51
Mutation Research, 56 (1977) 51--58 © Elsevier/North-Holland Biomedical Press
H U M A N SISTER CHROMATID EXCHANGE CAUSED BY M E T H Y L A Z O X Y M E T H A N O L ACETATE *
LEMUEL A. EVANS 1,2, MONICA J. KEVIN 3 and EDMUND C. JENKINS 1
1 N e w York State Institute for Basic Research in Mental Retardation, Staten Isand, New York 10314; 2 Present address: Department o f Natural Sciences, Medgar Evers College, City University o f New York, Brooklyn, N.Y. 11225; and 3 Department o f Biological Sciences, Fordham University, Bronx, N.Y. 10458 (U.S.A.) (Received 12 May, 1977) (Accepted 10 June, 1977)
Summary The incidence of sister chromatid exchange was determined in human leucocyte cultures treated with methylazoxymethanol acetate. In all individuals examined, treated cultures manifested a significantly higher frequency of sister chromatid exchanges than controls. Two concentrations of MAM AC were tested in blood cultures from nine individuals. The concentrations varied from individual to individual since they were determined by means of individual dose-response curves, which involved [3H]thymidine incorporation in PHA-stimulated short-term l y m p h o c y t e cultures versus MAM AC concentration. The lower concentration was less than the TDs0 dose. Compared to control cultures, the lower concentration caused a higher incidence of sister chromatid exchange in eight of nine individuals. The cumulative mean value for all control cultures was 5.32 exchanges per cell while that for cultures treated with the higher concentration was 10.73.
Introduction
Methylazoxymethanol, the aglycone of cycasin, and its more stable form, methylazoxymethanol acetate (MAM AC), have been established as mutagenic [10,12,19,25] and carcinogenic [11] agents. MAM AC has been shown to methylate nucleic acids at the N-7 position of guanine in vitro [16] and in vivo [18]. MAM AC has also been reported to alter cellular kinetics in HeLa cells [1]. We have previously reported the effects of MAM AC on PHA response in human short-term leucocyte cultures [5]. * This w o r k was supported in part by the National Fellowships F u n d and The N e w York State Department of Mental Hygiene.
52 Chromosomal aberrations were observed in root tips of Alliurn cepa exposed to the glucoside, cycasin [24]. Various mitotic abnormalities were reported in rat liver cells exposed to MAM AC [27,28]. The present study is concerned with the effects of MAM AC on the frequency of sister chromatid exchanges in human short-term leucocyte cultures. Materials and methods Peripheral blood from nine normal male individuals ranging in age from 25 to 38 was cultured in 4 ml aliquots of McCoy's 5A medium (modified, GIBCO 166-0) which was supplemented with 0.2% of PHA-P (Difco), 15% fetal calf serum (GIBCO 614), 100 units/ml of penicillin and 100 pg/ml of streptomycin (GIBCO 515), 2 mM of L-glutamine (GIBCO 503), and 16 units/ml of heparin (Scientific Products). All additions to the cultures were made in 0.04 ml volumes. The treated cultures were exposed continuously to MAM AC for 96 h at 36--36.5°C. Each variable was studied in six identical cultures. Two concentrations of MAM AC (Ash-Stevens) were tested. They were determined on the basis of dose-effect curves involving quantity of chemical versus blast transformation with PHA stimulation. PHA stimulation was measured in terms of [3H]thymidine uptake. The concentrations for each individual were determined by means of his dose-effect curve. The results [5] of the curves allowed the determination of the TDs0 dose for each individual, which referred to the concentration of MAM AC at which half as much label was incorporated as compared to control cultures which had no MAM AC. Nine individuals were examined for sister chromatid exchange frequency in a total of 162 cultures. The specific dosages for all individuals are given in Table 1. Control cultures contained no MAM AC and were referred to as the " A " series. The concentration of MAM AC in seven of the nine cases in the " B " series of cultures caused no significant difference in blast cell transformation when compared to PHA-containing control cultures which had no MAM AC [5]. The choice of dose in two of the nine cases was arbitrary. It caused a significantly lesser a m o u n t of tritium incorporation as compared to control cultures. In individual I the TDs0 dose was less than 5 pg/ml so that a concentration of I pg/ml was chosen. In individual III, a similar situation existed where the TDs0 dose was less than 20 pg/ml so that the lowest concentration in the dose-effect cuw,:e was employed. As seen in Table 1 the concentrations in the " C " series of cultures for individuals I and III were 5 and 12.5 pg/ml, respectively. These concentrations corresponded to the TDs0 dose. In the remaining seven cases for the " C " series of cultures the concentration was the same as the previously reported biologically active dose of 25 pg/ml [6]. 5-Bromodeoxyuridine (BrdU) (Nutritional Biochemicals) was added to all cultures for the last 24 h of incubation at a final concentration of 20 pM. Differential chromatid staining was carried out according to the method of Korenberg and Freedlender [10] as modified by Evans and Jenkins [4]. All slides were coded and read blindly. Wherever possible, at least 20 intact metaphase spreads per culture were scored for sister chromatid exchanges. Sister chromatid exchanges (SCE) were defined as those reciprocally stained
53
areas along the linear axis of the chromatids. The number of exchanges in a cell was the total number observed in all chromosomes in the complement. Results In all individuals examined, cultures exposed to MAM AC manifested a significantly higher frequency of sister chromatid exchange. Table 1 is a tabulation o f the mean n u m b e r o f exchanges per cell for culture series "A-C". TABLE 1 M E T H Y L A Z O X Y M E T H A N O L A C E T A T E (MAM A C ) C O N C E N T R A T I O N A N D S I S T E R C H R O M A T I D E X C H A N G E I N C I D E N C E IN S H O R T - T E R M H U M A N L E U C O C Y T E C U L T U R E S D E R I V E D FROM NINE INDIVIDUALS T h e M A M AC c o n c e n t r a t i o n s for i n d i v i d u a l s I a n d I I I in t h e " B " c u l t u r e series w e r e a r b i t r a r i l y c h o s e n . T h e y c a u s e d significantly less i n c o r p o r a t i o n o f t r i t i a t e d t h y m i d i n e t h a n t h e P H A - c o n t ~ i n i n g c o n t r o l c u l t u r e s ( " A " series). This o c c u r r e d b e c a u s e t h e T D s 0 d o s e ( " C " series) w a s 5 a n d 1 2 . 5 /~g/ml for individuals I and III, respectively. Individual
C u l t u r e series
I a
A b B c C c
II
Concentration o f M A M AC (}Jg/ml)
N u m b e r of metaphases analyzed
Mean number of SCE p e r metaphase
Standard error
Total range
0 1 5
166 132 113
2.93 3.71 6.08
-+0.44 -+0.44 -+0.83
1--10 1--10 1--15
A B C
0 10 25
104 37 137
5.78 10.01 8.96
-+1.20 -+1.31 -+0.69
1--10 1--19 2--25
In a
A B C
0 5 12.5
102 130 125
5.98 10.03 9.86
-+0.60 _+0.42 -+0.74
1--16 2--23 1--16
IV
A B C
0 10 25
121 128 5
5.09 10.73 10.20
-+0.51 -+0.51 0.00
2--14 4--22 2--34
V
A B C
0 20 25
139 112 .
-+1.26 -+1.26
1--12 3--29
.
5.07 11.04 .
VI
A B C
0 10 25
126 105 .
-+0.80 -+0.80
1--12 1--29
.
5.83 12.34 .
VII
A B C
0 10 25
156 87 27
6.06 12.95 14.43
-+0.71 -+0.78 -+0.54
1--14 6--21 7--28
Vlll
A B C
0 10 25
129 79 .
5.11 10.93 .
-+0.34 -+0,38
1--13 4--19
A B C
0 5 25
110 152 13
-+0.68 -+0.62 -+0.143
2--14 3--20 8--18
-+0.33 -+0.88 +-1.37
1--16 1--29 1--34
IX
Total
.
A 5.32 B 10.14 C 10.73 b B r d U a n d MAM AC.
.
.
2535
Cumulative m e a n
a BrdU only.
6.09 9.56 14.84
.
c B r d U a n d MAM AC.
,54
4
J¢ Fig. 1. A m e t a p h a s e derived f r o m c o n t r o l c u l t u r e s e x h i b i t i n g 5 sister c h r o m a t i d e x c h a n g e s . M a g n i f i c a t i o n X1800.
~
~i~i~ ¸
j
i~i
/
Fig. 2. E l e v e n sister c h r o m a t i d e x c h a n g e s were o b s e r v e d i n this cell w h i c h was e x p o s e d t o MAM AC. M a g n i f i c a t i o n X1 8 0 0 .
55 Fig. 1. is a metaphase spread which was derived from cultures exposed to BrdU only. Five exchanges were observed in the cell. Fig. 2, also derived from individual I, exhibited 11 sister chromatid exchanges. The concentration of MAM AC was 5#g/ml. In eight of nine individuals the lower concentration (Table 1) caused a higher incidence of sister chromatid exchange as compared to control cultures. In individual I, there was no significant difference between the lower concentration and the control while there was a significant difference at the higher concentration. In three individuals an insufficient number of cells did n o t allow sister chromatid exchange scoring at the higher drug dose. Forty-two of 162 cultures were not scored for incidence of SCE due to unavailability of mitotic figures. These 42 cultures were derived from eight of the nine individuals studied. Thirty-two cultures derived from seven individuals were exposed to the higher concentration ( " C " series}. Five " B " and five " A " series cultures were from five and three individuals, respectively. Of the 120 cultures scored the range of SCE frequency was from one to 34 (Table 1). The range and mean number of exchanges for series "A-C" are given in Table 1.
Discussion We have n o t only further tested the relatively new system of SCE but we have also enlarged the available information on MAM AC, a p o t e n t neuroteratogenic alkylating agent, which may serve as a model for certain types of mental deficiency. Since MAM AC is a k n o w n mutagen and since it increases the frequency of sister chromatid exchange in human short-term leucocyte cultures, we agree with suggestions made in previous reports [2,14,17,21] that sister chromatid exchange can be used as an index of mutagenicity. The effect of MAM AC on blastcell transformation was given in a previous report to determine the concentrations which were used to test for the effect of the drug upon sister chromatid exchange. The actual doses used in the present study were chosen in an a t t e m p t to distinguish between the effects of toxicity and the effects on sister chromatid exchange frequency. The lower concentration was always less than the TD 50 dose which was determined in d o s e ~ f f e c t curves described earlier by us [5]. The higher concentration not only caused a significant increase in the frequency of sister chromatid exchange as compared to control cultures but it was also toxic since a rather significant n u m b e r of cultures did not allow sufficient metaphase spreads to evaluate for sister chromatid exchange. In this case, as the dose became increasingly more toxic in terms of blast cell transformation, the effect on sister chromatid exchange remained unchanged. The cumulative mean values of both " B " and " C " series concentrations were significantly different from " A " as shown by the t test. Where the concentration was such that blast cell transformation was not affected ( " B " series) all but five cultures were readable.It is interesting to note that the same number of cultures were unreadable in the control ( " A " series), thus indicating the efficiency of the system, which was greater than 90%. As can be seen from Table 1 the SCE baseline values ( " A " series) in the experiments were between 2.93 and 6.09 with a cumulative mean of 5.32
56 (range 1--24). This is lower than all previously reported values except the 4.5 value reported by Beek and Obe [2]. One possible reason for variation might be the difference in exposure time to BrdU, which in our system was the last 24 h of culture as compared to most systems in which exposure was continuous. Of all agents investigated MAM AC was among the weakest in its effects on SCE rates. It is interesting to note that the other alkylating agents are bifunctional or trifunctional while MAM AC is believed to be monofunctional which may account for this difference. SCE is believed to involve interchange of DNA segments [22,23] ::~:tween chromatids followed by ligation [14] and post-replicative repair [3]. Wolff, Bodycote and Cleaver [26] have reported that current concepts regarding known DNA repair processes do not adequately explain SCE formation. SCE induction is nevertheless a simple and sensitive assay for chromosomal damage [17]. The methylation of nucleic acids at the N-7 position of guanine by MAM AC [15] results in a shift of electrons leading to cleavage of the glycosidic bond at nitrogen 9. Subsequently the phosphate is lost from the deoxyribose and a cyclic phosphate is formed. Alkylating agents such as MAM AC may cause cross-linking of the two DNA strands of a double helix with the result that either the molecule breaks apart or in the next DNA replication deleted molecules are produced [7]. The effects described in the present work may be the result of DNA alkylation thus accounting for an increase in SCE. This was not unexpected since previous reports have shown that alkylating agents effect higher frequencies of SCE [14,17,18,20,21]. Acknowledgments Thanks is due Dr. Nicholas Beratis for his constructive critique of the manuscript. Acknowledgment is also given to Dr. R.K. Haddad for helpful discussion. References 1 Bedford, A.J., E.H. C o o p e r a n d T.E. K e n n y , A kinetic analysis of d e a t h a n d survival of H e L a cells f o l l o w i n g e x p o s u r e t o m e t h y l a z o x y m e t h a n o l a c e t a t e , Eur. J. Cancer, 1 0 ( 1 9 7 4 ) 7 1 3 - - 7 2 0 . 2 Beck, B. a n d G. Obe, The h u m a n l e u c o c y t e test s y s t e m VI. The use o f sister c h r o m a t i d e x c h a n g e s as possible i n d i c a t o r s f o r m u t a g e n i c activities, H u m a n g e n e t i k , 29 ( 1 9 7 5 ) 1 2 7 - - 1 3 4 . 3 Bender, M.A., I n d u c e d lesions, D N A repair, a n d c h r o m o s o m e a b e r r a t i o n f o r m a t i o n , 7 t h A n n u a l Meeting of t h e E n v i r o n m e n t a l M u t a g e n S o c i e t y , A t l a n t a , Georgia, 1 9 7 6 . 4 Evans, L.A. a n d E.C. J e n k i n s , Differential sister c h r o m a t i d staining, L a n c e t , 11 ( 1 9 7 5 ) 1 7 8 - - 1 7 9 . 5 Evans, L.A. a n d E.C. J e n k i n s , P H A response a n d m e t h y l a z o x y m e t h a n o l acetate, Chem.-Biol. I n t e r a c t . , 14 ( 1 9 7 6 ) 1 3 5 - - 1 4 0 . 6 H a d d a d , R.K., A. R a b e , a n d R. Dumas, C o m p a r i s o n of effects of m e t h y l a z o x y m e t h a n o l a c e t a t e o n b r a i n d e v e l o p m e n t in d i f f e r e n t species, Fed. Proc., 31 ( 1 9 7 2 ) 1 5 2 0 - - 1 5 2 3 . 7 Harbers, E., G.F. D o m a q k , a n d W. Muller, I n t r o d u c t i o n t o Nucleic Acids, R e i n h o l d B o o k Corp., New Y o r k , 1 9 6 8 . 8 K i m , A., C h r o m a t i d a u s t a u s c h u n d H e t e r o c h r o m a t i n e r ~ / n d e r u n g e n m e n s c h l i c h e r c h r o m o s o m e n n a c h B u d R - M a r k i e r u n g , H u m a n g e n e t i k , 25 ( 1 9 7 4 ) 1 7 9 - - 1 8 8 . 9 K o b a y a s h i , A. a n d H. M a t s u m o t o , M e t h y l a z o x y m e t h a n o l t h e a g l y c o n e of c y c a s i n , Fed. Proc., 23 (1964) 1354. 1 0 K o r e n b e r g , J . R . a n d E. FreedlarAder, Giemsa t e c h n i q u e s f o r t h e d e t e c t i o n o f sister c h r o m a t i d e x c h a n g e s , C h r o m o s o m a , 48 ( 1 9 7 4 ) 355---63G. 11 L a q u e u r , G.L., O. Mickelson, M.G. W~iting a n d L.T. K u r l a n d , Carcinogenic p r o p e r t i e s of n u t s f r o m Cycas circinalis L. i n d i g e n o u s t o G u a m , J. Natl. C a n c e r Ins., 31 ( 1 9 6 3 ) 9 1 9 .
57 12 Laqueur, G.L., Carcinogenic effects of cycad meal and cycasin, m e t h y l a z o x y m e t h a n o l glycoside, in rats and effects of cycasin in germ-free rats, Fed. Proc., 23 (1964) 1386. 13 Latt, S.A., Sister c h r o m a t i d exchanges, indices of h u m a n c h r o m o s o m e damage and repair: de t e c t i on by fluorescence and i n d u c t i o n by m i t o m y e i n C, Proc. Natl. Acad. Sci., U.S.A., 71 (1974) 3162--3166. 14 Latt, S.A., G. Stetten, L.A. Juergens, G.R. Buchanan and P.S. Gerald, I n d u c t i o n by alkylating agents of sister c h r o m a t i d exchanges and c h r o m a t i d breaks in Fanconi's Anemia, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 4066---4070. 15 Matsumo to, H. and H.H. Higa, Studies on m e t h y l a z o x y m e t h a n o l , the aglycone of cycasin: methylation of nucleic acids in vitro, Biochem. J. 98 (1966) 20c--22c. 16 Natarajan, A.T., A.D. Tayes, P.P.W. VanBuul, M. Meijers and N. DeVogel, Cytogenetic effects of mutagens/ca~cinogens after activation in a microsomal system in vitro. I. I n d u c t i o n of c h r o m o s o m e aberrations and sister c h r o m a t i d exchanges by diethylrdtrosamine (DEN) and d i m e t h y i n i t r o s a m i n e (DMN) in CHO cells in the presence of rat-liver microsomes, Mutation Res., 37 (1976) 83--90. 17 Perry, P. and H.J. Evans, Cytological d e t e c t i o n of mutagen-carcinogen exposure by sister c h r o m a t i d exchange, Nature, 258 (1975) 121--125. 18 Shank, II.C. and P.N. Magee, Similarities b e t w e e n the action of cycasin and d i m e t h y l n i t r o s a m i n e , Biochem. J., 105 (1967) 521--527. 19 Smith, D.W.E., Mutagenicity of cycasin aglycone ( m e t h y l a z o x y m e t h a n o l ) a naturally occurring carcinogen, Science 152 (1966) 1273. 20 Solomon, E. and M. Bobrow, Sister c h r o m a t i d exchanges -- a sensitive assay of agents damaging h u m a n chro mo so mes, Mutation Res., 30 (1975) 273--278. 21 Stetka, D.G. and S. Wolff, Sister c h r o m a t i d 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. 22 Taylor, J.H., P.S. Woods and W.L. Hughers, The organization and dupl i c a t i on of c hromos ome s as revealed by autoradiographic studies using tritium-labeled t h y m i d i n e . Proc. Natl. Acad. Sci. 43 (1957) 122--128. 23 Taylor, J.H., Sister c h r o m a t i d exchanges in tritium-labeled chromosomes, Genetics, 43 (1958) 515-529. 24 Teas, H.J., H.J. Sax and K. Sax, Cycasin: r a d i o m i m e t i c effect, Science, 149 (1965) 541. 25 Teas, H.J. and J.G. Dyson, Mutation in Drosophila by m e t h y l a z o x y m e t h a n o l , the aglycone of cycasin, Proc. Soc. Exp. Biol. Med., 125 (1967) 988. 26 Wolff, S., J. Bodycote, G.H. Thomas and J.E. Cleaver, Sister c h r o m a t i d exchange in x e r o d e r m a p i g m e n t o s u m cells that are defective in DNA excision repair or post replication repair, Genetics, 81 (1975) 359--355. 27 Zedeck, M.S., S.S. Sternberg, X. Yataganos and J. McGowan, Early changes i nduc e d in rat liver by m e t h y l a z o x y m e t h a n o l acetate: Mitotic abnormalities and p o l y p l o i d y , J. Nat. Cancer Inst., 53 (1974) 719--723. 28 Zedeck, M.S., S.S. Sternberg, Megalocytosis and other abnormalities expressed during proliferation in regenerating liver of rats treated with m e t h y l a z o x y m e t h a n o l acetate prior to partial h e p a t e c t o m y , Cancer Res., 35 (1975) 2117--2122.
Mutation Research, 56 (1977) 51--58 © Elsevier/North-Holland Biomedical Press
H U M A N SISTER CHROMATID EXCHANGE CAUSED BY M E T H Y L A Z O X Y M E T H A N O L ACETATE *
LEMUEL A. EVANS 1,2, MONICA J. KEVIN 3 and EDMUND C. JENKINS 1
1 N e w York State Institute for Basic Research in Mental Retardation, Staten Isand, New York 10314; 2 Present address: Department o f Natural Sciences, Medgar Evers College, City University o f New York, Brooklyn, N.Y. 11225; and 3 Department o f Biological Sciences, Fordham University, Bronx, N.Y. 10458 (U.S.A.) (Received 12 May, 1977) (Accepted 10 June, 1977)
Summary The incidence of sister chromatid exchange was determined in human leucocyte cultures treated with methylazoxymethanol acetate. In all individuals examined, treated cultures manifested a significantly higher frequency of sister chromatid exchanges than controls. Two concentrations of MAM AC were tested in blood cultures from nine individuals. The concentrations varied from individual to individual since they were determined by means of individual dose-response curves, which involved [3H]thymidine incorporation in PHA-stimulated short-term l y m p h o c y t e cultures versus MAM AC concentration. The lower concentration was less than the TDs0 dose. Compared to control cultures, the lower concentration caused a higher incidence of sister chromatid exchange in eight of nine individuals. The cumulative mean value for all control cultures was 5.32 exchanges per cell while that for cultures treated with the higher concentration was 10.73.
Introduction
Methylazoxymethanol, the aglycone of cycasin, and its more stable form, methylazoxymethanol acetate (MAM AC), have been established as mutagenic [10,12,19,25] and carcinogenic [11] agents. MAM AC has been shown to methylate nucleic acids at the N-7 position of guanine in vitro [16] and in vivo [18]. MAM AC has also been reported to alter cellular kinetics in HeLa cells [1]. We have previously reported the effects of MAM AC on PHA response in human short-term leucocyte cultures [5]. * This w o r k was supported in part by the National Fellowships F u n d and The N e w York State Department of Mental Hygiene.
52 Chromosomal aberrations were observed in root tips of Alliurn cepa exposed to the glucoside, cycasin [24]. Various mitotic abnormalities were reported in rat liver cells exposed to MAM AC [27,28]. The present study is concerned with the effects of MAM AC on the frequency of sister chromatid exchanges in human short-term leucocyte cultures. Materials and methods Peripheral blood from nine normal male individuals ranging in age from 25 to 38 was cultured in 4 ml aliquots of McCoy's 5A medium (modified, GIBCO 166-0) which was supplemented with 0.2% of PHA-P (Difco), 15% fetal calf serum (GIBCO 614), 100 units/ml of penicillin and 100 pg/ml of streptomycin (GIBCO 515), 2 mM of L-glutamine (GIBCO 503), and 16 units/ml of heparin (Scientific Products). All additions to the cultures were made in 0.04 ml volumes. The treated cultures were exposed continuously to MAM AC for 96 h at 36--36.5°C. Each variable was studied in six identical cultures. Two concentrations of MAM AC (Ash-Stevens) were tested. They were determined on the basis of dose-effect curves involving quantity of chemical versus blast transformation with PHA stimulation. PHA stimulation was measured in terms of [3H]thymidine uptake. The concentrations for each individual were determined by means of his dose-effect curve. The results [5] of the curves allowed the determination of the TDs0 dose for each individual, which referred to the concentration of MAM AC at which half as much label was incorporated as compared to control cultures which had no MAM AC. Nine individuals were examined for sister chromatid exchange frequency in a total of 162 cultures. The specific dosages for all individuals are given in Table 1. Control cultures contained no MAM AC and were referred to as the " A " series. The concentration of MAM AC in seven of the nine cases in the " B " series of cultures caused no significant difference in blast cell transformation when compared to PHA-containing control cultures which had no MAM AC [5]. The choice of dose in two of the nine cases was arbitrary. It caused a significantly lesser a m o u n t of tritium incorporation as compared to control cultures. In individual I the TDs0 dose was less than 5 pg/ml so that a concentration of I pg/ml was chosen. In individual III, a similar situation existed where the TDs0 dose was less than 20 pg/ml so that the lowest concentration in the dose-effect cuw,:e was employed. As seen in Table 1 the concentrations in the " C " series of cultures for individuals I and III were 5 and 12.5 pg/ml, respectively. These concentrations corresponded to the TDs0 dose. In the remaining seven cases for the " C " series of cultures the concentration was the same as the previously reported biologically active dose of 25 pg/ml [6]. 5-Bromodeoxyuridine (BrdU) (Nutritional Biochemicals) was added to all cultures for the last 24 h of incubation at a final concentration of 20 pM. Differential chromatid staining was carried out according to the method of Korenberg and Freedlender [10] as modified by Evans and Jenkins [4]. All slides were coded and read blindly. Wherever possible, at least 20 intact metaphase spreads per culture were scored for sister chromatid exchanges. Sister chromatid exchanges (SCE) were defined as those reciprocally stained
53
areas along the linear axis of the chromatids. The number of exchanges in a cell was the total number observed in all chromosomes in the complement. Results In all individuals examined, cultures exposed to MAM AC manifested a significantly higher frequency of sister chromatid exchange. Table 1 is a tabulation o f the mean n u m b e r o f exchanges per cell for culture series "A-C". TABLE 1 M E T H Y L A Z O X Y M E T H A N O L A C E T A T E (MAM A C ) C O N C E N T R A T I O N A N D S I S T E R C H R O M A T I D E X C H A N G E I N C I D E N C E IN S H O R T - T E R M H U M A N L E U C O C Y T E C U L T U R E S D E R I V E D FROM NINE INDIVIDUALS T h e M A M AC c o n c e n t r a t i o n s for i n d i v i d u a l s I a n d I I I in t h e " B " c u l t u r e series w e r e a r b i t r a r i l y c h o s e n . T h e y c a u s e d significantly less i n c o r p o r a t i o n o f t r i t i a t e d t h y m i d i n e t h a n t h e P H A - c o n t ~ i n i n g c o n t r o l c u l t u r e s ( " A " series). This o c c u r r e d b e c a u s e t h e T D s 0 d o s e ( " C " series) w a s 5 a n d 1 2 . 5 /~g/ml for individuals I and III, respectively. Individual
C u l t u r e series
I a
A b B c C c
II
Concentration o f M A M AC (}Jg/ml)
N u m b e r of metaphases analyzed
Mean number of SCE p e r metaphase
Standard error
Total range
0 1 5
166 132 113
2.93 3.71 6.08
-+0.44 -+0.44 -+0.83
1--10 1--10 1--15
A B C
0 10 25
104 37 137
5.78 10.01 8.96
-+1.20 -+1.31 -+0.69
1--10 1--19 2--25
In a
A B C
0 5 12.5
102 130 125
5.98 10.03 9.86
-+0.60 _+0.42 -+0.74
1--16 2--23 1--16
IV
A B C
0 10 25
121 128 5
5.09 10.73 10.20
-+0.51 -+0.51 0.00
2--14 4--22 2--34
V
A B C
0 20 25
139 112 .
-+1.26 -+1.26
1--12 3--29
.
5.07 11.04 .
VI
A B C
0 10 25
126 105 .
-+0.80 -+0.80
1--12 1--29
.
5.83 12.34 .
VII
A B C
0 10 25
156 87 27
6.06 12.95 14.43
-+0.71 -+0.78 -+0.54
1--14 6--21 7--28
Vlll
A B C
0 10 25
129 79 .
5.11 10.93 .
-+0.34 -+0,38
1--13 4--19
A B C
0 5 25
110 152 13
-+0.68 -+0.62 -+0.143
2--14 3--20 8--18
-+0.33 -+0.88 +-1.37
1--16 1--29 1--34
IX
Total
.
A 5.32 B 10.14 C 10.73 b B r d U a n d MAM AC.
.
.
2535
Cumulative m e a n
a BrdU only.
6.09 9.56 14.84
.
c B r d U a n d MAM AC.
,54
4
J¢ Fig. 1. A m e t a p h a s e derived f r o m c o n t r o l c u l t u r e s e x h i b i t i n g 5 sister c h r o m a t i d e x c h a n g e s . M a g n i f i c a t i o n X1800.
~
~i~i~ ¸
j
i~i
/
Fig. 2. E l e v e n sister c h r o m a t i d e x c h a n g e s were o b s e r v e d i n this cell w h i c h was e x p o s e d t o MAM AC. M a g n i f i c a t i o n X1 8 0 0 .
55 Fig. 1. is a metaphase spread which was derived from cultures exposed to BrdU only. Five exchanges were observed in the cell. Fig. 2, also derived from individual I, exhibited 11 sister chromatid exchanges. The concentration of MAM AC was 5#g/ml. In eight of nine individuals the lower concentration (Table 1) caused a higher incidence of sister chromatid exchange as compared to control cultures. In individual I, there was no significant difference between the lower concentration and the control while there was a significant difference at the higher concentration. In three individuals an insufficient number of cells did n o t allow sister chromatid exchange scoring at the higher drug dose. Forty-two of 162 cultures were not scored for incidence of SCE due to unavailability of mitotic figures. These 42 cultures were derived from eight of the nine individuals studied. Thirty-two cultures derived from seven individuals were exposed to the higher concentration ( " C " series}. Five " B " and five " A " series cultures were from five and three individuals, respectively. Of the 120 cultures scored the range of SCE frequency was from one to 34 (Table 1). The range and mean number of exchanges for series "A-C" are given in Table 1.
Discussion We have n o t only further tested the relatively new system of SCE but we have also enlarged the available information on MAM AC, a p o t e n t neuroteratogenic alkylating agent, which may serve as a model for certain types of mental deficiency. Since MAM AC is a k n o w n mutagen and since it increases the frequency of sister chromatid exchange in human short-term leucocyte cultures, we agree with suggestions made in previous reports [2,14,17,21] that sister chromatid exchange can be used as an index of mutagenicity. The effect of MAM AC on blastcell transformation was given in a previous report to determine the concentrations which were used to test for the effect of the drug upon sister chromatid exchange. The actual doses used in the present study were chosen in an a t t e m p t to distinguish between the effects of toxicity and the effects on sister chromatid exchange frequency. The lower concentration was always less than the TD 50 dose which was determined in d o s e ~ f f e c t curves described earlier by us [5]. The higher concentration not only caused a significant increase in the frequency of sister chromatid exchange as compared to control cultures but it was also toxic since a rather significant n u m b e r of cultures did not allow sufficient metaphase spreads to evaluate for sister chromatid exchange. In this case, as the dose became increasingly more toxic in terms of blast cell transformation, the effect on sister chromatid exchange remained unchanged. The cumulative mean values of both " B " and " C " series concentrations were significantly different from " A " as shown by the t test. Where the concentration was such that blast cell transformation was not affected ( " B " series) all but five cultures were readable.It is interesting to note that the same number of cultures were unreadable in the control ( " A " series), thus indicating the efficiency of the system, which was greater than 90%. As can be seen from Table 1 the SCE baseline values ( " A " series) in the experiments were between 2.93 and 6.09 with a cumulative mean of 5.32
56 (range 1--24). This is lower than all previously reported values except the 4.5 value reported by Beek and Obe [2]. One possible reason for variation might be the difference in exposure time to BrdU, which in our system was the last 24 h of culture as compared to most systems in which exposure was continuous. Of all agents investigated MAM AC was among the weakest in its effects on SCE rates. It is interesting to note that the other alkylating agents are bifunctional or trifunctional while MAM AC is believed to be monofunctional which may account for this difference. SCE is believed to involve interchange of DNA segments [22,23] ::~:tween chromatids followed by ligation [14] and post-replicative repair [3]. Wolff, Bodycote and Cleaver [26] have reported that current concepts regarding known DNA repair processes do not adequately explain SCE formation. SCE induction is nevertheless a simple and sensitive assay for chromosomal damage [17]. The methylation of nucleic acids at the N-7 position of guanine by MAM AC [15] results in a shift of electrons leading to cleavage of the glycosidic bond at nitrogen 9. Subsequently the phosphate is lost from the deoxyribose and a cyclic phosphate is formed. Alkylating agents such as MAM AC may cause cross-linking of the two DNA strands of a double helix with the result that either the molecule breaks apart or in the next DNA replication deleted molecules are produced [7]. The effects described in the present work may be the result of DNA alkylation thus accounting for an increase in SCE. This was not unexpected since previous reports have shown that alkylating agents effect higher frequencies of SCE [14,17,18,20,21]. Acknowledgments Thanks is due Dr. Nicholas Beratis for his constructive critique of the manuscript. Acknowledgment is also given to Dr. R.K. Haddad for helpful discussion. References 1 Bedford, A.J., E.H. C o o p e r a n d T.E. K e n n y , A kinetic analysis of d e a t h a n d survival of H e L a cells f o l l o w i n g e x p o s u r e t o m e t h y l a z o x y m e t h a n o l a c e t a t e , Eur. J. Cancer, 1 0 ( 1 9 7 4 ) 7 1 3 - - 7 2 0 . 2 Beck, B. a n d G. Obe, The h u m a n l e u c o c y t e test s y s t e m VI. The use o f sister c h r o m a t i d e x c h a n g e s as possible i n d i c a t o r s f o r m u t a g e n i c activities, H u m a n g e n e t i k , 29 ( 1 9 7 5 ) 1 2 7 - - 1 3 4 . 3 Bender, M.A., I n d u c e d lesions, D N A repair, a n d c h r o m o s o m e a b e r r a t i o n f o r m a t i o n , 7 t h A n n u a l Meeting of t h e E n v i r o n m e n t a l M u t a g e n S o c i e t y , A t l a n t a , Georgia, 1 9 7 6 . 4 Evans, L.A. a n d E.C. J e n k i n s , Differential sister c h r o m a t i d staining, L a n c e t , 11 ( 1 9 7 5 ) 1 7 8 - - 1 7 9 . 5 Evans, L.A. a n d E.C. J e n k i n s , P H A response a n d m e t h y l a z o x y m e t h a n o l acetate, Chem.-Biol. I n t e r a c t . , 14 ( 1 9 7 6 ) 1 3 5 - - 1 4 0 . 6 H a d d a d , R.K., A. R a b e , a n d R. Dumas, C o m p a r i s o n of effects of m e t h y l a z o x y m e t h a n o l a c e t a t e o n b r a i n d e v e l o p m e n t in d i f f e r e n t species, Fed. Proc., 31 ( 1 9 7 2 ) 1 5 2 0 - - 1 5 2 3 . 7 Harbers, E., G.F. D o m a q k , a n d W. Muller, I n t r o d u c t i o n t o Nucleic Acids, R e i n h o l d B o o k Corp., New Y o r k , 1 9 6 8 . 8 K i m , A., C h r o m a t i d a u s t a u s c h u n d H e t e r o c h r o m a t i n e r ~ / n d e r u n g e n m e n s c h l i c h e r c h r o m o s o m e n n a c h B u d R - M a r k i e r u n g , H u m a n g e n e t i k , 25 ( 1 9 7 4 ) 1 7 9 - - 1 8 8 . 9 K o b a y a s h i , A. a n d H. M a t s u m o t o , M e t h y l a z o x y m e t h a n o l t h e a g l y c o n e of c y c a s i n , Fed. Proc., 23 (1964) 1354. 1 0 K o r e n b e r g , J . R . a n d E. FreedlarAder, Giemsa t e c h n i q u e s f o r t h e d e t e c t i o n o f sister c h r o m a t i d e x c h a n g e s , C h r o m o s o m a , 48 ( 1 9 7 4 ) 355---63G. 11 L a q u e u r , G.L., O. Mickelson, M.G. W~iting a n d L.T. K u r l a n d , Carcinogenic p r o p e r t i e s of n u t s f r o m Cycas circinalis L. i n d i g e n o u s t o G u a m , J. Natl. C a n c e r Ins., 31 ( 1 9 6 3 ) 9 1 9 .
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