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Diphenylhydantoin: Effect on the chromosomes of human leukocytes
Volume Number
109 6
Communications
Table I. Distribution of plasma total ascorbic acid levels (in milligrams period of reproductive cycle Weeks of gestation
14-16 17-19 20-22 23-26
22 29 33 40
0.72 0.75 0.69 0.67
0.09 0.08 0.06 0.06
<0.40 27 24 21 33
30-32 27-29 33-35 36 and over
37 33 43 9
0.58 t2 0.06 0.59 0.07 0.54 + 0.06 0.33 f. 0.10
43 37 47 78
*Values
arc f standard
Mean*
f 2 + +
1
No. of Mean*
124 122 difference;
1
>o.ao 32 i9 27 :: 23 11
per 100 ml.) by
Per
samples
Second Third are f standard
of samples
0.40-0.80 41 38 46 40
error.
Trimester of pregnancy
tSigni6cant
cent
42 35 30 11
Table II. Distribution of plasma total ascorbic acid levels (in milligrams trimester of pregnancy
*Values
961
per 100 ml.) by Per
No. of samples
in brief
0.71 f. 0.04t 0.55 k 0.03
<0.40 27 45
1
cent
of samples
0.40-0.80 41 34
(
>0.80 32 21
error. 224 df, t = 3.333,
p < 0.001.
REFERENCES
1. Macy, I. G., Moyer, H. C., DiLoreto, P. Nutr. 50 (Supp. 1): 2. Lowry, 0. H., Lopez, J. Biol. Chem. 160:
E. Z., Kelly, H. J., Mack, C., and Pratt, J. P.: J. 1, 1954. J. A., and Bessey, 0. A.: 609, 1945.
Diphenylhydantoin: Effect on the chromosomes of human leukocytes MORTON A. STENCHEVER, JANE A. JARVIS, M.T.
M.D.
Department of Reproductive Biology, Case Western Reserve University, School of Medicine, Cleveland, Ohio
DIPHENYLHYDANTOIN (Dilantin) is a widely used anticonvulsant drug and as such is frequently taken by pregnant women who have convulsive disorders. It has been shown to be teratogenic in mice19 2 although no such data
land,
Supported Ohio,
Reprint Salt Lake
by grants from and the National requests to: Dr. City, Utah 84112.
the Brush Foundation, ClewFoundation-March of Dimes. Stenchever,
50 N. Medical
Dr.,
have as yet been offered concerning human subjects. Because there is definitely a human population at risk exposed to the drug, we undertook a study of the effects of diphenylhydantoin on the chromosomes of human leukocytes in vitro. From each of two individuals (a 25-year-old female laboratory technician and a 24-year-old male medical student), 20 ml. samples of heparinized venous blood were drawn. Both subjects were in good health, had had no recent exposures to agents known to break chromosomes, and had been used in previous experiments involving agents which cause chromosome breakage. Each blood sample was treated with phytohemagglutinin, and centrifuged at 500 r.p.m. for five minutes. The plasma containing leukocytes was removed and divided equally into five tissue culture flasks for each sample. Dulbecco’s modified Eagles Medium (GIBCO) with penicillin and streptomycin was added to each flask to make a total volume of 10 ml. In each case, one flask served as a control culture and the other four were treated at their initiation with diphenylhydantoin to make final concert& trations of 100.0, 10.0, 1.0, and 0.1 pg per milli-
962
Communications
in brief
Table I. Chromosome with diphenylhydantoin Concentration (CcJml.) Subject
gap, break, and abnormal
of cells
sco7ed
Cells No.
with
March 15, 1971 J. Obstet. Gym.
form data for human leukocytes treated
gaps
1 Per cent
Cells 1
with
No.
4N cells
breaks 1 Per cent
No.
Per cent
I
(female) 100.0 10.0 1.0 0.1 o.o* Subject
No.
Amer.
2 (male) 100.0 10.0 1.0
0.1 o.o*
29 208 204 232 214
3.4 4.3 5.3 4.3 5.6
0
0
1.9 0 0 0.9
0.4 0.4 0.8 0
212 200 208 206 208
0.9 3.5 1.4 3.4 0.9
0
liter, respectively, of culture fluid. All flasks were incubated at 37O C. for 72 hours. TWO hours before harvesting, each culture was treated with 0.4 pg of colcemid. Harvesting was carried out by an air-dry method, and slides were prepared, coded, and scored for chromatid and isochromatid gaps and breaks and for abnormal forms by methods previously described.3 Individuals doing the scoring did not know from which culture the slides were obtained. Table I summarizes the data for both subjects. Since only small numbers of gaps and breaks were observed, no attempt was made to separate chromatid and isochromatid gaps or breaks. No significant differences were noted between the data for the various concentrations and the controls. The 100.0 pg concentration culture grew poorly in both cases, but even here no significant abnormalities were noted. Four N figures were noted in small numbers in the series from the female subject, but the significance of so few such forms could not be evaluated and their occurrence has been noted from time to time in certain cultures in our laboratory. Gap rates of 0 to 8 per cent and breakage
AR 0 0
rates of 0 to 3 per cent are standard in cultures grown in our laboratory. It is comforting to note that such a widely used amd necessary drug does not appear to cause obvious chromosome damage. But it must be remembered that diphenylhydantoin does seem to be teratogenic in mice and that it may, under certain conditions, have a similar effect in human beings. While chromosome breakage may be associated with embryonal damage, it is only one parameter and an agent could certainly cause defects by a pathway other than chromosome damage. Thus, negative data such as these, while reassuring, should not be taken as evidence for absolute safety, and a drug should be used in pregnancy onty when the needs of the mother outweigh the theoretical risks to the fetus. REFERENCES
1. Massey, K. M.: Oral Ther. 2: 38, 1966. 2. Gibson, J. E., and Becker, B. A.: Proc. Sot. Exp. Biol. Med. 128: 905, 1968. 3. Stenchever, M. A., Frankel, R. S., Jarvis, J. A., and Veress, K.: Aman. J. OBSTET. GYNEC. 103: 836, 1969.
109 6
Communications
Table I. Distribution of plasma total ascorbic acid levels (in milligrams period of reproductive cycle Weeks of gestation
14-16 17-19 20-22 23-26
22 29 33 40
0.72 0.75 0.69 0.67
0.09 0.08 0.06 0.06
<0.40 27 24 21 33
30-32 27-29 33-35 36 and over
37 33 43 9
0.58 t2 0.06 0.59 0.07 0.54 + 0.06 0.33 f. 0.10
43 37 47 78
*Values
arc f standard
Mean*
f 2 + +
1
No. of Mean*
124 122 difference;
1
>o.ao 32 i9 27 :: 23 11
per 100 ml.) by
Per
samples
Second Third are f standard
of samples
0.40-0.80 41 38 46 40
error.
Trimester of pregnancy
tSigni6cant
cent
42 35 30 11
Table II. Distribution of plasma total ascorbic acid levels (in milligrams trimester of pregnancy
*Values
961
per 100 ml.) by Per
No. of samples
in brief
0.71 f. 0.04t 0.55 k 0.03
<0.40 27 45
1
cent
of samples
0.40-0.80 41 34
(
>0.80 32 21
error. 224 df, t = 3.333,
p < 0.001.
REFERENCES
1. Macy, I. G., Moyer, H. C., DiLoreto, P. Nutr. 50 (Supp. 1): 2. Lowry, 0. H., Lopez, J. Biol. Chem. 160:
E. Z., Kelly, H. J., Mack, C., and Pratt, J. P.: J. 1, 1954. J. A., and Bessey, 0. A.: 609, 1945.
Diphenylhydantoin: Effect on the chromosomes of human leukocytes MORTON A. STENCHEVER, JANE A. JARVIS, M.T.
M.D.
Department of Reproductive Biology, Case Western Reserve University, School of Medicine, Cleveland, Ohio
DIPHENYLHYDANTOIN (Dilantin) is a widely used anticonvulsant drug and as such is frequently taken by pregnant women who have convulsive disorders. It has been shown to be teratogenic in mice19 2 although no such data
land,
Supported Ohio,
Reprint Salt Lake
by grants from and the National requests to: Dr. City, Utah 84112.
the Brush Foundation, ClewFoundation-March of Dimes. Stenchever,
50 N. Medical
Dr.,
have as yet been offered concerning human subjects. Because there is definitely a human population at risk exposed to the drug, we undertook a study of the effects of diphenylhydantoin on the chromosomes of human leukocytes in vitro. From each of two individuals (a 25-year-old female laboratory technician and a 24-year-old male medical student), 20 ml. samples of heparinized venous blood were drawn. Both subjects were in good health, had had no recent exposures to agents known to break chromosomes, and had been used in previous experiments involving agents which cause chromosome breakage. Each blood sample was treated with phytohemagglutinin, and centrifuged at 500 r.p.m. for five minutes. The plasma containing leukocytes was removed and divided equally into five tissue culture flasks for each sample. Dulbecco’s modified Eagles Medium (GIBCO) with penicillin and streptomycin was added to each flask to make a total volume of 10 ml. In each case, one flask served as a control culture and the other four were treated at their initiation with diphenylhydantoin to make final concert& trations of 100.0, 10.0, 1.0, and 0.1 pg per milli-
962
Communications
in brief
Table I. Chromosome with diphenylhydantoin Concentration (CcJml.) Subject
gap, break, and abnormal
of cells
sco7ed
Cells No.
with
March 15, 1971 J. Obstet. Gym.
form data for human leukocytes treated
gaps
1 Per cent
Cells 1
with
No.
4N cells
breaks 1 Per cent
No.
Per cent
I
(female) 100.0 10.0 1.0 0.1 o.o* Subject
No.
Amer.
2 (male) 100.0 10.0 1.0
0.1 o.o*
29 208 204 232 214
3.4 4.3 5.3 4.3 5.6
0
0
1.9 0 0 0.9
0.4 0.4 0.8 0
212 200 208 206 208
0.9 3.5 1.4 3.4 0.9
0
liter, respectively, of culture fluid. All flasks were incubated at 37O C. for 72 hours. TWO hours before harvesting, each culture was treated with 0.4 pg of colcemid. Harvesting was carried out by an air-dry method, and slides were prepared, coded, and scored for chromatid and isochromatid gaps and breaks and for abnormal forms by methods previously described.3 Individuals doing the scoring did not know from which culture the slides were obtained. Table I summarizes the data for both subjects. Since only small numbers of gaps and breaks were observed, no attempt was made to separate chromatid and isochromatid gaps or breaks. No significant differences were noted between the data for the various concentrations and the controls. The 100.0 pg concentration culture grew poorly in both cases, but even here no significant abnormalities were noted. Four N figures were noted in small numbers in the series from the female subject, but the significance of so few such forms could not be evaluated and their occurrence has been noted from time to time in certain cultures in our laboratory. Gap rates of 0 to 8 per cent and breakage
AR 0 0
rates of 0 to 3 per cent are standard in cultures grown in our laboratory. It is comforting to note that such a widely used amd necessary drug does not appear to cause obvious chromosome damage. But it must be remembered that diphenylhydantoin does seem to be teratogenic in mice and that it may, under certain conditions, have a similar effect in human beings. While chromosome breakage may be associated with embryonal damage, it is only one parameter and an agent could certainly cause defects by a pathway other than chromosome damage. Thus, negative data such as these, while reassuring, should not be taken as evidence for absolute safety, and a drug should be used in pregnancy onty when the needs of the mother outweigh the theoretical risks to the fetus. REFERENCES
1. Massey, K. M.: Oral Ther. 2: 38, 1966. 2. Gibson, J. E., and Becker, B. A.: Proc. Sot. Exp. Biol. Med. 128: 905, 1968. 3. Stenchever, M. A., Frankel, R. S., Jarvis, J. A., and Veress, K.: Aman. J. OBSTET. GYNEC. 103: 836, 1969.