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Mutagenicity tests with griseofulvin
225
Mutation Research, 68 (1979) 225--234 © Elsevier/North-Holland Biomedical Press
MUTAGENICITY TESTS WITH GRISEOFULVIN
A. L I ~ O N A R D a, F. P O N C E L E T
b, G. G R U T M A N
c, E. C A R B O N E L L E
c and L. F A B R Y
a
a Mammalian Genetics Laboratory, Department ofRadiobiology, C.E.N.-S.C.K., B 2400 Mol (Belgium), b Laboratory of Biotoxicology, Catholic University of Louvain, Pharmacy School, U C L 7369, B 1200 Brussels (Belgium) and c Catholic University of Louvain, B 1348 Louvain-la-Neuve (Belgium) (Received 21 February 1979) (Revision received 5 June 1979) (Accepted 14 June 1979)
Summary Griseofulvin was studied for its ability to induce structural chromosomal aberrations in germ and somatic cells of the male mouse. It was also tested for its capacity to produce his + revertants in Salmonella typhimurium. All tests yielded negative results, whereas highly significant effects were recorded in control assays with thio-TEPA.
Griseofulvin (C17H17C106) is a mould metabolite produced by Penicillium griseofulvum Dierckx and by P. Janczewskii Zal. This antibiotic is widely used for the t r e a t m e n t of human and animal dermatophytic infections. Owing to its specifical localization and concentration in the keratinized cells of the skin, hair and nails, it inhibits the growth of dermatophytes parasitizing these tissues, the infected tissue being removed by desquamation and the growth of new epidermal layers [9]. Over 20 years ago Paget and Walpole [26] discovered that griseofulvin is a spindle poison and causes disorientation of the spindle, scattering of the chromosomes and inhibition of chromosome movement at anaphase in rat cells and in growing roots of Vicia faba. Subsequent observations on the effects of griseofulvin on the spindle microtubules have been published in animals [6,14, 20--23,25,29,31] and in plants [9--13,15,27]. The antimitotic properties of griseofulvin have been studied and discussed by several investigators [8,24,33,34]. It is probable that griseofulvin, like other C-mitotic drugs, inhibits tubulin polymerization but, according to Weber et.al. [33], it is unclear whether griseofulvin acts on tubulin itself or on a tuhulinassociated protein. Because certain results [8,12,35] have led to the suggestion that griseofulvin
226 may also induce structural chromosomal aberrations, the present experiments were performed to study its clastogenic properties in somatic and germ cells of the male mouse and to detect its genetic effects on S. typhimurium. Materials and m e t h o d s Tests with mouse Tests were performed with 12-week-old BALB/c mice. The effect of griseofulvin on bone-marrow chromosomes was studied [17] by analysing 400 metaphases from 4 mice which, 48 h before, had been injected i.p. with griseofulvin (Leo Pharmaceutical Products, Denmark) at 0, 0.5, 1.0, 1.5 or 2.0 g/kg in 0.5 ml aqueous solution. The possible influence of the time interval between treatment and observation was followed by investigating bone marrow 1, 2, 4 or 9 days after i.p. injection of 1.5 g griseofulvin per kg b o d y weight. The production of micronuclei in polychromatic erythrocytes [28] was studied, for the different doses, 30 h after i.p. injection of 8-week-old mice. Griseofulvin is an antibiotic used orally, b u t histological studies of Paget and Walpole [26] have demonstrated that this c o m p o u n d is readily absorbed after intraperitoneal administration. The ability of griseofulvin to induce chromosomal aberrations in male germ cells was studied by the dominant-lethal test [3] in the post-meiotic stages i.e. 1--3 weeks after administration of griseofulvin (1.5 g/kg) and by examination of dividing spermatocytes [16] for the presence of reciprocal translocations induced in the pre-meiotic cells. 6 h after injection, each male was caged with 3 virgin females from the same strain which were replaced after 7 and 14 days. The females were dissected 17 days after mating had started, and pre- and postimplantation losses were determined b y conventional methods. After t w o months, the males were killed for spermatocyte scoring. Untreated animals and mice given thio-TEPA [tris(1-aziridinyl) phosphine sulphide] were utilized as controls. The dose of thio-TEPA, kindly provided by Dr. A.M. Malashenko (Moscow), used for the dominant-lethal test and the examination of bone-marrow chromosomes at different intervals of time was 5 mg/kg and that for dose--response relationship studies was 1.25 to 20 mg/kg. The doses of griseofulvin and thio-TEPA were chosen on the basis of experiments reported in the literature [1,5,18,19,30,32,35]. Tests with S. typhimurium Strains of S. typhimurium TA1530, TA1535, TA100, TA98, TA1978, TA1950, TA1538, TA1532, TA1537, T A 1 9 7 5 and G46 were kindly provided b y Professor B.N. Ames. Animals Adult male Wistar rats (200--250 g) were fed a RAL diet. The following pretreatments were applied to the animals. Aroclor-1254 diluted in corn oil (200 mg/ml) and injected i.p. (500 mg/kg) 5 days before the preparation of the liver fractions. Phenobarbital: 0.1% drinking water given ad libitum during 7 days before the rats were killed.
227
Metabolic activating system The post-mitochondrial fractions ($9) were from 3 pooled rat livers, the homogenate (3 ml of 0.15 M KC1/g wet liver) of which was centrifuged as described [2]. Preparation of $9 mix was made according to Ames et al. [2]. 100 pl (25 mg wet liver)/ml or 300 pl (75 mg wet liver)/ml mix of $9 were utilized (0.5 ml $9 mix/plate). Spot tests and plate-incorporation assays were performed according to Ames and coworkers [2] by using dilutions of griseofulvin in dimethyl sulphoxide (DMSO) (0.1 ml/plate) and with a bacterial inoculum of 2--8 X 107 viable bacteria from an overnight culture in nutrient broth (Difco)/plate. Bacterial fluctuation tests were performed in triplicate by using a modification of the method proposed by Green et al. [7]. Into a tube kept in an icewater bath, were successively introduced: liquid minimal glucose medium (Vogel--Bonnet E medium) supplemented with histidine and biotin (0.005 mM) (4 ml), 2--7 X 107 bacterial cells from an overnight culture in nutrient broth (Difco), substrate diluted in DMSO (0.1 ml), $9 mix (300 pl S9/ml mix) (1 ml). After homogenization, the mixture was distributed into 50 tubes (0.1 ml/tube). The racks of 50 tubes were incubated at 37°C in the dark for 3 h, then 2 ml of histidine-biotin-supplemented liquid minimal glucose medium containing a pH indicator (BCP) (5 ~g/ml) were added to each tube. The incubation of the racks was then carried on at 37°C for a total incubation time of 72 h, after which the Immbers of positive growing tubes (yellow)/rack were counted. Results and discussion
All tests with griseofulvin on somatic cells in vivo (Tables 1--3) yielded negative results, the incidence of erythrocytes with micronuclei as well as of bonemarrow cells carrying chromosomal aberrations being not statistically different from control values. Injection of griseofulvin did not produce reciprocal translocations in the preTABLE 1 C H R O M O S O M A L A N O M A L I E S IN M O U S E B O N E - M A R R O W C E L L S A F T E R D I F F E R E N T i.p. D O S E S OF GRISEOFULVIN AND THIO-TEPA Treatment (mg/kg)
Animals examined
Cells analysed
Cells w i t h c h r o m o s o m a l a n o m a l i e s Total
%
Controls
8
600
6
1.0
Griseofulvin 500 1000 1500 2000
4 4 4 4
400 400 400 400
0 2 1 2
0 0.5 0.25 0.5
Thio-TEPA 1.25 2.50 5 10
4 4 4 4
200 200 200 200
16 61 111 174
8.0 30.5 55.5 87.0
228
TABLE 2 CHROMOSOMAL ANOMALIES IN MOUSE BONE-MARROW CELLS AT DIFFERENT INTERVALS O F T I M E A F T E R i.p. T R E A T M E N T W I T H G R I S E O F U L V I N ( 1 5 0 0 m g ] k g ) O R T H I O - T E P A (5 m g / k g ) Treatment
Interval (days)
Animals examined
Cells analysed
A b n o r m a l cells Total
Control
%
8
600
6
1.0
Griseo fulvin
1 2 4 9
4 4 4 4
400 400 400 400
1 1 1 1
0.25 0.25 0.25 0.25
Thio-TEPA
1 2 4 9
4 4 4 4
200 200 200 150
161 111 19 11
80.50 55.50 9.50 0.73
TABLE 3 RESULTS OF THE MICRONUCLEUS Treatment (mg/kg)
Animals examined
TEST IN THE MOUSE Cells analysed
Cells w i t h m i c r o n u c l e i Total
Controls
10
10 0 0 0
%
36
0.36
Griseofulvin 500 1000 1500 2000
5 5 5 5
5 5 5 5
000 000 000 000
13 13 16 21
0.26 0.26 0.32 0.42
Thio-TEPA 2.5 5 10 20
5 5 5 5
5 5 5 5
000 000 000 000
296 279 247 163
5.92 5.58 4.94 3.26
TABLE 4 RESULTS OF MALE MICE
THE
EXAMINATION
OF
SPERMATOCYTES
I FROM
Treatment
Animals examined
Cells analysed
Anomalies
Control Grise o fulvin (1.5 g / k g ) Thio-TEPA
20 15
2000 1500
0 0
7
1000
3 CIV 1 CIII + I
CONTROL
AND TREATED
136 1.89 + 0.16
33.4 12.3
21.1
Dead embryos Total Per female
T o t a l loss (%) Pre-implantation
loss (%) Post-implantation
loss (%)
428 5.94 + 0.29
Live e m b r y o s Total Per female
21.2
32.4 11.2
70 1.84 ± 0.22
223 5.87 ± 0.39
293 7.71 + 0.45
15.2
32.5 17.3
29 1.32 + 0.24
129 5.86 + 0.52
158 7.18 ± 0.57
17.0
27.4 10.4
106 1.51 + 0.15
452 6.46 + 0.30
558 7.97 + 0.34
623 8.90 + 0.36
70 72.9 + 8.7
564 7.83 + 0.33
191 8.68 + 0.63
22 29.3 + 6.3
Implantations Total Per female
330 8.68 + 0.47
+ 8.6
643 8.93 ~ 0.35
38 52.8
Corpora lutea Total Per female
+ 8.8
72 75.0
96
72
96
Total females
Pregnant females Total %
75
Controls
1500 mg/kg
Controls
3000 mg/kg
2nd week
1st week
Observations
+ 8.1
15.9
23.7 7.8
45 1.32 + 0.20
216 6.35 + 0.43
261 7.68 ± 0.47
283 8.32 ± 0.49
34 47.2
72
1500 mg/kg
+ 7.7
19.7
36.4 16.7
53 1.61 + 0.22
171 5.18 + 0.40
224 6.79 ± 0.45
269 8.15 + 0.50
33 44.0
75
3000 mgfkg
-+ 7.9
16.9
29.8 16.9
85 1.49 ± 0.16
353 6.19 + 0.33
438 6 . 7 9 -+ 0 . 3 7
503 8.82 + 0.39
57 59.4
96
Controls
3rd week
R E S U L T S O F T H E D I S S E C T I O N O F F E M A L E M I C E M A T E D W I T H M A L E S G I V E N i.p. I N J E C T I O N S O F G R I S E O F U L V I N
TABLE 5
± 8.1
17.4
34.4 17.0
51 1.50 + 0.21
193 5.68 + 0.41
244 7.18 ± 0.46
294 8.65 ± 0.50
34 47.2
72
1500 mg/kg
+ 6.5
21.9
35.0 13.1
45 1.88 ± 0.28
134 5.58 + 0.48
179 7.45 + 0.56
206 8.58 + 0.60
24 32.0
75
3000 mg/kg
b~ CD
12
8 8 156 30 26 10 27 14
14 98 33 46 26 24 11 13 7 6
d d d d d d d d
7 d
11 7 118 21 44 7 35 12
17 109 31 31 19 29 6 6 3 2
b
d d d d d d d d d d
c
d d d d d d
12 d
12 8 94 22 25 51
a
d d d d d d d d
4 d
10 7 84 29 31 14 40 7 13 100 39 47 17 51 14 5 5 4
b
pl S 9 / m l m i x ; b, $ 9 m i x A x o c l o r 1 2 5 4 : 3 0 0
10
15 18 106 42 31 15
a
c
a
b
1000
0
750
ASSAYS ON SALMONELLA
D o s e s o f g r i s e o f u l v i n (/ag/plate)
a, $9 m i x A r o c l o r 1 2 5 4 : 1 0 0
TA1530 TA1535 TA100 TA98 TA1978 TA1950 TA1538 TA1532 TA1537 TA1975 G46
Strain
RESULTS OF PLATE INCORPORATION
TABLE 6
10 7 157 38 34 27
d d d d d d
a
5 d
7 10 d 82 20 22 12 38 24 d
b
9 76 56 40 22 48 30 17 5 6 12
16 6 165 30 44 19
c
5
16 12 103 38 32 7 51 24
a
250 b
13 97 31 30 14 37 14 14 6 8
22
7 15 134 21 40 16
c
a
7
9 8 77 30 37 10 32 22
100
12 134 35 37 17 35 20 19 1 8
b
c
19
13 11 86 24 32 19
pl S 9 / m l m i x ; c, $ 9 m i x P B : 1 0 0 p l / m l m i x ; d, S i m u l t a n e o u s t o x i c e f f e c t .
d d d d d d d d d d
c
500
TYPHIMURIUM
to
231 TABLE 7 RESULTS OF BACTERIAL FLUCTUATION Strain
$9 m i x
/~g/ml
T E S T S (S.
TYPHIMURIUM)
Number of Expts.
Average number of positive tubes per rack Control
Treated
Significance (P)
TA1538
Arochlor 1254
5 2.5 1
3
24
13.3 19 16.6
N.S. N.S. N.S.
TA98
Arochlor 1254
5 2.5 1
3
25.3
22 32.3 28.3
N.S. N.S. N.S.
meiotic cells (Table 4) and did not affect significantly (chi-square test) the preand post-implantation losses (Table 5). The incidence of pregnancy in females was significantly lower (P < 0.001) after treatment of the males with griseofulvin. This effect can be explained by a great depression in motional, and presumably also sexual, activity for several days after injection. Because griseofulvin is a spindle poison interfering with tubulin polymerization and/or the function of microtubules [8,12,24,33--35] it seemed unlikely that it could produce mutations in prokaryotic cells such as those of S. typhimurium, and that explains our negative findings with this organism (Tables 6--7). It is also probable that the abnormalities in sperm morphology observed by Wyrobek and Bruce [35] in mice given griseofulvin were not due to genetic changes in the genes responsible for spermatogenesis as claimed by the authors. It was also expected that chromosomal aberrations, if produced at all by such a substance, would be only of numerical type. Our observations demonstrate, indeed, that this compound did not produce, in vivo, structural chromosomal aberrations in somatic or in germ cells of the male mouse. Negative results have also been reported by Epstein et al. [5] in the dominant-lethal test with male mice given i.p. injection of griseofulvin (750--3000 mg/kg) and mated during 3--8 weeks. Positive results, however, have been published on plant material and insects. Heymer [12] showed, for instance, that griseofulvin used within the clinical concentration range caused fragmentation and chromosomal bridges in dividing cells of Vicia faba, whereas Bhunya and Manna [4] found an increase in the incidence of structural chromosomal aberrations (gaps, breaks, despiralization) in the spermatocytes of the grasshopper, Spathosternum pasiniferum, fed with griseofulvin mixed with Drosophila culture medium. It is impossible to decide whether these effects reflect variations in sensitivity of the chromosomes from different species, whether they are due to the route of administration or whether they indicate that the active substance is a metabolic conversion product of griseofulvin. From our results and from the data published in the literature we may conclude, however, that acute i.p. doses of griseofulvin do not produce, in vivo, structural chromosomal aberrations in the somatic cells or in the germ cells of the male mouse. Administration of thio-TEPA increased significantly (P < 0.0001) the yield
TABLE 8
T o t a l loss (%) Pre-implantation loss (%) Post-implantation loss (%)
Total Per female
Dead embryos
Total Per female
Live e m b r y o s
Total Per female
Implantations
Total Per female
Corpora lutea
Total %
Total females Pregnant females
Observations
± 9.5
± 6.3
66.7 34.7
32.0
20.0
48 3.0
50 3.1
± 0.43
± 0.44
98 6.12 • 0.62
150 9.37 ± 0.77
16 25.4
63
32.3 12.3
68 1.89 ± 0.23
231 6.42 ± 0.42
299 8.31 ± 0.48
341 9.47 ± 0.51
36 57.1
63
21.5
34.6 13.1
80 1.90 ±
244 5.81 ±
324 7.71 ±
0.21
0.37
0.43
0.46
± 10.3
373 8.88 ±
42 66.7
63
Controls
Controls
Treated
2nd week
1st w e e k
36.4
81.8 45.4
4 4.0 ± 1
2 2 . 0 ± 1.41
6 6 . 0 ± 2.4
11 1 1 . 0 ± 3.3
1 1.6 ± 1.6
63
Treated
20.5
35.6 15.1
86 1.87 ±
269 5.85 ±
355 7.72 ±
0.20
0.36
0.41
0.44
± 10.7
418 9.09 ±
46 73.0
63
Controls
3rd week
± 6.9
18.6
65.9 47.3
31 1.6
57 3.0
88 4.6
± 0.29
± 0.40
± 0.49
167 8.79 ± 0.68
19 30.2
63
Treated
20.9
33.6 12.7
76 1.40 ±
241 6.03 ±
317 7.92 ±
0.22
0.39
0.44
0.48
-+ 1 0 . 0
363 9.08 ±
4O 63.5
63
Controls
4th week
R E S U L T S O F T H E D I S S E C T I O N O F F E M A L E M I C E M A T E D W I T H M A L E S G I V E N i.p. I N J E C T I O N S O F 5 m g T H I O - T E P A P E R k g
± 2.7
0.3
3.4
66.7 63.3
1
10 3.3
-+ 0 . 3
± 1.05
11 3 . 7 -+ 1 . 1 0
30 1 0 . 0 ± 1.82
3 4.8
63
Treated
c~
233 of micronuclei in the erythrocytes and of chromosomal aberrations (gaps, breaks, exchanges) in bone-marrow cells (Tables 1--3). It also induced some (<1%) reciprocal translocations in spermatogonia (Table 4) and was highly effective in the production of dominant lethals (Table 8). 2 and 4 weeks after treatment too few females became pregnant to permit us to draw valid conclusions for this period. These results confirm the numerous similar data published in the literature on the effects of thio-TEPA and related compounds in small mammals [1,18,19,30,32]. Acknowledgement Part of this investigation was supported by grants from the "Fonds de la Recherche Fondamentale Collective". References 1 A d l e r , I.D., G. R a m a r a o a n d S.S. E p s t e i n , In viv0 c y t o g e n e t i c e f f e c t s o f t r i m e t h y l p h o s p h a t e a n d T E P A o n b o n e m a r r o w cells o f m a l e r a t s , M u t a t i o n R e s . , 1 3 ( 1 9 7 1 ) 2 6 3 - - 2 7 3 . 2 A m e s , B . N . , J. 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234
21 M a l a w i s t a , S.E., H. S a t o a n d K . G . B e n s e h , V i n b l a s t i n e a n d g r i s e o f u l v i n r e v e r s i b l y d i s r u p t t h e living spindle, Science, 160 (1968) 770--772. 2 2 M a l a w i s t a , S . E . , H. S a t o a n d W.A. C r e a s e y , D i s s o c i a t i o n o f t h e m i t o t i c s p i n d l e in o o c y t e s e x p o s e d t o g r i s e o f u l v i n a n d v i n b l a s t i n e , E x p t l . Cell R e s . , 9 9 ( 1 9 7 6 ) 1 9 3 - - 1 9 6 . 2 3 M a r g u l i s , L., J . A . N e v i a c k a s a n d S. B a n e r j e e , Cilia r e g e n e r a t i o n in S t e n t o r : I n h i b i t i o n , d e l a y a n d a b n o r m a l i t i e s i n d u c e d b y g r i s e o f u l v i n , J. P r o t o z o o L , 1 6 ( 1 9 6 9 ) 6 6 0 - - 6 6 7 . 2 4 Mir, L., M . L . A u s t r i n , P. L e c o i n t e a n d M. W r i g h t , C o r r e l a t i o n b e t w e e n t h e in vivo e f f e c t s o f s o m e griseofulvin derivatives and their in vitro interactions with mammalian microtubules, FEBS Lett., 88 (1978) 259--263. 2 5 N o r d b e r g , B., C y t o p l a s m i c m i c r o t u b u l e s a n d r a d i o s e g m e n t e d n u c l e i ( R i e d e r cells), E f f e c t s o f o s m o larity, ionic strength, pH, penetrating non-electrolytes, griseofulvin and podophylline derivative, S c a n d . J. H a e m a t o l . , 7 ( 1 9 7 0 ) 3 4 9 - - 3 5 6 . 26 Paget, G.E., and A.L. Walpole, Some cytological effects of griseofulvin, Nature (London), 182 (1958) 1320--1321. 27 P a g e t , G.E., a n d A . L . W a l p o l e , T h e e x p e r i m e n t a l t o x i c o l o g y o f g r i s e o f u l v i n , A r c h . D e r m a t o l . , 81 (1960) 750--757. 2 8 S c h m i d , W., T h e m i c r o n u c l e u s t e s t f o r c y t o g e n e t i c a n a l y s i s , i n : A. H o l l a e n d e r ( E d . ) , C h e m i c a l M u t a ? g e n s , V o l . 4, P l e n u m , N e w Y o r k , 1 9 7 6 , p p . 3 1 - - 5 3 . 2 9 S c h w a r z , J., a n d J . K . L o u t z e n h i s e r , L a b o r a t o r y e x p e r i m e n t s w i t h g r i s e o f u l v i n , J. I n v e s t . D e r m a t o l . , 3 4 (1960) 295--299. 3 0 S r a m , R . J . , a n d Z. Z u d o v a , T h e r e l a t i o n s h i p o f m u t a g e n i c a c t i v i t y a n d t h e c h e m i c a l s t r u c t u r e o f a l k y l a m i n o a z i r i d i n e s in m i c e , F o l i a Biol. ( P r a h a ) , 2 0 ( 1 9 7 4 ) 1 - - 1 3 . 31 S t r a u k a , P., a n d T. N a s e m a n , I n h i b i t i o n o f m i t o s i s i n H e L a cell c u l t u r e , H a u t a r z t , 1 2 ( 1 9 6 1 ) 4 6 8 - 469. 3 2 S u r k h o v a , N . T . , a n d A . M . M a l a s h e n k o , M u t a g e n i c e f f e c t o f t h i o - T E P A in l a b o r a t o r y m i c e , II. C y t o g e n e t i c a n a l y s i s o f d a m a g e s in b o n e m a r r o w cells, G e n e t i k a ( M o s c o w ) , 1 0 ( 1 9 7 4 ) 8 1 - - 8 7 . 3 3 W e b e r , K., J. W e h l a n d a n d W. H e r z a g , G r i s e o f u l v i n i n t e r a c t s w i t h m i c r o t u b u l e s b o t h in vivo a n d i n v i t r o , J. Mol. Biol., 1 0 2 ( 1 9 7 6 ) 8 1 7 - - 8 2 9 . 3 4 W i l s o n , L., J . R . B a m b u r g , S.B. Mizel, L.M. G r i s h a m a n d K.M. C r e s w e l l , I n t e r a c t i o n o f d r u g s i w i t h m i e r o t u b u l e p r o t e i n s , F e d . P r o c . , 3 3 ( 1 9 7 4 ) 1 5 8 - - 1 6 6 ; W.A. C r e a s e y , V i n c a a l k a l o i d s a n d c o l c h i c i n e , Hanb. Exp. Pharmakol., 38 (1975) 670--694. 3 5 W y r o b e k , A . J . , a n d W . R . B r u c e , C h e m i c a l i n d u c t i o n o f s p e r m a b n o r m a l i t i e s in m i c e , P r o c . N a t l . A c a d . Sei. ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 4 4 2 5 - - 4 4 2 9 .
Mutation Research, 68 (1979) 225--234 © Elsevier/North-Holland Biomedical Press
MUTAGENICITY TESTS WITH GRISEOFULVIN
A. L I ~ O N A R D a, F. P O N C E L E T
b, G. G R U T M A N
c, E. C A R B O N E L L E
c and L. F A B R Y
a
a Mammalian Genetics Laboratory, Department ofRadiobiology, C.E.N.-S.C.K., B 2400 Mol (Belgium), b Laboratory of Biotoxicology, Catholic University of Louvain, Pharmacy School, U C L 7369, B 1200 Brussels (Belgium) and c Catholic University of Louvain, B 1348 Louvain-la-Neuve (Belgium) (Received 21 February 1979) (Revision received 5 June 1979) (Accepted 14 June 1979)
Summary Griseofulvin was studied for its ability to induce structural chromosomal aberrations in germ and somatic cells of the male mouse. It was also tested for its capacity to produce his + revertants in Salmonella typhimurium. All tests yielded negative results, whereas highly significant effects were recorded in control assays with thio-TEPA.
Griseofulvin (C17H17C106) is a mould metabolite produced by Penicillium griseofulvum Dierckx and by P. Janczewskii Zal. This antibiotic is widely used for the t r e a t m e n t of human and animal dermatophytic infections. Owing to its specifical localization and concentration in the keratinized cells of the skin, hair and nails, it inhibits the growth of dermatophytes parasitizing these tissues, the infected tissue being removed by desquamation and the growth of new epidermal layers [9]. Over 20 years ago Paget and Walpole [26] discovered that griseofulvin is a spindle poison and causes disorientation of the spindle, scattering of the chromosomes and inhibition of chromosome movement at anaphase in rat cells and in growing roots of Vicia faba. Subsequent observations on the effects of griseofulvin on the spindle microtubules have been published in animals [6,14, 20--23,25,29,31] and in plants [9--13,15,27]. The antimitotic properties of griseofulvin have been studied and discussed by several investigators [8,24,33,34]. It is probable that griseofulvin, like other C-mitotic drugs, inhibits tubulin polymerization but, according to Weber et.al. [33], it is unclear whether griseofulvin acts on tubulin itself or on a tuhulinassociated protein. Because certain results [8,12,35] have led to the suggestion that griseofulvin
226 may also induce structural chromosomal aberrations, the present experiments were performed to study its clastogenic properties in somatic and germ cells of the male mouse and to detect its genetic effects on S. typhimurium. Materials and m e t h o d s Tests with mouse Tests were performed with 12-week-old BALB/c mice. The effect of griseofulvin on bone-marrow chromosomes was studied [17] by analysing 400 metaphases from 4 mice which, 48 h before, had been injected i.p. with griseofulvin (Leo Pharmaceutical Products, Denmark) at 0, 0.5, 1.0, 1.5 or 2.0 g/kg in 0.5 ml aqueous solution. The possible influence of the time interval between treatment and observation was followed by investigating bone marrow 1, 2, 4 or 9 days after i.p. injection of 1.5 g griseofulvin per kg b o d y weight. The production of micronuclei in polychromatic erythrocytes [28] was studied, for the different doses, 30 h after i.p. injection of 8-week-old mice. Griseofulvin is an antibiotic used orally, b u t histological studies of Paget and Walpole [26] have demonstrated that this c o m p o u n d is readily absorbed after intraperitoneal administration. The ability of griseofulvin to induce chromosomal aberrations in male germ cells was studied by the dominant-lethal test [3] in the post-meiotic stages i.e. 1--3 weeks after administration of griseofulvin (1.5 g/kg) and by examination of dividing spermatocytes [16] for the presence of reciprocal translocations induced in the pre-meiotic cells. 6 h after injection, each male was caged with 3 virgin females from the same strain which were replaced after 7 and 14 days. The females were dissected 17 days after mating had started, and pre- and postimplantation losses were determined b y conventional methods. After t w o months, the males were killed for spermatocyte scoring. Untreated animals and mice given thio-TEPA [tris(1-aziridinyl) phosphine sulphide] were utilized as controls. The dose of thio-TEPA, kindly provided by Dr. A.M. Malashenko (Moscow), used for the dominant-lethal test and the examination of bone-marrow chromosomes at different intervals of time was 5 mg/kg and that for dose--response relationship studies was 1.25 to 20 mg/kg. The doses of griseofulvin and thio-TEPA were chosen on the basis of experiments reported in the literature [1,5,18,19,30,32,35]. Tests with S. typhimurium Strains of S. typhimurium TA1530, TA1535, TA100, TA98, TA1978, TA1950, TA1538, TA1532, TA1537, T A 1 9 7 5 and G46 were kindly provided b y Professor B.N. Ames. Animals Adult male Wistar rats (200--250 g) were fed a RAL diet. The following pretreatments were applied to the animals. Aroclor-1254 diluted in corn oil (200 mg/ml) and injected i.p. (500 mg/kg) 5 days before the preparation of the liver fractions. Phenobarbital: 0.1% drinking water given ad libitum during 7 days before the rats were killed.
227
Metabolic activating system The post-mitochondrial fractions ($9) were from 3 pooled rat livers, the homogenate (3 ml of 0.15 M KC1/g wet liver) of which was centrifuged as described [2]. Preparation of $9 mix was made according to Ames et al. [2]. 100 pl (25 mg wet liver)/ml or 300 pl (75 mg wet liver)/ml mix of $9 were utilized (0.5 ml $9 mix/plate). Spot tests and plate-incorporation assays were performed according to Ames and coworkers [2] by using dilutions of griseofulvin in dimethyl sulphoxide (DMSO) (0.1 ml/plate) and with a bacterial inoculum of 2--8 X 107 viable bacteria from an overnight culture in nutrient broth (Difco)/plate. Bacterial fluctuation tests were performed in triplicate by using a modification of the method proposed by Green et al. [7]. Into a tube kept in an icewater bath, were successively introduced: liquid minimal glucose medium (Vogel--Bonnet E medium) supplemented with histidine and biotin (0.005 mM) (4 ml), 2--7 X 107 bacterial cells from an overnight culture in nutrient broth (Difco), substrate diluted in DMSO (0.1 ml), $9 mix (300 pl S9/ml mix) (1 ml). After homogenization, the mixture was distributed into 50 tubes (0.1 ml/tube). The racks of 50 tubes were incubated at 37°C in the dark for 3 h, then 2 ml of histidine-biotin-supplemented liquid minimal glucose medium containing a pH indicator (BCP) (5 ~g/ml) were added to each tube. The incubation of the racks was then carried on at 37°C for a total incubation time of 72 h, after which the Immbers of positive growing tubes (yellow)/rack were counted. Results and discussion
All tests with griseofulvin on somatic cells in vivo (Tables 1--3) yielded negative results, the incidence of erythrocytes with micronuclei as well as of bonemarrow cells carrying chromosomal aberrations being not statistically different from control values. Injection of griseofulvin did not produce reciprocal translocations in the preTABLE 1 C H R O M O S O M A L A N O M A L I E S IN M O U S E B O N E - M A R R O W C E L L S A F T E R D I F F E R E N T i.p. D O S E S OF GRISEOFULVIN AND THIO-TEPA Treatment (mg/kg)
Animals examined
Cells analysed
Cells w i t h c h r o m o s o m a l a n o m a l i e s Total
%
Controls
8
600
6
1.0
Griseofulvin 500 1000 1500 2000
4 4 4 4
400 400 400 400
0 2 1 2
0 0.5 0.25 0.5
Thio-TEPA 1.25 2.50 5 10
4 4 4 4
200 200 200 200
16 61 111 174
8.0 30.5 55.5 87.0
228
TABLE 2 CHROMOSOMAL ANOMALIES IN MOUSE BONE-MARROW CELLS AT DIFFERENT INTERVALS O F T I M E A F T E R i.p. T R E A T M E N T W I T H G R I S E O F U L V I N ( 1 5 0 0 m g ] k g ) O R T H I O - T E P A (5 m g / k g ) Treatment
Interval (days)
Animals examined
Cells analysed
A b n o r m a l cells Total
Control
%
8
600
6
1.0
Griseo fulvin
1 2 4 9
4 4 4 4
400 400 400 400
1 1 1 1
0.25 0.25 0.25 0.25
Thio-TEPA
1 2 4 9
4 4 4 4
200 200 200 150
161 111 19 11
80.50 55.50 9.50 0.73
TABLE 3 RESULTS OF THE MICRONUCLEUS Treatment (mg/kg)
Animals examined
TEST IN THE MOUSE Cells analysed
Cells w i t h m i c r o n u c l e i Total
Controls
10
10 0 0 0
%
36
0.36
Griseofulvin 500 1000 1500 2000
5 5 5 5
5 5 5 5
000 000 000 000
13 13 16 21
0.26 0.26 0.32 0.42
Thio-TEPA 2.5 5 10 20
5 5 5 5
5 5 5 5
000 000 000 000
296 279 247 163
5.92 5.58 4.94 3.26
TABLE 4 RESULTS OF MALE MICE
THE
EXAMINATION
OF
SPERMATOCYTES
I FROM
Treatment
Animals examined
Cells analysed
Anomalies
Control Grise o fulvin (1.5 g / k g ) Thio-TEPA
20 15
2000 1500
0 0
7
1000
3 CIV 1 CIII + I
CONTROL
AND TREATED
136 1.89 + 0.16
33.4 12.3
21.1
Dead embryos Total Per female
T o t a l loss (%) Pre-implantation
loss (%) Post-implantation
loss (%)
428 5.94 + 0.29
Live e m b r y o s Total Per female
21.2
32.4 11.2
70 1.84 ± 0.22
223 5.87 ± 0.39
293 7.71 + 0.45
15.2
32.5 17.3
29 1.32 + 0.24
129 5.86 + 0.52
158 7.18 ± 0.57
17.0
27.4 10.4
106 1.51 + 0.15
452 6.46 + 0.30
558 7.97 + 0.34
623 8.90 + 0.36
70 72.9 + 8.7
564 7.83 + 0.33
191 8.68 + 0.63
22 29.3 + 6.3
Implantations Total Per female
330 8.68 + 0.47
+ 8.6
643 8.93 ~ 0.35
38 52.8
Corpora lutea Total Per female
+ 8.8
72 75.0
96
72
96
Total females
Pregnant females Total %
75
Controls
1500 mg/kg
Controls
3000 mg/kg
2nd week
1st week
Observations
+ 8.1
15.9
23.7 7.8
45 1.32 + 0.20
216 6.35 + 0.43
261 7.68 ± 0.47
283 8.32 ± 0.49
34 47.2
72
1500 mg/kg
+ 7.7
19.7
36.4 16.7
53 1.61 + 0.22
171 5.18 + 0.40
224 6.79 ± 0.45
269 8.15 + 0.50
33 44.0
75
3000 mgfkg
-+ 7.9
16.9
29.8 16.9
85 1.49 ± 0.16
353 6.19 + 0.33
438 6 . 7 9 -+ 0 . 3 7
503 8.82 + 0.39
57 59.4
96
Controls
3rd week
R E S U L T S O F T H E D I S S E C T I O N O F F E M A L E M I C E M A T E D W I T H M A L E S G I V E N i.p. I N J E C T I O N S O F G R I S E O F U L V I N
TABLE 5
± 8.1
17.4
34.4 17.0
51 1.50 + 0.21
193 5.68 + 0.41
244 7.18 ± 0.46
294 8.65 ± 0.50
34 47.2
72
1500 mg/kg
+ 6.5
21.9
35.0 13.1
45 1.88 ± 0.28
134 5.58 + 0.48
179 7.45 + 0.56
206 8.58 + 0.60
24 32.0
75
3000 mg/kg
b~ CD
12
8 8 156 30 26 10 27 14
14 98 33 46 26 24 11 13 7 6
d d d d d d d d
7 d
11 7 118 21 44 7 35 12
17 109 31 31 19 29 6 6 3 2
b
d d d d d d d d d d
c
d d d d d d
12 d
12 8 94 22 25 51
a
d d d d d d d d
4 d
10 7 84 29 31 14 40 7 13 100 39 47 17 51 14 5 5 4
b
pl S 9 / m l m i x ; b, $ 9 m i x A x o c l o r 1 2 5 4 : 3 0 0
10
15 18 106 42 31 15
a
c
a
b
1000
0
750
ASSAYS ON SALMONELLA
D o s e s o f g r i s e o f u l v i n (/ag/plate)
a, $9 m i x A r o c l o r 1 2 5 4 : 1 0 0
TA1530 TA1535 TA100 TA98 TA1978 TA1950 TA1538 TA1532 TA1537 TA1975 G46
Strain
RESULTS OF PLATE INCORPORATION
TABLE 6
10 7 157 38 34 27
d d d d d d
a
5 d
7 10 d 82 20 22 12 38 24 d
b
9 76 56 40 22 48 30 17 5 6 12
16 6 165 30 44 19
c
5
16 12 103 38 32 7 51 24
a
250 b
13 97 31 30 14 37 14 14 6 8
22
7 15 134 21 40 16
c
a
7
9 8 77 30 37 10 32 22
100
12 134 35 37 17 35 20 19 1 8
b
c
19
13 11 86 24 32 19
pl S 9 / m l m i x ; c, $ 9 m i x P B : 1 0 0 p l / m l m i x ; d, S i m u l t a n e o u s t o x i c e f f e c t .
d d d d d d d d d d
c
500
TYPHIMURIUM
to
231 TABLE 7 RESULTS OF BACTERIAL FLUCTUATION Strain
$9 m i x
/~g/ml
T E S T S (S.
TYPHIMURIUM)
Number of Expts.
Average number of positive tubes per rack Control
Treated
Significance (P)
TA1538
Arochlor 1254
5 2.5 1
3
24
13.3 19 16.6
N.S. N.S. N.S.
TA98
Arochlor 1254
5 2.5 1
3
25.3
22 32.3 28.3
N.S. N.S. N.S.
meiotic cells (Table 4) and did not affect significantly (chi-square test) the preand post-implantation losses (Table 5). The incidence of pregnancy in females was significantly lower (P < 0.001) after treatment of the males with griseofulvin. This effect can be explained by a great depression in motional, and presumably also sexual, activity for several days after injection. Because griseofulvin is a spindle poison interfering with tubulin polymerization and/or the function of microtubules [8,12,24,33--35] it seemed unlikely that it could produce mutations in prokaryotic cells such as those of S. typhimurium, and that explains our negative findings with this organism (Tables 6--7). It is also probable that the abnormalities in sperm morphology observed by Wyrobek and Bruce [35] in mice given griseofulvin were not due to genetic changes in the genes responsible for spermatogenesis as claimed by the authors. It was also expected that chromosomal aberrations, if produced at all by such a substance, would be only of numerical type. Our observations demonstrate, indeed, that this compound did not produce, in vivo, structural chromosomal aberrations in somatic or in germ cells of the male mouse. Negative results have also been reported by Epstein et al. [5] in the dominant-lethal test with male mice given i.p. injection of griseofulvin (750--3000 mg/kg) and mated during 3--8 weeks. Positive results, however, have been published on plant material and insects. Heymer [12] showed, for instance, that griseofulvin used within the clinical concentration range caused fragmentation and chromosomal bridges in dividing cells of Vicia faba, whereas Bhunya and Manna [4] found an increase in the incidence of structural chromosomal aberrations (gaps, breaks, despiralization) in the spermatocytes of the grasshopper, Spathosternum pasiniferum, fed with griseofulvin mixed with Drosophila culture medium. It is impossible to decide whether these effects reflect variations in sensitivity of the chromosomes from different species, whether they are due to the route of administration or whether they indicate that the active substance is a metabolic conversion product of griseofulvin. From our results and from the data published in the literature we may conclude, however, that acute i.p. doses of griseofulvin do not produce, in vivo, structural chromosomal aberrations in the somatic cells or in the germ cells of the male mouse. Administration of thio-TEPA increased significantly (P < 0.0001) the yield
TABLE 8
T o t a l loss (%) Pre-implantation loss (%) Post-implantation loss (%)
Total Per female
Dead embryos
Total Per female
Live e m b r y o s
Total Per female
Implantations
Total Per female
Corpora lutea
Total %
Total females Pregnant females
Observations
± 9.5
± 6.3
66.7 34.7
32.0
20.0
48 3.0
50 3.1
± 0.43
± 0.44
98 6.12 • 0.62
150 9.37 ± 0.77
16 25.4
63
32.3 12.3
68 1.89 ± 0.23
231 6.42 ± 0.42
299 8.31 ± 0.48
341 9.47 ± 0.51
36 57.1
63
21.5
34.6 13.1
80 1.90 ±
244 5.81 ±
324 7.71 ±
0.21
0.37
0.43
0.46
± 10.3
373 8.88 ±
42 66.7
63
Controls
Controls
Treated
2nd week
1st w e e k
36.4
81.8 45.4
4 4.0 ± 1
2 2 . 0 ± 1.41
6 6 . 0 ± 2.4
11 1 1 . 0 ± 3.3
1 1.6 ± 1.6
63
Treated
20.5
35.6 15.1
86 1.87 ±
269 5.85 ±
355 7.72 ±
0.20
0.36
0.41
0.44
± 10.7
418 9.09 ±
46 73.0
63
Controls
3rd week
± 6.9
18.6
65.9 47.3
31 1.6
57 3.0
88 4.6
± 0.29
± 0.40
± 0.49
167 8.79 ± 0.68
19 30.2
63
Treated
20.9
33.6 12.7
76 1.40 ±
241 6.03 ±
317 7.92 ±
0.22
0.39
0.44
0.48
-+ 1 0 . 0
363 9.08 ±
4O 63.5
63
Controls
4th week
R E S U L T S O F T H E D I S S E C T I O N O F F E M A L E M I C E M A T E D W I T H M A L E S G I V E N i.p. I N J E C T I O N S O F 5 m g T H I O - T E P A P E R k g
± 2.7
0.3
3.4
66.7 63.3
1
10 3.3
-+ 0 . 3
± 1.05
11 3 . 7 -+ 1 . 1 0
30 1 0 . 0 ± 1.82
3 4.8
63
Treated
c~
233 of micronuclei in the erythrocytes and of chromosomal aberrations (gaps, breaks, exchanges) in bone-marrow cells (Tables 1--3). It also induced some (<1%) reciprocal translocations in spermatogonia (Table 4) and was highly effective in the production of dominant lethals (Table 8). 2 and 4 weeks after treatment too few females became pregnant to permit us to draw valid conclusions for this period. These results confirm the numerous similar data published in the literature on the effects of thio-TEPA and related compounds in small mammals [1,18,19,30,32]. Acknowledgement Part of this investigation was supported by grants from the "Fonds de la Recherche Fondamentale Collective". References 1 A d l e r , I.D., G. R a m a r a o a n d S.S. E p s t e i n , In viv0 c y t o g e n e t i c e f f e c t s o f t r i m e t h y l p h o s p h a t e a n d T E P A o n b o n e m a r r o w cells o f m a l e r a t s , M u t a t i o n R e s . , 1 3 ( 1 9 7 1 ) 2 6 3 - - 2 7 3 . 2 A m e s , B . N . , J. 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