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Effect of anticonvulsants on human chromosomes
Mutation Research, 204 (1988) 623-626
623
Elsevier MTR 01262
Effect of anticonvulsants on h u m a n chromosomes 2. In vitro studies J.M. Garcia Sagredo Seroicio de Gen~tica M~dica, Hospital Rambn y Cajal, Madrid (Spain)
(Received25 June 1987) (Revision received31 August 1987) (Accepted 5 October 1987)
Keywords: Anticonvulsantdrugs; Chromosomalaberrations; Diphenylhydantoin;Ethosuximide; Phenobarbital.
Summary The effects of 3 anticonvulsant drugs (diphenylhydantoin, ethosuximide, and phenobarbital) on human peripheral lymphocytes in vitro were studied. The rate of chromosomal aberrations induced by the 3 anticonvulsants was significantly increased from the first concentration analyzed, similar to half the therapeutic serum concentration. These findings are compared with other previous reports.
In 1972 Grosse et al., in a wide study of 32 epileptic women who were treated with various combinations of anticonvulsant drugs and their 33 children (aged between 0 and 3) exposed to the drug in utero, found a significant increase of chromosomal aberrations (CAs). Later, Ayrand et al. (1974) described similar findings in 45 epileptic patients and 15 children exposed in utero. Herha and Obe (1976) also found an increase of CAs in 2 groups of epileptic patients on monotherapy with diphenylhydantoin (DPH) and carbamazepine. With the exception of Garcla Sagredo (1987) who reported chromosomal damage in epileptic patients, other later reports show no chromosomal effects on peripheral lymphocytes in epileptic patients treated with anticonvulsants (Alving et al., 1977; Knuutila et al., 1977; Esser et al., 1981). Correspondence: Dr. J.M. Garcia Sagredo,Serviciode Gen&ica M&tica, Hospital Ram6n y Cajal, Crtra. de Colmenar Kin. 9.100, 28034 Madrid (Spain).
At the same time, negative results have been obtained in experiments performed in vitro to assess the capability of anticonvulsants to produce CAs in human peripheral lymphocytes (Brogger, 1970; Stenchever et al., 1973; Bishun et al., 1975; Alving et al., 1976; L6onard et al., 1984). In the present study, we report on the results of chromosome investigations in human peripheral lymphocytes treated in vitro with 3 commonly used anticonvulsants: DPH, ethosuximide (ETX), and phenobarbital (PB).
Material and methods A group of 10 healthy, normal individuals was used in this study. All cultures were carried out as follows: heparinised whole blood was cultivated for 72 h in 5 ml RPMI-1640 (Gibco) medium and 20% fetal calf serum. Phytohaemagglutinin M (Gibco) was used as a stimulator and Colcemid as an inhibitor. The preparations were carried out according to Moorhead et al. (1960).
0165-1218/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
624
Ten cultures were done of each donor. One of the cultures was the control, and in the other 9 cultures the anticonvulsant drug was added for the last 22 h of culture. We studied 3 anticonvulsant drugs at 3 different concentrations (with the lowest concentration half the therapeutic serum concentration and the highest 3 times the therapeutic concentration). In this way, a set of cultures from one donor is composed of 10 cultures as follows: (A) control; (B) DPH-I 10/tg/ml; (C) DPH-II 40 txg/ml; (D) DPH-III 6 0 /,g / m l ; (E) ETX-I 30 #g/ml; (F) ETX-II 120/,g/ml; (G) ETX-III 180/*g/ml; (H) PB-I 10 #g/ml; (I) PB-II 4 0 / , g / m l ; (J) PB-III 60/~g/ml. From each culture 100 metaphases were studied in a blind fashion in order to evaluate CAs. CAs were classified into mono-chromatid breaks, iso-chromatid breaks, dicentrics and others. Gaps were ignored. The criterion for a break was a discontinuity in the chromosome arm with either a dislocation of the fragment or a separation between the 2 fragments equal to or more than the width of the arm. Acentric fragments were considered as iso-breaks.
Since all samples tested had simultaneous exposed and unexposed cultures, each donor served as his own control, and the statistical analysis was made by 2-tailed paired t test and Friedman test.
Results Table 1 illustrates the results observed in the control cultures and in the cultures exposed to the action of the anticonvulsants studied: DPH, ETX, and PB. The CAs observed increased progressively with the anticonvulsant concentration, and in all experimental cultures the level of CAs was higher than in the controls. As in the cultures with the highest concentration of PB (60/xg/ml) the mitotic index was very low, it was impossible to evaluate CAs. The difference between the percentage of induced CAs in cultures exposed to the anticonvulsants and the spontaneous aberration in control cultures was significant (Table 2), except between the cultures with 120 # g / m l ETX and controls. With the 3 anticonvulsants studied, the CAs increased progressively from control to the highest concentration culture (Fig. 1); significant differences were found with DPH, not only between controls and the experimental cultures, but also between experimental cultures (see Table 2), and this progressive increase of CAs was globally sig-
TABLE 1 CHROMOSOMAL ABERRATIONS F O U N D IN C O N T R O L A N D E X P E R I M E N T A L C U L T U R E S W I T H A N T I C O N VULSANT D R U G S Culture
Number of cells studied
MB
Control
985
49
DPH-I DPH-II DPH-III
828 900 797
ETX-I ETX-II ETX-III
800 711 736
PB-I PB-II PB-III
IB
dic
other
Aberrations
8
-
2
60
(6.33)
77 93 93
11 16 23
1 1 3
2 4 5
91 114 124
(10.99) (12.67) (15.56)
75 60 72
15 18 23
2 1
5 2 1
97 80 97
(12.12) (11.25) (13.18)
700 83 700 88 very low mitotic index
17 17
1 1
4 2
105 108
(15.0) (15.43)
Total
MB: mono-breaks; IB: iso-breaks, dic: dicentrics.
(%)
625
TABLE 2 RESULTS OF PAIRED t TEST COMPARING CONTROL CULTURES WITH EXPERIMENTAL CULTURES Control
DPH-I
DPH-II
DPH-III
Control DPH-I DPH-II DPH-III
0
3.1837 * 0
3.3768 * * 0.5106 0
Control
ETX-I
ETX-II
Control ETX-I ETX-II ETX-III
0
3.8188 * 0
1.8847 0.0515 0
Control
PB-I
PB-II
Control PB-I PB-II
0
2.8721 * 0
6.0764 * * * 2.5871 * 2.7512 * 0 F r i e d m a n test X 2 = 12.9332 * * ETX-III
2.8438 * 0.8534 1.8228 0 F r i e d m a n test X 2 = 6.3375
4.5341 * * 0.2367 0 F r i e d m a n test X 2 = 5.4286
• p < 0.05; * * p < 0 . 0 1 ; * * *
p < 0.001.
Z~
o+_.._~ R ~ A T
/
[~
_j---+ [~
..
I
II
IIl
Fig. 1. M e a n of c h r o m o s o m a l a b e r r a t i o n s f o r c o n t r o l a n d e x p e r i m e n t a l c u l t u r e s w i t h p r o g r e s s i v e c o n c e n t r a t i o n s of D P H , E T X , a n d PB.
nificant when evaluated by means of a Friedman test (X 2 = 12.9333, p < 0.01). In the case of ETX and PB, although the increase was progressive the Friedman test did not reach a significant level (Table 2).
Discussion These results show that the three anticonvulsants studied, DPH, ETX, and PB, have the capacity of inducing significant chromosomal damage in human chromosomes in vitro. In contrast to the reports of Brogger (1970), Stenchever et al. (1973), Bishun et al. (1975), A1ving et al. (1976), and Lronard et al. (1984) who found no chromosomal damage with anticonvulsants in vitro, our results are in consonance with in vivo reports in epileptic patients describing an increased level of chromosomal damage (Grosse et al., 1972; Ayraud et al., 1974; Herha and Obe, 1976; Garcia Sagredo, 1987). Nevertheless, studies exist of epileptic patients in which no chromosomal effect was reported (Alving et al., 1977; Knuutila et al., 1977; Esser et al., 1981). It is difficult to explain these different results, but in vitro some differences can arise from individual variability of the donors (principally if there are few donors) and from the total number of cells studied. Therefore the controversy about the effect of anticonvulsants on human chromosomes is not yet closed. At another level of analysis, when the rate of production of sister-chromatid exchanges (SCE) was studied, Habedank et al. (1982), Conde Guerri (1984), Kulkarni et al. (1984) and Shauman et al. (1985) reported an increase of SCE in epileptic patients treated with anticonvulsants, and Maurya and Goyle (1985) reported an increase of SCE in vitro with DPH. Therefore, further studies are necessary to elucidate the effect of anticonvulsants on human chromosomes.
Acknowledgement This study was supported in part by a grant from the Ministerio de Educacirn de Espaha.
626
References Alving, J., M.K. Jensen and H. Meyer (1976) Diphenylhydantoin and chromosome morphology in man and rat. A negative report, Mutation Res., 40, 173-176. Alving, J., M.K. Jensen and H. Meyer (1977) Chromosome studies of bone marrow cells and peripheral blood lymphocytes from dyphenylhydantoin-treated patients, Mutation Res., 48, 361-366. Ayraud, N., C. Cantrelle and G. Darcourt (1974) Action des mt,dicaments anticonvulsivants sur les chromosomes de lymphocytes humains, C.R. Soc. Biol. (Paris), 168, 573-577. Bishum, N.P., N.S. Smith and D.C. Williams (1975) Chromosomes and anticonvulsivant drugs, Mutation Res., 28, 141-143. Brogger, A. (1970) Anticonvulsivant drugs and chromosomes, Lancet, ii, 979. Conde Guerri, B. (1984) Fenobarbital: estudio 'in vivo' de sus posibles interacciones a nivel immtmoltgico y citogen~tico, Med. Clin., 82, 57-61. Esser, K.J., F. Kotlarek, M. Habedank, U. Miihler and E. Mtihler (1981) Chromosomal investigations in epileptic children during long-term therapy with phenytoin or primidone, Hum. Genet., 56, 345-348. Garcia Sagredo, J.M. (1987) Efecto de los anticonvulsivantes sobre los cromosomas humanos: 1. Estudio 'in vivo', Rev. Clin. Esp., 181, 361-367. Grosse, K.P., G. Schwanitz, H.O. Rott and H.F. Wismtiller (1972) Chromosomenuntersuchungen bei Behandlung mit Anticonvulsiva, Humangenetik, 16, 209-216. Habedank, M., J.J. Esser, D. Briill, F. Kotlarek and C. Stumpf
(1982) Increased sister chromatid exchanges in epileptic children during long-term therapy with phenytoin, Ham. Genet., 61, 71-72. Herha, J., and G. Obe (1976) Chromosomal damage in epileptics on monotherapy with Carbamazepine and Diphenylhydantoin, Hum. Genet., 34, 255-263. Knuutila, S., M. Slimes, O. Simello, P. Tammisto and T. Weber (1977) Long-term use of phenytoin: effects on bone-marrow chromosomes in man, Mutation Res., 43, 309-312. Kulkarni, P.S., V.P. Mondkar, A.B. Sonawalla and L.M. Ambani (1984) Chromosomal studies of peripheral blood from epileptic patients treated with phenobarbital and/or diphenylhydantoin, Food Chem. Toxicol., 22, 1009-1012. Ltonard, A., C. de Meester, L. Fabry, L. de Saint-Georges and P. Dumont (1984) Lack of mutagenicity of diphenylhydantoin in in vitro short-term tests, Mutation Res., 137, 79-88. Maurya, A.K., and S. Goyle (1985) Mutagenic potential of anticonvulsant diphenylhydantoin (DPH) on human lymphocytes in vitro, Meth. and Find. Exptl. Clin. Pharmacol., 7, 109-112. Moorhead, P.S., P.C. Nowell, W.J. Mellman, D.M. Battips and D.A. Hungerford (1960) Chromosome preparations of leukocytes cultured from human peripheral blood, Exp. Cell Res., 20, 613-616. Shaumann, B., S.B. Johnson, N. Wang and S.V. Brunt (1985) Sister chromatid exchanges in adult epileptic patients on phenytoin therapy, Environ. Mutagen., 7, 711-714. Stenchever, M.A., and M. Allen (1973) The effect of selected antiepileptic drugs on the chromosomes of human lymphocytes in vitro, Am. J. Obstet. Gynecol., 116, 867-870.
623
Elsevier MTR 01262
Effect of anticonvulsants on h u m a n chromosomes 2. In vitro studies J.M. Garcia Sagredo Seroicio de Gen~tica M~dica, Hospital Rambn y Cajal, Madrid (Spain)
(Received25 June 1987) (Revision received31 August 1987) (Accepted 5 October 1987)
Keywords: Anticonvulsantdrugs; Chromosomalaberrations; Diphenylhydantoin;Ethosuximide; Phenobarbital.
Summary The effects of 3 anticonvulsant drugs (diphenylhydantoin, ethosuximide, and phenobarbital) on human peripheral lymphocytes in vitro were studied. The rate of chromosomal aberrations induced by the 3 anticonvulsants was significantly increased from the first concentration analyzed, similar to half the therapeutic serum concentration. These findings are compared with other previous reports.
In 1972 Grosse et al., in a wide study of 32 epileptic women who were treated with various combinations of anticonvulsant drugs and their 33 children (aged between 0 and 3) exposed to the drug in utero, found a significant increase of chromosomal aberrations (CAs). Later, Ayrand et al. (1974) described similar findings in 45 epileptic patients and 15 children exposed in utero. Herha and Obe (1976) also found an increase of CAs in 2 groups of epileptic patients on monotherapy with diphenylhydantoin (DPH) and carbamazepine. With the exception of Garcla Sagredo (1987) who reported chromosomal damage in epileptic patients, other later reports show no chromosomal effects on peripheral lymphocytes in epileptic patients treated with anticonvulsants (Alving et al., 1977; Knuutila et al., 1977; Esser et al., 1981). Correspondence: Dr. J.M. Garcia Sagredo,Serviciode Gen&ica M&tica, Hospital Ram6n y Cajal, Crtra. de Colmenar Kin. 9.100, 28034 Madrid (Spain).
At the same time, negative results have been obtained in experiments performed in vitro to assess the capability of anticonvulsants to produce CAs in human peripheral lymphocytes (Brogger, 1970; Stenchever et al., 1973; Bishun et al., 1975; Alving et al., 1976; L6onard et al., 1984). In the present study, we report on the results of chromosome investigations in human peripheral lymphocytes treated in vitro with 3 commonly used anticonvulsants: DPH, ethosuximide (ETX), and phenobarbital (PB).
Material and methods A group of 10 healthy, normal individuals was used in this study. All cultures were carried out as follows: heparinised whole blood was cultivated for 72 h in 5 ml RPMI-1640 (Gibco) medium and 20% fetal calf serum. Phytohaemagglutinin M (Gibco) was used as a stimulator and Colcemid as an inhibitor. The preparations were carried out according to Moorhead et al. (1960).
0165-1218/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (BiomedicalDivision)
624
Ten cultures were done of each donor. One of the cultures was the control, and in the other 9 cultures the anticonvulsant drug was added for the last 22 h of culture. We studied 3 anticonvulsant drugs at 3 different concentrations (with the lowest concentration half the therapeutic serum concentration and the highest 3 times the therapeutic concentration). In this way, a set of cultures from one donor is composed of 10 cultures as follows: (A) control; (B) DPH-I 10/tg/ml; (C) DPH-II 40 txg/ml; (D) DPH-III 6 0 /,g / m l ; (E) ETX-I 30 #g/ml; (F) ETX-II 120/,g/ml; (G) ETX-III 180/*g/ml; (H) PB-I 10 #g/ml; (I) PB-II 4 0 / , g / m l ; (J) PB-III 60/~g/ml. From each culture 100 metaphases were studied in a blind fashion in order to evaluate CAs. CAs were classified into mono-chromatid breaks, iso-chromatid breaks, dicentrics and others. Gaps were ignored. The criterion for a break was a discontinuity in the chromosome arm with either a dislocation of the fragment or a separation between the 2 fragments equal to or more than the width of the arm. Acentric fragments were considered as iso-breaks.
Since all samples tested had simultaneous exposed and unexposed cultures, each donor served as his own control, and the statistical analysis was made by 2-tailed paired t test and Friedman test.
Results Table 1 illustrates the results observed in the control cultures and in the cultures exposed to the action of the anticonvulsants studied: DPH, ETX, and PB. The CAs observed increased progressively with the anticonvulsant concentration, and in all experimental cultures the level of CAs was higher than in the controls. As in the cultures with the highest concentration of PB (60/xg/ml) the mitotic index was very low, it was impossible to evaluate CAs. The difference between the percentage of induced CAs in cultures exposed to the anticonvulsants and the spontaneous aberration in control cultures was significant (Table 2), except between the cultures with 120 # g / m l ETX and controls. With the 3 anticonvulsants studied, the CAs increased progressively from control to the highest concentration culture (Fig. 1); significant differences were found with DPH, not only between controls and the experimental cultures, but also between experimental cultures (see Table 2), and this progressive increase of CAs was globally sig-
TABLE 1 CHROMOSOMAL ABERRATIONS F O U N D IN C O N T R O L A N D E X P E R I M E N T A L C U L T U R E S W I T H A N T I C O N VULSANT D R U G S Culture
Number of cells studied
MB
Control
985
49
DPH-I DPH-II DPH-III
828 900 797
ETX-I ETX-II ETX-III
800 711 736
PB-I PB-II PB-III
IB
dic
other
Aberrations
8
-
2
60
(6.33)
77 93 93
11 16 23
1 1 3
2 4 5
91 114 124
(10.99) (12.67) (15.56)
75 60 72
15 18 23
2 1
5 2 1
97 80 97
(12.12) (11.25) (13.18)
700 83 700 88 very low mitotic index
17 17
1 1
4 2
105 108
(15.0) (15.43)
Total
MB: mono-breaks; IB: iso-breaks, dic: dicentrics.
(%)
625
TABLE 2 RESULTS OF PAIRED t TEST COMPARING CONTROL CULTURES WITH EXPERIMENTAL CULTURES Control
DPH-I
DPH-II
DPH-III
Control DPH-I DPH-II DPH-III
0
3.1837 * 0
3.3768 * * 0.5106 0
Control
ETX-I
ETX-II
Control ETX-I ETX-II ETX-III
0
3.8188 * 0
1.8847 0.0515 0
Control
PB-I
PB-II
Control PB-I PB-II
0
2.8721 * 0
6.0764 * * * 2.5871 * 2.7512 * 0 F r i e d m a n test X 2 = 12.9332 * * ETX-III
2.8438 * 0.8534 1.8228 0 F r i e d m a n test X 2 = 6.3375
4.5341 * * 0.2367 0 F r i e d m a n test X 2 = 5.4286
• p < 0.05; * * p < 0 . 0 1 ; * * *
p < 0.001.
Z~
o+_.._~ R ~ A T
/
[~
_j---+ [~
..
I
II
IIl
Fig. 1. M e a n of c h r o m o s o m a l a b e r r a t i o n s f o r c o n t r o l a n d e x p e r i m e n t a l c u l t u r e s w i t h p r o g r e s s i v e c o n c e n t r a t i o n s of D P H , E T X , a n d PB.
nificant when evaluated by means of a Friedman test (X 2 = 12.9333, p < 0.01). In the case of ETX and PB, although the increase was progressive the Friedman test did not reach a significant level (Table 2).
Discussion These results show that the three anticonvulsants studied, DPH, ETX, and PB, have the capacity of inducing significant chromosomal damage in human chromosomes in vitro. In contrast to the reports of Brogger (1970), Stenchever et al. (1973), Bishun et al. (1975), A1ving et al. (1976), and Lronard et al. (1984) who found no chromosomal damage with anticonvulsants in vitro, our results are in consonance with in vivo reports in epileptic patients describing an increased level of chromosomal damage (Grosse et al., 1972; Ayraud et al., 1974; Herha and Obe, 1976; Garcia Sagredo, 1987). Nevertheless, studies exist of epileptic patients in which no chromosomal effect was reported (Alving et al., 1977; Knuutila et al., 1977; Esser et al., 1981). It is difficult to explain these different results, but in vitro some differences can arise from individual variability of the donors (principally if there are few donors) and from the total number of cells studied. Therefore the controversy about the effect of anticonvulsants on human chromosomes is not yet closed. At another level of analysis, when the rate of production of sister-chromatid exchanges (SCE) was studied, Habedank et al. (1982), Conde Guerri (1984), Kulkarni et al. (1984) and Shauman et al. (1985) reported an increase of SCE in epileptic patients treated with anticonvulsants, and Maurya and Goyle (1985) reported an increase of SCE in vitro with DPH. Therefore, further studies are necessary to elucidate the effect of anticonvulsants on human chromosomes.
Acknowledgement This study was supported in part by a grant from the Ministerio de Educacirn de Espaha.
626
References Alving, J., M.K. Jensen and H. Meyer (1976) Diphenylhydantoin and chromosome morphology in man and rat. A negative report, Mutation Res., 40, 173-176. Alving, J., M.K. Jensen and H. Meyer (1977) Chromosome studies of bone marrow cells and peripheral blood lymphocytes from dyphenylhydantoin-treated patients, Mutation Res., 48, 361-366. Ayraud, N., C. Cantrelle and G. Darcourt (1974) Action des mt,dicaments anticonvulsivants sur les chromosomes de lymphocytes humains, C.R. Soc. Biol. (Paris), 168, 573-577. Bishum, N.P., N.S. Smith and D.C. Williams (1975) Chromosomes and anticonvulsivant drugs, Mutation Res., 28, 141-143. Brogger, A. (1970) Anticonvulsivant drugs and chromosomes, Lancet, ii, 979. Conde Guerri, B. (1984) Fenobarbital: estudio 'in vivo' de sus posibles interacciones a nivel immtmoltgico y citogen~tico, Med. Clin., 82, 57-61. Esser, K.J., F. Kotlarek, M. Habedank, U. Miihler and E. Mtihler (1981) Chromosomal investigations in epileptic children during long-term therapy with phenytoin or primidone, Hum. Genet., 56, 345-348. Garcia Sagredo, J.M. (1987) Efecto de los anticonvulsivantes sobre los cromosomas humanos: 1. Estudio 'in vivo', Rev. Clin. Esp., 181, 361-367. Grosse, K.P., G. Schwanitz, H.O. Rott and H.F. Wismtiller (1972) Chromosomenuntersuchungen bei Behandlung mit Anticonvulsiva, Humangenetik, 16, 209-216. Habedank, M., J.J. Esser, D. Briill, F. Kotlarek and C. Stumpf
(1982) Increased sister chromatid exchanges in epileptic children during long-term therapy with phenytoin, Ham. Genet., 61, 71-72. Herha, J., and G. Obe (1976) Chromosomal damage in epileptics on monotherapy with Carbamazepine and Diphenylhydantoin, Hum. Genet., 34, 255-263. Knuutila, S., M. Slimes, O. Simello, P. Tammisto and T. Weber (1977) Long-term use of phenytoin: effects on bone-marrow chromosomes in man, Mutation Res., 43, 309-312. Kulkarni, P.S., V.P. Mondkar, A.B. Sonawalla and L.M. Ambani (1984) Chromosomal studies of peripheral blood from epileptic patients treated with phenobarbital and/or diphenylhydantoin, Food Chem. Toxicol., 22, 1009-1012. Ltonard, A., C. de Meester, L. Fabry, L. de Saint-Georges and P. Dumont (1984) Lack of mutagenicity of diphenylhydantoin in in vitro short-term tests, Mutation Res., 137, 79-88. Maurya, A.K., and S. Goyle (1985) Mutagenic potential of anticonvulsant diphenylhydantoin (DPH) on human lymphocytes in vitro, Meth. and Find. Exptl. Clin. Pharmacol., 7, 109-112. Moorhead, P.S., P.C. Nowell, W.J. Mellman, D.M. Battips and D.A. Hungerford (1960) Chromosome preparations of leukocytes cultured from human peripheral blood, Exp. Cell Res., 20, 613-616. Shaumann, B., S.B. Johnson, N. Wang and S.V. Brunt (1985) Sister chromatid exchanges in adult epileptic patients on phenytoin therapy, Environ. Mutagen., 7, 711-714. Stenchever, M.A., and M. Allen (1973) The effect of selected antiepileptic drugs on the chromosomes of human lymphocytes in vitro, Am. J. Obstet. Gynecol., 116, 867-870.