3 7 ° C in the dark. PBL, h a r v e s t e d a f t e r g e n t l e c e n t r i f u g a t i o n (1000 rpm, 10 min) a n d d i s c a r d i n g the s u p e r n a t a n t , w e r e r e s u s p e n d e d in 0.075 M KCI solution for h y p o t o n i c t r e a t m e n t for 20 min, cent r i f u g e d again, and finally fixed in m e t h a n o l acetic acid solution ( 3 : 1 ) t h r e e times. A few d r o p s of the cell s u s p e n s i o n w e r e d r o p p e d o n t o a clean slide t a k e n from icy w a t e r a n d f r a m e - d r i e d . T h e slides w e r e i n c u b a t e d in 2 × SSC solution in p e t r i dishes at 70 ° C a n d i r r a d i a t e d s i m u l t a n e ously u n d e r a U V l a m p (30 W ) at a d i s t a n c e of 10 cm for 20 min. T h e p r e p a r a t i o n s w e r e s t a i n e d in 3 % G i e m s a solution for 10 min a f t e r b e i n g r i n s e d with t a p w a t e r (Xing, 1990; Z h a n g , 1991). S C E analysis was c a r r i e d out on cells having r e p l i c a t e d for two cycles in the p r e s e n c e o f B r d U . T e n m e t a p h a s e s w e r e s c o r e d for each t r e a t m e n t .
Hordeum culgare SCE test S e e d s w e r e g e r m i n a t e d on m o i s t e n e d filter p a p e r in p e t r i dishes at 25 ° C. W h e n they were
a b o u t 1 cm long, the roots of the g e r m i n a t e d seeds w e r e t r a n s f e r r e d into an a q u e o u s solution c o n t a i n i n g B r d U (5 × 10 4 M), 5 - f l u o r o d e oxyuridine ( F d U ) (10 - s M) and u r i d i n e ( U r d ) (10 (' M), a n d i n c u b a t e d at 2 5 ° C in the d a r k for 12 h. T h e seedlings w e r e w a s h e d and t r a n s f e r r e d e i t h e r into an a q u e o u s solution c o n t a i n i n g thymid i n e (Thd) (10 4 M) a n d U r d (10 ~' M) (for control) or into an a q u e o u s solution c o n t a i n i n g T h d (10 4 M), U r d (10 ~' M) and A A and incub a t e d for a n o t h e r 12 h at 2 5 ° C in the dark. T h e n the s e e d l i n g s were w a s h e d , chilled (0 ° C , 36 h) for p r e t r e a t m e n t and fixed in m e t h a n o l - a c e t i c acid solution ( 3 : 1 ) for 24 h. A f t e r being r i n s e d with t a p water, the m c r i s t e m a t i c regions w e r e excised a n d d i g e s t e d in a mixture of cellulase a n d p e c t i n a s e (2.5% each) for 5 h at 25 ° C b e f o r e the cells u n d e r w e n t h y p o t o n i c t r e a t m e n t in distilled w a t e r for 1 h. T h e w a t e r was then d i s c a r d e d and the r o o t tips w e r e p o u n d e d into a paste. Cell s u s p e n s i o n s w e r e m a d e by a d d i n g a p r o p e r a m o u n t of fixative to the paste. A few d r o p s of the s u s p e n s i o n were s u b s e q u e n t l y d r o p p e d o n t o a clean slide t a k e n from icy w a t e r a n d f l a m e - d r i e d . T h e slides w e r e i n c u b a t e d in 2 × SSC solution in petri dishes at 80 ° C a n d i r r a d i a t e d s i m u l t a n e ously u n d e r a U V l a m p (30 W ) at a d i s t a n c e of 10 cm for 45 min. T h e p r e p a r a t i o n s w e r e s t a i n e d in 3% G i e m s a solution for 10 min a f t e r b e i n g rinsed with t a p w a t e r (Xing, 199(/; Z h a n g , 1991). Results
The effects of AAs on SCE in PBL T e n A A s w e r e involved in the test and d i v i d e d into two g r o u p s with e a c h g r o u p consisting of five A A s . A r a n d o m i z e d c o m p l e t e - b l o c k design was used to avoid the i n f l u e n c e of individual differ-
TABLE 1 EFFECTS OF Ala, Gly, Thr, lie AND Lys ON SCE IN PBL Concentrations (/xg/ml)
Amino acid Ala
Gly
Thr
lie
Lys
0 10 50 100
9.(1_+3.0 9.3 _+2.3 11.4+2.9 * 12.2_+3.7 **
9.2 + 2.7 1(I.9+ 3.1 10.7+3.7 11.9_+3.7 **
9.3 + 2.3 10.3 + 3.5 10.4+3.5 1(I.2+_2.9
9.5 ± 2.3 10.5 + 2.9 9.9-+3.4 10.1+3.5
10.0 + 2.5 9.8 ± 2.8 10.1 +3.9 9.5+3.(I
* P < 0.05: ** P
279
MUTGEN 01817
Effects of amino acids on sister-chromatid exchanges Zili Zhang
a
and Jie Yang b
a Department of Biology, Nankai University, Tianjin 300071, China and ~ Gynecology-Obstetrics Department, Second Affiliated Hospital of Tianjin Medical College, Tianjin 300211, China (Received 17 December 1991) (Revision received 23 March 1992) (Accepted 4 May 1992)
Keywords." Sister-chromatid exchange; Amino acids; Human Bromodeoxyuridine
peripheral blood lymphocytes; Hordeum
vulgare; Lysine;
Summary
The effects of 10 amino acids on sister-chromatid exchange (SCE) frequency in human peripheral blood lymphocytes (PBL) and six amino acids on the SCE frquency in root tip cells of Hordeum vulgare were studied. Alanine (Ala), glycine (Gly), phenylalanine (Phe), valine (Val), histidine (His) and serine (Ser) induced a significant increase in SCE in PBL but threonine (Thr), isoleucine (lie), lysine (Lys) and arginine (Arg) did not. Ala, Gly, Thr, lie and Val induced a significant increase in SCE in root tip cells of Hordeum vulgare but Lys did not. The effect of Lys and bromodeoxyuridine (BrdU) on SCE levels in PBL and the interaction between them were also studied. The results show that Lys can inhibit the SCE induced by BrdU.
The existence of sister-chromatid exchanges (SCE) was first detected by J. Herbert Taylor and his colleagues in 1957 (Taylor et al., 1957). In the following years, scientific interest in the nature, significance and utility of this cytogenetically observed phenomenon expanded (Latt, 1974). SCE are now known to be associated with chromosome damage a n d / o r repair. Many researchers discovered that numerous mutagens and carcinogens can induce SCE and the concentration for inducing SCE is far lower than that needed to induce a significant increase in chromosomal aberrations. This has resulted in SCE being widely
Correspondence: Dr. Zili Zhang, Department of Biology, Nankai University, Tianjin 300071, China.
accepted as an indicator of mutagenic and carcinogenic potency (Tice et al., 1984; Carrano et al., 1978; Kato, 1975; Latt et al., 1975; Perry and Evans, 1975; Solomon and Bobrow, 1975). Few contributions have been published about the effects of chemical agents such as amino acids (AAs) which are often used and are thought to be non-genotoxic. Thus, we evaluated whether AAs can induce SCE in human peripheral blood lymphocytes (PBL) and root tip cells of Hordeum vulgate.
Materials and methods
PBL SCE test The whole-blood microculture technique was used. 0.4 ml of venous whole blood was inocu-
280 l a t e d to 5 ml of the c u l t u r e m e d i u m ( p H 7.0) c o n t a i n i n g 41.6 mg of R P M I 1640 ( J R Scientific Inc., W o o d l a n d , CA), 1 ml of fetal calf s e r u m ( p u r c h a s e d from T i a n j i n M e d i c a l College, China), 1.5 mg of P H A ( p r o d u c e d by G u a n g d o n g Biological P r o d u c t Institute, China), 10,000 IU each o f penicillin a n d s t r e p t o m y c i n , and 0.1 ml of 5 % N a H C O 3. T h e mixture was i n c u b a t e d for 24 h. B r d U , at 10 p , g / m l , was a d d e d at 24 h of culture. A t 48 h of culture, A A s (dissolved in saline solution) or saline solution (for c o n t r o l ) was a d d e d a n d the c u l t u r e s w e r e i n c u b a t e d for a n o t h e r 24 h. P B L were h a r v e s t e d at the e n d of the 24-h exposure p e r i o d (total c u l t u r e time = 72 h) with 0.05 / x g / m l colchicine b e i n g a d d e d 2 h b e f o r e harvesting. All c u l t u r e s w e r e i n c u b a t e d at 3 7 ° C in the dark. PBL, h a r v e s t e d a f t e r g e n t l e c e n t r i f u g a t i o n (1000 rpm, 10 min) a n d d i s c a r d i n g the s u p e r n a t a n t , w e r e r e s u s p e n d e d in 0.075 M KCI solution for h y p o t o n i c t r e a t m e n t for 20 min, cent r i f u g e d again, and finally fixed in m e t h a n o l acetic acid solution ( 3 : 1 ) t h r e e times. A few d r o p s of the cell s u s p e n s i o n w e r e d r o p p e d o n t o a clean slide t a k e n from icy w a t e r a n d f r a m e - d r i e d . T h e slides w e r e i n c u b a t e d in 2 × SSC solution in p e t r i dishes at 70 ° C a n d i r r a d i a t e d s i m u l t a n e ously u n d e r a U V l a m p (30 W ) at a d i s t a n c e of 10 cm for 20 min. T h e p r e p a r a t i o n s w e r e s t a i n e d in 3 % G i e m s a solution for 10 min a f t e r b e i n g r i n s e d with t a p w a t e r (Xing, 1990; Z h a n g , 1991). S C E analysis was c a r r i e d out on cells having r e p l i c a t e d for two cycles in the p r e s e n c e o f B r d U . T e n m e t a p h a s e s w e r e s c o r e d for each t r e a t m e n t .
Hordeum culgare SCE test S e e d s w e r e g e r m i n a t e d on m o i s t e n e d filter p a p e r in p e t r i dishes at 25 ° C. W h e n they were
a b o u t 1 cm long, the roots of the g e r m i n a t e d seeds w e r e t r a n s f e r r e d into an a q u e o u s solution c o n t a i n i n g B r d U (5 × 10 4 M), 5 - f l u o r o d e oxyuridine ( F d U ) (10 - s M) and u r i d i n e ( U r d ) (10 (' M), a n d i n c u b a t e d at 2 5 ° C in the d a r k for 12 h. T h e seedlings w e r e w a s h e d and t r a n s f e r r e d e i t h e r into an a q u e o u s solution c o n t a i n i n g thymid i n e (Thd) (10 4 M) a n d U r d (10 ~' M) (for control) or into an a q u e o u s solution c o n t a i n i n g T h d (10 4 M), U r d (10 ~' M) and A A and incub a t e d for a n o t h e r 12 h at 2 5 ° C in the dark. T h e n the s e e d l i n g s were w a s h e d , chilled (0 ° C , 36 h) for p r e t r e a t m e n t and fixed in m e t h a n o l - a c e t i c acid solution ( 3 : 1 ) for 24 h. A f t e r being r i n s e d with t a p water, the m c r i s t e m a t i c regions w e r e excised a n d d i g e s t e d in a mixture of cellulase a n d p e c t i n a s e (2.5% each) for 5 h at 25 ° C b e f o r e the cells u n d e r w e n t h y p o t o n i c t r e a t m e n t in distilled w a t e r for 1 h. T h e w a t e r was then d i s c a r d e d and the r o o t tips w e r e p o u n d e d into a paste. Cell s u s p e n s i o n s w e r e m a d e by a d d i n g a p r o p e r a m o u n t of fixative to the paste. A few d r o p s of the s u s p e n s i o n were s u b s e q u e n t l y d r o p p e d o n t o a clean slide t a k e n from icy w a t e r a n d f l a m e - d r i e d . T h e slides w e r e i n c u b a t e d in 2 × SSC solution in petri dishes at 80 ° C a n d i r r a d i a t e d s i m u l t a n e ously u n d e r a U V l a m p (30 W ) at a d i s t a n c e of 10 cm for 45 min. T h e p r e p a r a t i o n s w e r e s t a i n e d in 3% G i e m s a solution for 10 min a f t e r b e i n g rinsed with t a p w a t e r (Xing, 199(/; Z h a n g , 1991). Results
The effects of AAs on SCE in PBL T e n A A s w e r e involved in the test and d i v i d e d into two g r o u p s with e a c h g r o u p consisting of five A A s . A r a n d o m i z e d c o m p l e t e - b l o c k design was used to avoid the i n f l u e n c e of individual differ-
TABLE 1 EFFECTS OF Ala, Gly, Thr, lie AND Lys ON SCE IN PBL Concentrations (/xg/ml)
Amino acid Ala
Gly
Thr
lie
Lys
0 10 50 100
9.(1_+3.0 9.3 _+2.3 11.4+2.9 * 12.2_+3.7 **
9.2 + 2.7 1(I.9+ 3.1 10.7+3.7 11.9_+3.7 **
9.3 + 2.3 10.3 + 3.5 10.4+3.5 1(I.2+_2.9
9.5 ± 2.3 10.5 + 2.9 9.9-+3.4 10.1+3.5
10.0 + 2.5 9.8 ± 2.8 10.1 +3.9 9.5+3.(I
* P < 0.05: ** P
