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The mutagenicity of azathioprine in mice drosophila melanogaster and neurospora crassa
Mutation Research, 28 (1975) 87~99 © Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands
87
T H E MUTAGENICITY OF A Z A T H I O P R I N E IN MICE, DROSOPHILA M E L A N O G A S T E R AND NEUROSPORA CRASSA
J. M. CLARK School of Biological Sciences, F~inders University of South Australia, Bedford Park (South Australia, 5042) (Received October ist, 1974)
SUMMARY
The chemotherapeutic compound azathioprine was tested for possible mutagenicity in Swiss Albino mice, Drosophila melanogaster and Neurospora crassa. Utilizing the dominant-lethal assay it was found that acute oral doses of azathioprine (2 × 25 mg/kg body weight), induced dominant-lethal mutations in mouse spermatocytes. Chronic oral doses of azathioprine (2 × 25 mg/kg body weight/week for IO weeks) resulted in a greater rate of dominant-lethality. This increase was not permanent, and by week 4 of gamete sampling there was no significant increase in dominant-lethal mutations. Histological sections showed that chronic treatment of male mice with azathioprine caused pyknosis of spermatocyte nuclei and depletion of the spermatid population. Both acute and chronic doses of azathioprine caused a temporary reduction in sperm viability. Oral treatment of male Canton-S D. melanogaster with azathioprine caused an increase in dominant-lethality in broods assumed to correspond to spermatid and spermatocyte stages. Azathioprine also increased the rate of non-disjunction of the X and Y chromosomes, loss of the long arm of the Y chromosome, and loss of the X or Y chromosome in treated male R(r)2, v//B~Yy+D, melanogaster. Since sex-ratio deviation did not occur in progeny from treated rod-X (yv/B,yy+) male D. melanogaster, it was concluded that the observed sex-ratio deviation in the treated ring-X stock was the result of induced ring-X lethality. Azathioprine induced recessive-lethal mutations in the ad-3 region of a N. crassa heterokaryon. In the host-mediated assay using this same heterokaryon and male Swiss Albino mice as host, the mutagenic activity of azathioprine did not appear to be potentiated or detoxified by the host. The results show that azathioprine has a deleterious effect on reproduction in mice and probably induces mutational events in mice, D. melanogaster and N. crassa.
INTRODUCTION
Azathioprine (6-(i-methyl-4-nitro-5-imidazolyl) thiopurine, Imuran ®) is a drug which has been, and still is used extensively for the chemotherapy of a number of condiPresent Address: Institute cf Genetics, University of Glasgow, Glasgow (Scotland).
88
J . M . CI.AI{K
tions. For example, in Australian general hospitals, Io8 kg of azathioprine were consumed in over Io 6 tablets for the year I971-72 (ref. 5). This drug is used as an anti-inflammatory agent and a general imnmnosuppressor in organ transplantation and autoimmune diseases ; as an antineoplastic agent for the treatment of general malignancies and leukemia; and for the treatment of chronic hepatitis, glomerulo-nephritis, rheumatoid arthritis, gout, leprosy and psoriasis 1. The therapeutic dose is usually in the lower region of the range o.5 IOO mg/kg body weight/day 1. In view of the fact that azathioprine and its metabolites can cause reversible inhibition of DNA synthesis ~7and incorporation of nucleotide analogues into DNA '-'°,it is possible that treatment with azathiporine could cause both somatic and germ cell mutation. The available literature on the mutagenicity of azathioprine is conflicting. KROGHJENSEN 15 found an increase in chromosome aberrations in human bone-marrow cells treated i,~ vilro and i,t vivo. Similarly, HAMPEL ct al. la found a substantial increase in chromosome breakage after treatment with both azathioprine and a metabolite, 6-mercaptopurine. In contrast to these findings, GA.XNEReta[. ~2showed that azathioprine did not cause chromosome damage in bone-marrow cells. Chromosome aberrations in lvmphocytes from the same patients were attributed to the long life-span of these cells and the accumulation of cells with chromosomal abnormalities 12.The possibility of cytogenetic inertness of azathioprine was proposed by FRIEDRICH and ZEUTHEN 11,2'', who attributed chromosome damage during azathioprine treatment to uraemia rather than a direct action of the drug. EBERI.E7 also did not find evidence that azathioprine was clastogenic. It appears as if azathioprine only causes a significant increase in chromosome aberrations in vitro, and at levels exceeding normal therapeutic concentrationsl~, '-'~. As a result of the conflicting evidence on the mutagenie status of azathioprine, and since people of reproductive age are being treated with this conlpound, this study was undertaken to examine the potential genetic risks associated with its use. Currently available methods using mice, Drosophila melalzogaster and Ncurospora crassa make it possible to detect a wide spectrum of mutational and clastogenic events in these species, as well as reducing the problenls of sex, age and individual sensitivity which may be difficult to account for in clinical tests. MATERIALS AND METHODS
The azathioprine, originally from Burroughs-Wellcome, was a gift from Professor A. W. Murray, Flinders University, South Australia. The methods used have been described in greater detail by CI.ARK~. (±) Azathioprine i~ mice The effects of azathioprine in mice were observed using the dominant-lethal assay, sperm viability tests, and testis histological sections. Inbred, 8-week-old Swiss Albino mice were used for all mammalian systems. In the dominant-lethal assay, 8-week-old male mice were orally dosed with 2 × 25 mg/kg body weight azathioprine over 2 days (acute administration) or 2 x 25 mg/kg body weight/week for IO weeks (chronic administration). The chronic administration doses were given on the first and fourth day of the week. Each dose of azathioprine was suspended in o.5 ml physiological saline, pH 7.o and administered at IO:OO a.m. Control mice received saline only. After a recovery period of 24 h, the males were
M U T A G E N I C I T Y OF A Z A T H I O P R I N E
89
sequentially mated with groups of untreated virgin, 8-week-old females. Each male was caged with 3 virgin females/week for 6 weeks. Uterine contents of all females were examined 12 days after the mid-week of their caging and presumptive mating. For each brood, the number of pregnant females and the number of dead and live implants/fertilized female were calculated. Sperm viability in four males/brood was also determined by the supravital staining method of ELIASSONANn TREICHL9. Eosin Y (o. 5 % in o. 15 M phosphate buffer, pH 7.4) stained inviable sperm yellowish and viable sperm blue. Percentage sperm viability was calculated on the basis of 200 sperm/male. At the end of chronic treatment with azathioprine for IO weeks, the testes of IO control and IO treated males were removed and immediately weighed. Normal histological procedures were adopted for the examination of the testis. From both groups, 60 transverse sections of seminiferous tubules on coded slides were selected at random from different regions of the testes and photographed. The number of sectioned spermatogonia, spermatocytes and spermatids were counted in those tubules which were sectioned perpendicular to their axis. (2) A zathioprine in Drosophila A description of the stocks and marked chromosomes used can be found in LINDSLEY AND GRELLTM. Azathioprine was fed to the Drosophila by placing a drop of saline containing the suspension on the surface of the usual treacle-meal-agar medium. Ten starved 36-h old male flies were allowed to feed on 5 mg azathioprine per tube. Treated male Canton-S flies were mated sequentially with a brood interval of 3 days to 3-day-old virgin Canton-S females. The proportion of unhatched eggs from inseminated females was used as an indicator of dominant-lethality. Any tubes where all eggs failed to hatch were discarded. The ability of azathioprine to cause partial or total chromosome loss and nondisjunction was determined by treating R(r)2, vfiB ~Yy+ males and mating with 3-day brood intervals to 3-day-old virgin yw afemales. Exceptional progeny, indicating loss of the long arm of the Y chromosome, loss of the X or Y chromosome, non-disjunction or X-Y exchange were scored. The phenotypes of flies resulting from these events are described by BRINK3. The frequency of loss of Y chromosome segments was calculated on the basis of the number of male progeny. The rate of X or Y chromosome loss and nondisjunction were calculated on the basis of total number of progeny. Sex-ratios were recorded in the progeny of treated ring-X (R(~)2, vf/B~Yy+) and rod-X (yv/BsYy+) flies when mated to virgin yw ~females with 3-day brood intervals. All mosaic flies were tested for fertility.
(3) A zathioprine in Neurospora and the host-mediated assay Neurospora heterokaryon 12 (obtained from Dr. F. J. de Serres, Natl. Inst. of Environmental Health Sciences, Research Triangle Park, N,C. 27709. U.S.A.) was used for the host-mediated assay 8in male Swiss Albino mice. The in vitro dose of azathioprine was 12o mg in ioo ml of Hanks' Balanced Salt Solution treating 2. lO 7 conidia with stirring for 24 h at 37 °. Control conidia were kept under the same conditions but without azathioprine. Survival and forward mutation frequency at the ad-3 locus were determined as outlined by CLARK4. Forward mutation frequency at the ad-3 locus and survival of conidia in the intraperitoneal cavity of treated and control mice were also
9°
j . M . CI.ARK
d e t e r m i n e d . T h e i n i t i a l d o s e of a z a t h i o p r i n e (25 m g / k g b o d y w e i g h t ) w a s o r a l l y a d m i n i s t e r e d 3 h b e f o r e 2. IO 7 w a s h e d c o n i d i a w e r e i n j e c t e d in I m l of H a n k s ' B a l a n c e d S a l t S o l u t i o n i n t o t h e i n t r a p e r i t o n e a l c a v i t y , A s i m i l a r d o s e of a z a t h i o p r i n e w a s a d m i n i s t e r e d IO h a f t e r t h e c o n i d i a h a d b e e n i n j e c t e d . A f t e r 24 h, t h e c o n i d i a w e r e r e c o v e r e d a n d t h e mutation frequency was determined*. RESULTS
(±) M o u s e Dominant-lethal
a s s a y . T h e r e s u l t s for tile d o m i n a n t - l e t h a l a s s a y in m i c e a f t e r
a c u t e a n d c h r o n i c t r e a t m e n t w i t h a z a t h i o p r i n e a p p e a r in T a b l e s I a n d I I r e s p e c t i v e l y . I t is a p p a r e n t t h a t b o t h t r e a t m e n t s c a u s e d a r e d u c t i o n in t h e f e r t i l i t y i n d e x ( % m a t i n g s r e s u l t i n g in p r e g n a n c y ) . T h i s r e d u c t i o n , w h i c h w a s g r e a t e s t a f t e r t h e c h r o n i c t r e a t m e n t , c o u l d b e t h e r e s u l t of t w o f a c t o r s a r e d u c t i o n in l i b i d o , or a r e d u c t i o n in p h y s i o l o g i c a l f e r t i l i t y . A t t h i s s t a g e it is n o t p o s s i b l e t o d e c i d e w h i c h is m o r e i m p o r t a n t .
TABLE I R E S U L T S OF T H E D O M I N A N T - L E T H A L
ASSAY AFTER ACUTE TREATMENT
O F M A L E SV¢ISS A L B I N O M I C E
WITH AZATHIOPRINE
T, treated; C, control; 1, implants; LI, live inlplants; DI, dead implants. Fertility index is the percentage rate of pregnancy. Week •
2
3
"t
5
6
Number T of females C
3° 3°
3° 3°
3° 3°
3° 3°
3o 3o
3° 3o
Number pregnant
T C
18 24
I9 23
21 2o
20 22
20
2I
24
23
Fertility T index (o{~) C
60 80
63 77
7° 67
67 73
67 80
7o 77
i51 207
156 195
168 175
158 183
i68 206
174 195
Nunlber of [
T C
I/fenlale
T C
8.39 8.62
8.21 8.48
8.oo 8.75
7.go
8.40
8.2 9
8.32
8.58
8.48
T C T C
16 22 lO.6O lO.63
16 21 lO.26 lO.77
18 ~8 lO.71 lO.29
28 2o 17.72 to.93
42 23 25.00 1 ].16
33 21 18.97 1o.77
Number of DI °o DI
DI/ T femalea C
0.89--0.37 0.92 4_ o.35
o.84~o.39 o.91 -k o.32
0.86±0.34 o.9o 4_ o.33
r.4o:t 0.75 o.91±o.32
2.to:j 0.98 o.96~o.36
1.574-o.81 o.9o--o.35
LI/ female
7.5 o 7.5 °
7.37 7.57
7.I4 7.g5
6.5o 7.41
6.30 7.62
6.72 7..57
o.oo
o.o2
o.o2
3.23
12.33
4.94
z22DI, I
T C
a DI/female are means m s.d.
I n b o t h c a s e s a z a t h i o p r i n e c a u s e d a r e d u c t i o n b e l o w t h e c o n t r o l v a l u e in t h e n u m b e r of i m p l a n t s / f e m a l e . W i t h r e s p e c t t o t o t a l i m p l a n t s a n d live i m p l a n t s , t h e a c u t e a n d c h r o n i c c o n t r o l s w e r e h o m o g e n e o u s (Z5" - - o.o5 a n d o.13 r e s p e c t i v e l y ) . B o t h a c u t e a n d c h r o n i c t r e a t m e n t i n c r e a s e d t h e n u m b e r of d e a d i m p l a n t s / f e m a l e . A f t e r a c u t e
91
MUTAGENICITY OF AZATHIOPRINE TABLE II
RESULTS OF THE DOMINANT-LETHAL ASSAY AFTER CHRONIC TREATMENT OF MALE SWISS ALBINO MICE WITH AZATHIOPRINE T, t r e a t e d ; C, c o n t r o l ; I, i m p l a n t s ; LI, live i m p l a n t s ; D I, d e a d i m p l a n t s . F e r t i l i t y i n d e x is t h e p e r c e n t a g e r a t e of p r e g n a n c y . i
2
3
4
5
6
N u m b e r of T females C
Week
3° 30
3° 3°
3° 3°
3° 3°
3° 3°
3° 3°
Number T pregnantC
16 22
14 24
17 21
19 25
17 22
18 21
Fertility T index(%) C
53 73
47 80
57 7°
63 83
57 73
60 7°
126 189
114 212
139 177
161 221
146 192
155 175
Number of I
T C
I/female
T C
Number
T C
37 21
24 23
27 18
24 23
23 20
23 18
T
29.36 I I.I I
21.05 lO.84
19.42 lO.16
14.9o lO.4O
15.75 lO.41
14.83 lO.28
of DI % DI
C
DI/
T
femalea C LI/ female
)¢22 DI, I
T C
7.87 8.59
8.14 8.83
8.18 8.43
8.47 8.84
8.59 8.73
8.61 8.33
2.3Iii.o2 0.95 4-o.39
1.71-t-o.87 0.96 4- 0.40
1.59±o.81 0.86 4- 0.34
1.26io.59 o.92 ~:o.35
1.35-t-o.55 o.91 4- o.35
1.284-o.5o 0.86 4- o.31
5.56 7.64
6.43 7.87
6-59 7-57
7 .21 7-92
7.24 7.82
7.33 7.47
16.76
6.26
5.46
1.75
2.13
1.57
a D I / f e m a l e a r e m e a n s 4- s.d.
t r e a t m e n t the m a x i m u m sensitivity to the induction of dominant-lethal mutations occurred at week 5 (Z2~ = 12.33 ; t = 5.16; 38 degrees of freedom), which is assumed to represent the sampling of treated spermatocytes 1°. There was no significant increase in the induction of dominant-lethal mutations in post-meiotic stages. After completion of treatment with azathioprine for IO weeks, the m a x i m u m increase in dead implants/female was observed in week I 0C2~ = 16.76; t = 5.74; 28 degrees of freedom). B y week 4 of gamete sampling this increase had fallen and the difference from the control series was not significant. A sample correlation coefficient was calculated for the relationship between dead implants/female and the week of gamete sampling after chronic treatment. A linear regression found r = 0.899, a log transformation followed b y a linear regression found r = 0.902. The difference between the two p r o d u c t - m o m e n t correlation coefficients is small, and hence it is not possible to say whether the decrease in dead implants/female after cessation of chronic t r e a t m e n t is linear or exponential. F r o m the data, it is concluded t h a t azathioprine did not cause a permanent increase in the n u m b e r of dead implants/female. The value " n u m b e r of live implants/female" expresses both the a m o u n t of preimplantation egg loss and dead implantation. This value can be used to calculate the actual rate of dominant lethal mutation induction. E m b r y o s can die before or after implantation as a consequence of one or severallethal hits. The induced post-implantation dominant-lethality can be expressed as:
92
J.M. CLARK •
N u m b e r of living e m b r y o s / a l l i m p l a n t s in t r e a t e d group N u m b e r of living e m b r y o s / a l l i m p l a n t s in control group
a n d i n d u c e d pre- a n d p o s t - i m p l a n t a t i o n d o m i n a n t - l e t h a l i t y as: of living e m b r y o s / f e m a l e in t r e a t e d group
I--Number
N u m b e r of living e m b r y o s / f e m a l e in control group Hence the p r e - i m p l a n t a t i o n egg loss is found to be the difference between the induced pre- a n d p o s t - i m p l a n t a t i o n d o n l i n a n t - l e t h a l i t y and the i n d u c e d p o s t - i m p l a n t a t i o n d o m i n a n t - l e t h a l i t y 19. On the a s s u m p t i o n of a Poisson d i s t r i b u t i o n , the m u t a t i o n r a t e (p) in lethal hits per g a m e t e can be c a l c u l a t e d according to the m e t h o d described b y EDWARDS AND SEARLE s, where: # - - --log~ ( I - - i n d u c e d pre- a n d p o s t - i m p l a n t a t i o n d o m i n a n t lethality) The m a x i m u m p o s t - i m p l a n t a t i o n d o m i n a n t - l e t h a l i t y after acute t r e a t m e n t , occurring at week 5, was o.156. At the same stage, the i n d u c e d p r e - a n d p o s t - i n t p l a n t a t i o n domin a n t - l e t h a l i t y was o.173. Thus the i n d u c e d p r e - i m p l a n t a t i o n egg loss was 1.76°/o . The m a x i m u m true m u t a t i o n r a t e i n d u c e d b y acute t r e a t m e n t w i t h a z a t h i o p r i n e (at week 5) is o.19o lethal h i t s / g a m e t e . The m a x i m u m i n d u c e d p r e - i m p l a n t a t i o n egg loss after chronic t r e a t m e n t was 6.69% at week I. This gave the m a x i m u m true m u t a t i o n r a t e to be o.317 lethal h i t s / g a m e t e . The d a t a show t h a t c o n t i n u e d t r e a t m e n t with a z a t h i o p r i n e is more n m t a g e n i c in the d o m i n a n t - l e t h a l assay t h a n acute t r e a t m e n t . S p e r m z,iabili(v. The effects of acute a n d chronic t r e a t m e n t with a z a t h i o p r i n e on s p e r m v i a b i l i t y are shown in Table I I I . In both cases the s p e r m v i a b i l i t y r e t u r n s to within the control range b y week 5 (t values are found in the table). This r e d u c t i o n in s p e r m v i a b i l i t y could cause a r e d u c t i o n in the a p p a r e n t fertility of the animal. The p a t t e r n of i n d u c e d d o m i n a n t - l e t h a l s e n s i t i v i t y seen in Table I I suggests t h a t the initial decrease in s p e r m v i a b i l i t y is not r e l a t e d to the d o m i n a n t - l e t h a l m u t a t i o n s recovered. E f f e c t on s p e r m a t o g e n e s i s . A t the 5 % level of significance, chronic t r e a t m e n t with a z a t h i o p r i n e caused a r e d u c t i o n in testes weight from o. 1988 ~ o.o226 g for b o t h testes in the control to o x 7 5 4 ! o.o232 g in the t r e a t e d animals (t 2.29; I8 degrees of freedom). Comparison of b o d y - - t e s t e s weight i n d i c a t e d t h a t there was a general loss of weight from 27.36 ! 1.79 g in t h e control to 24.98 ! •.89 g in the t r e a t e d animals (significant at the 1% level; t 2.89; 18 degrees of freedom). The effect of a z a t h i o p r i n e on the m o r p h o l o g y of a section of seminiferous t u b u l e TABLE II[ THE EFFECT ALBINO
MICE
t6 ( P =
o.oi)
OF ACUTE
=
AND CHRONIC
DOSES
OF AZATHIOPRINE
ON SPERM
VIABILITY
IN FOUR
3.71 .
Acute treatment Control t(n-1) Chronic t r e a t m e n t Control t(n--1)
Percentage viability at week • 2 3
4
5
65.2
57-5
61.0
62.2
69.5
7 5 .2
74.7 3.52 53-2 73.0
75 .2 7.26 54.5 74.0 7.31
76.0 6.24 6I .2 71.7 3.49
76'5 5-55 65.2 75.7 5.14
77 .0 2"67 68.2 74 "a 1.95
76"2
7-75
6
0"47 70.2 74-7 1.62
S\VISS
MUTAGENICITY OF AZATHIOPRINE
93
Fig. ia. T y p i c a l histological section of a region of s e m i n i f e r o u s t u b u l e from a m a l e Swiss Albino m o u s e t r e a t e d w i t h chronic levels of azathioprine. N o t e p y k n o s i s of s p e r m a t o c y t e nuclei. Stained b y t h e F e u l g e n r e a c t i o n w i t h light green as c o u n t e r s t a i n , original m a g n i f i c a t i o n × iooo.
Fig. Ib. T y p i c a l histological section of a region of s e m i n i f e r o u s t u b u l e f r o m a n u n t r e a t e d m a l e Swiss Albino mouse. S t a i n e d b y t h e F e u l g e n reaction w i t h light green as c o u n t e r s t a i n , original m a g n i f i c a t i o n × iooo.
94
j.M. CLARK
TABLE IV THE PERCENTAGE OF THE THREE MAIN CELL TYPES IN 4 ° SEMINIFEROUS TUBULE T H R E E S~,VISS A L B I N O M I C E F O L L O W I N G C H R O N I C T R E A T M E N T W I T H A Z A T H I O P R I N E
SECTIONS
FROM
Values are means ± standard deviation.
Control
Treated
Number of cells
% Spermatogonia
% Spermatocytes
ql, Spermatids
IO32t 5823
15-6 ~h o.6 22.6 ~h 7.2
29.9 ± 2.2 45.8 q_ I3.5
54.5 7[ 3.0 31.6 ~: 2o.o
can be seen in Fig. I. T r e a t m e n t caused extreme pyknosis of p r i m a r y spermat,,mytes in m a n y tubules. Since differentiation into spermatozoa appeared unaffected, it is suggested t h a t this pyknosis resulted in a depletion of the spermatid population. In a sample of 4o seminiferous tubules the n u m b e r of cells present was a p p r o x i m a t e l y one half t h a t in the control tubules. This could account for some of the loss in weight. The percentages of the various cell types in a sample of 4o tubules are shown in Table IV. This a t t e m p t to q u a n t i t a t e the effect of azathioprine in the testis, d e m o n s t r a t e s a reduction in cell n u m b e r occurring at the spermatid stage and an increase in the n u m b e r of spermatocytes in the tubules. Many of these spermatocytes p r o b a b l y did not complete the meiotic divisions. The effect of the azathioprine was often highly localized to a section of a tubule. The localization of effects (evident front the large s t a n d a r d deviations) p r o b a b l y accounts for the fact t h a t the fertility of the mice was not reduced as m u c h as would be expected front the decrease in the n u m b e r of spermatids.
(2) Drosophila Dominant-lethal mutations. The effects on egg hatch of oral t r e a t m e n t of C a n t o n - S males with azathioprine are shown in Table V. At the 1% level of significance there was an increase in the proportion of u n h a t c h e d eggs in broods 2, 3 a n d 4. This increase in the proportion of u n h a t c h e d eggs was a t t r i b u t e d to genetic damage in the form of d o m i n a n t lethal m u t a t i o n s . Chromosome breakage, loss and non-disjunction. The effect of azathioprine oll chromosome breakage, loss a n d n o n - d i s j u n c t i o n in male D. melanogaster is seen in Table VI a n d Fig. 2. The m a x i m u m increases in the loss of the X or Y chromosome, loss of the long a r m of the Y chromosome (yL), a n d n o n - d i s j u n c t i o n were significant at the 1% level (Z1-+ 8.08 at brood 4, 8.58 at brood 3 a n d 8.52 at brood 4, respectively). There TABLE \: THE EFFECT OF AZATHIOPRINE TREATMENT OF MALE CANTON-.~ D. OF UNHATCHED EGGS (ll.h.), \VITH 3-DAY BROOD INTERVAL
melanogaster ON
Brood
Control
Treated
Zt ~
t. Total
3675
3473
~!~ u . h .
2. Total ~o u.h. 3. Total ~'o u . h .
4. Total o~ u.h. 5. Total °' o u . h .
13.83 ± 0.57
3824 14.8o }: 0.57 3438 I5.IO
-~: o . 6 I
2980 15.o3 ~ 0.65 2173 13-25 i
0.72
3.08
1 5 . 2 9 ~2 O . 6 1
3627 19.O8 ± 0.65 335° I9-I9
24.27 2o.o8
]c o.68
2993 19.14 ± o.71 2305 1 5 . 8 4 ~ 0"7.5
17-81 5-97
THE PROPORTION
95
MUTAGENICITY OF AZATHIOPRINE TABLE
VI
THE EFFECT OF AZATHIOPRINE TREATMENT OF RING-X ( R ( I ) 2 , R ( I ) 2 , v f / B S y y +) AND R o D - X (yv/-yv/ B s Y y +) MALE D. melanogaster ON PROGENY SEX-RATIO, LOSS OF X OR ~tr CHROMOSOMEN, LOSS OF THE LONG ARM OF THE Y CHROMOSOME ( y L ) , LOSS OF THE SHORT ARM OF THE Y CHROMOSOME ( y s ) , NON-DISJUNCTION AND EXCHANGE WHEN MATED TO WWa FEMALES WITH 3-DAY BROOD INTERVAL Numbers
in p a r e n t h e s e s
Brood
represent mosaic events.
•
2
3
4
Control
4327 3937 o.9o8 8 3 3 7
3541 3539 o.999 b IO
2417 2430 I.OO5b 12(2)
1473 1431 o.97Ia I4a
7629 6583 o.863 28
Ring-X Females Males Male/female sex-ratio L o s s of X o r Y L o s s of y L L o s s of Y s Non-disj unction X-Y Exchange
3
5(I) a
2
2
4
4(1)
1
4
7
4
--
I
7a
9
--
--
2402 229 o 0.954
4957 4789 0.950
Rod-X Females Males Male/female sex-ratio
3723 353 ° 0.948
2971 2868 0-965
3 6 9 5
3547 0.960
a p < o.oi. b p < 0.005.
ring-X 1.0 .o
- 0.9
0.5
04
> o
0.3
y
E
as~
c
g
o.2
Lc
0.1
IF-~
c Brood F i g . 2. T h e e f f e c t o f a z a t h i o p r i n e t r e a t m e n t o f y / R ( I ) 2 , v f / B S y y + m a l e D. melanogaster o n l o s s of the X or Y chromosome (-A--A-) ; loss of the short segment of the Y chromosome, ys (-O--O-), l o s s of t h e l o n g s e g m e n t o f t h e Y c h r o m o s o m e , y L ( - - © - - G - - ) , and non-disjunction of the X and Y c h r o m o s o m e ( - m - - m - ) , w h e n m a t e d t o yw a f e m a l e s . I n a d d i t i o n a r e i n c l u d e d t h e s e x - r a t i o s o f t h e p r o g e n y f r o m t h e a b o v e r i n g - X m a t i n g , a s w e l l a s t h o s e f r o m m a t i n g t r e a t e d r o d - X , y/yv/B s y y + males to yw a females. C refers to control values.
96
.I.M. {;LARK
was little increase in the m a x i m u m rate of loss of the short a r m of tile Y c h r o m o s o m e (Y~), (Zt ~ 2.26 at brood 3). The p e a k rate of non-disjunction in b r o o d 4 implies t h a t this brood represents s a m p l i n g of t r e a t e d s p e r m a t o c y t e s . Only 4 mosaic flies were o b s e r v e d a m o n g s t 95 e x c e p t i o n a l p r o g e n y in the t r e a t e d series. These mosaic i n d i v i d u a l s were s u b s e q u e n t l y found to 1)e fertile. Of ~4 p r o g e n y with p a r t i a l chromosome losses in the t r e a t e d series, 5 were fertile, 2 of which were mosaics. T h u s m a r k e r loss in 3 flies was not a c c o m p a n i e d b y loss of f e r t i l i t y factors. This would i m p l y t h a t a m u t a t i o n a l event involving a smaller segment of chrom(~sonae h a d occurred. The m a x i n m m ring-X m a l e / f e m a l e sex-ratio which occurred in brood 3 (see Table VI) differed significantly from t h a t of its control (ZI2 23.t)o). The rod--X m a l e / female sex-ratio was g r e a t e s t at brm)d 3 b u t did not differ significantly from its control (Zg~ - - o.23). Frnnl this it is inferred t h a t m u c h of tile sex-ratio shift o b s e r v e d in t h e p r o g e n y of t r e a t e d r i n g - X flies can be a t t r i b u t e d to events associated with the r i n g - X chromosome.
(3) Neuro@ora and the hosl-mediatect assay The results for tile h o s t - m e d i a t e d a s s a y a p p e a r in Table VI I. A z a t h i o p r i n e caused a reduction in the n u m b e r of survivors b o t h in vitro a n d in vivo. Using the tables described b y KASTENBAUM AND BOWMANI~ for testing the significance of m u t a t i o n frequencies, a z a t h i o p r i n e was seen to be m u t a g e n i c at the I °/o level of significance in the ad-2 region of N e u r o s p o r a conidia u n d e r b o t h the in vitro a n d in ",,ivo conditions. TABLI: POOLED
V[I RESULTS
AS INDICATOR
AND
OF
FOUR
MALE
REPLICATE
HOST-MEI)IATEI)
SV~rlSS ALBINO
MICE
N u m b e r of conidia
N u m b e r oj survivors
(. xo°)
(. ion)
18.05 I8.72
13.24 25.53
ASSAYS
USING *'\;~. crassa
HETEROKARYON
12
AS HOST
?o survivors
N u m b e r of mutants
Mutants/io ~ survivors
17.86 I3.87
99 74
lo 34
o. 5 {) 2.45
9.26 9.o~
7° 58
5° ~43
It* vitro Control Treated
I n vivo Control Treated
5.4 ° 25 .88
DISCUSSION
As an initial point, it should be n o t e d t h a t when the g r e a t e r m e t a b o l i c r a t e a n d small mass of a mouse are t a k e n into account, the a z a t h i o p r i n e dose was well with.in the range used for the t r e a t m e n t of humans. The d a t a p r e s e n t e d in this p a p e r suggest t h a t a z a t h i o p r i n e is m u t a g e n i c in mice, D. melanogasler a n d N. crassa. The d o m i n a n t - l e t h a l a s s a y in mice showed t h a t s p e r m a t o c y t e s were most sensitive to the i n d u c t i o n of domin a n t - l e t h a l m u t a t i o n s . The pre-meiotic stages did not a p p e a r to be sensitive, a n d perm a n e n t d a m a g e to the s t e m cells did not occur after chronic t r e a t m e n t . Selection a g a i n s t d a m a g e d s p e r m a t o c y t e s a n d s p e r m a t i d s p r o b a b l y resulted in u n d e r e s t i m a t e s of the m a x i m u m rates of d o m i n a n t - l e t h a l i t y . Tile a p p e a r a n c e of sortie s p e r m a t o c y t e
M U T A G E N I C I T Y OF A Z A T H I O P R I N E
97
nuclei in treated seminiferous tubules (see Fig. I) suggests that cell killing effects probably occur. It is not possible to say whether the pyknotic nuclei are related to the dominant-lethality observed in some implanted eggs, but it is likely that they represent one end of a spectrum of cytotoxic effects induced by azathioprine and probably do not differentiate into spermatids. The dominant-lethal assay in mice suggests that azathioprine is mutagenic. Continued administration resulted in greater dominant-lethality than acute adminstration. Since the total doses were not equivalent, the mutagenic effects of chronic and acute treatment are not strictly comparable .However, for initial screening, it was considered more appropriate to select a chronic level which represented continued administration of the acute dose--a situation often encountered in the therapeutic use of azathioprine. The m a x i m u m rate of induction of dominant-lethal mutations of o. 317 lethal hits/gamete was much less than that observed after, for example, treatment with the strong mutagen Trenimon. R/JHRBORN'S data TM show that Trenimon induced 0.978 lethal hits/gamete in mouse spermatids. Although the dose of azathioprine was greater and the mutagenic effects therefore not strictly comparable with that after Trenimon treatment, these figures illustrate that it is possible to induce much greater rates of dominant-lethality with selected compounds. On the basis of comparative induction of lethal hits in sampled gametes, it is concluded that azathioprine is a relatively weak mutagen. It should be noted that a high incidence of dominant-lethality does not prove the mutagenicity of azathioprine, since the effect produced m a y always result in lethality and never in mutation. Treatment of male D. melanogaster with azathioprine produced a different dominant-lethal sensitivity pattern to that observed in mice. Young spermatozoa and spermatids appear to be the most sensitive, whereas in mice the spermatocytes were the most sensitive. Such an effect m a y be due to the reproductive differences between mice and Drosophila. Inactivation of Drosophila sperm m a y lead to the laying of unfertilized eggs, and hence a greater proportion of unhatched eggs, but in mice the dominant-lethality is expressed as a fraction of fertilized eggs. It is possible that the mouse dominant-lethal sensitivity pattern m a y b e similar to that in Drosophila if preimplantation egg loss is considered. Alternative explanations for the difference in sensitivity pattern could lie in differences in the metabolic fate of azathioprine or in the mixing of germinal cell types. Azathioprine was shown to be capable of breaking chromosomes in Drosophila. Breakage was maximal in the spermatid stage. At this stage loss of the long arm of the Y chromosome occurred more frequently than loss of the short arm. Such an apparent difference in trequency of loss probably reflects a greater probability of breaks in the longer chromosome segment. The observation that azathioprine caused loss of Y chromosome markers in 3 out of 5 exceptional flies without loss of fertility, suggests that mutational events involving relatively small segments of chromatin can occur. Translocation of Y markers m a y result in an underestimate of loss, however, it is expected that such events are relatively infrequent. As expected, the rate of chromosome loss in Drosophila was maximal in spermatocytes, where a significant contribution would have been made by non-disjunction. The studies on sex-ratio shift in Drosophila after treatment with azathioprine demonstrate that ring-X chromosomes are in some way more sensitive to the establishment of lethal events than rod-X chromosomes. Although peak chromosome loss occurs
98
J . M . CLARK
in the same brood as m a x i m u m sex-ratio deviation, chromosome loss and tile occurrence of XO males account for only a fraction of the sex-ratio deviation. It has been suggested by BAKER2 that breakage of the ring-X chromosome is the type of primary lesion which causes both ctiromosome loss and dominant-lethality, these being expressed as XO males and sex-ratio shift. If lethality of the ring-X chromosome was responsible for the sex-ratio deviation in brood 3, then 12. 1% of the ring-X chromosomes would need to be lethal. When this lethality is interpreted as breakage it must be concluded t h a t the ring-X chromosome is more susceptible to the establishment of breaks than the Y chromosome, since the rate of breakage of the Y chromosome is o.45% when both y s and yL loss are considered (excluding the possibility of multiple hits). Sucll an interpretation supports the observations by BRINK a using heliotrine, and CLARK~using DDT, of the differing susceptibility of the ring-X and Y chromosomes to breaks and lethals. The data presented does not give any indication as to the mechanism of induction of ring-X lethality by azathioprine, however, breakage of the chromosome with the formation of a large dicentric and subsequent unbalanced gametes could account for much of the lethality. Also, breaks in the ring-X chromosome probably have a reduced chance of restitution to the original form. The host-mediated assay demonstrates t h a t the murine host did not detoxify or potentiate the mutagenic capabilities of azathioprine. The fact t h a t this compound caused recessive-lethal mutations in the a&2 region of Neurospora suggests that it is capable of producing interactions with genetic material resulting in m u t a t i o n at specific loci. These events nlay be the result of a n u m b e r of possibilities such as point mutation, deletion, or small rearrangements. Although the heterokaryon contained sufficient markers for a more detailed s t u d y and characterization of the ad-:,," locus mutants, this was not performed. The conclusion t h a t can be drawn from these separate assay systems is t h a t azathioprine is a mutagenic compound. Its effects include small events resulting in recessive-lethal mutations in Neurospora crassa and the induction of dominant-lethality in Drosophila melanogaster and mice. Although azathioprine has not been shown to cause point mutations in mammals, the mutagenic effects of this drug in mice and Drosophila are sufficient to say that it m a y have genetically deleterious effects on these organisms. The relevance of these data to man is uncertain. The fact t h a t azathioprine is nmtagcnic in such evolutionarv distant species such as mice, Drosophila and Neurospora suggests that it could be nmtagenic in man. Such a finding m a y indicate that caution is required in the use of this drug particularly in patients of reproductive age. ACKNO'WLEDGEMENTS
Tile a u t h o r is most grateful for the guidance given b y Dr. N. G. BRINK and Dr. D. E. A. CATCHESIDE during the course of this work ; and also wishes to t h a n k Dr. F. J. DE SERRES for N. crassa heterokaryon 12, and Professor A. W. MURRAY for the azathioprine. REFERENCES I ANO>-.,Australian Drug Compendiun~, 6th, ed., Seaforth, New South \Vales, I97 I. 2 BAKER,\¥. K., Induced loss of a ring and a telomeric chromosome in Drosophila mdanogaster, Genetics, 42 (I957) 735-748.
MUTAGENICITY OF AZATHIOPRINE
99
3 BRINK, N. G., T h e m u t a g e n i c a c t i v i t y of t h e pyrrolizidine alkaloid heliotrine in Drosophila melanogaster, Mutation Res., 8 (1969) I 3 9 - I 4 6 . 4 CLARK, J. M., T h e m u t a g e n i c i t y of D D T in mice, Drosophila melanogaster a n d Neurospora crassa, Aust. J. Biol. Sci., 27 (I974) 427-44 o. 5 CRAFTER, K., Director, Medical Services, Flinders Univ. of S o u t h Australia, 1973 (personal communication). 6 DE SERRES, F. J., AND H. V. MALLING, M e a s u r e m e n t of recessive lethal d a m a g e over t h e entire g e n o m e a n d a t two specific loci in t h e ad-3 region of a t w o - c o m p o n e n t h e t e r o k a r y o n of Neurospora crassa, in A. HOLLAENDER (Ed.), Chemical Mutagens; Principles and Methods for their Detection, Vol. 2, P l e n u m , N e w York, 1971, pp. 311-342. 7 EBERLE, P., W. HUNSTEIN AND E. PERINGS, C h r o m o s o m e s in p a t i e n t s t r e a t e d w i t h I m u r a n , Humangenetik, 6 (1968) 69-73. 8 EDWARDS, R. G., AND A. G. SEARLE, Genetic r a d i o s e n s i t i v i t y of specific p o s t - d i c t a t e stages in m o u s e oocytes, Genet. Res., 4 (1963) 389-398. 9 ELIASSON, R., AND L. TREICHL, S u p r a v i t a l s t a i n i n g of h u m a n s p e r m a t o z o a , Fertil. Steril., 22 (1971 ) 134-137. io EPSTEIN, S. S., AND G. ROHRBORN, R e c o m m e n d e d p r o c e d u r e s for t e s t i n g genetic h a z a r d s f r o m chemicals, b a s e d on t h e i n d u c t i o n of d o m i n a n t lethal m u t a t i o n s in m a m m a l s , Nature, 230 (1971 ) 459-460. i i FRIEDRICH, U., AND E. ZEUTHEN, C h r o m o s o m e n a b n o r m i t R t e n u n d B e h a n d l u n g m i t I m u r a n (Azathioprin) n a c h N i e r e n t r a n s p l a n t a t i o n e n , Humangenetih, 8 (197 o) 289-294. i2 GANNER, E., J. OSMENT, P. DITTRICH AND H. HUBER, C h r o m o s o m e s in p a t i e n t s t r e a t e d w i t h a z a t h i o p r i n e , Humangenetik, 18 (1973) 231-236. 13 HAMPEL, I{. E., A. LACKNER, G. SCHULZ AND V. BUSSE, C h r o m o s o m e n m u t a t i o n e n d u t c h A z a t h i o p r i n bei m e n s c h l i c h e n L e u k o z y t e n in vitro, Z. Gastroenterol., 9 (1971) 47-51. 14 KASTENBAUM,M. A., AND I42. O. BOWMAN,Tables for d e t e r m i n i n g t h e s t a t i s t i c a l significance of m u t a t i o n frequencies, Mutation Res., 9 (197 o) 527-549. 15 KROGH-JENSEN, M., Effect of a z a t h i o p r i n e on t h e c h r o m o s o m e c o m p l e m e n t of h u m a n bone m a r r o w cells, Intern. J. Cancer, 5 (197 o) 147-15116 LINDSLEY, D. L., AND E. H. GRELL, Genetic v a r i a t i o n of Drosophila melanogaster, Carnegie Inst. Wash. Publ., 627 (1967). 17 MALAMUD, D., E. M. GONZALEZ, H. CHIU AND R. A. MALT, I n h i b i t i o n of cell proliferation b y a z a t h i o p r i n e , Cancer Res., 32 (1972) 1226-1229. 18 OBE, G., Die W i r k u n g y o n 6 - M e r c a p t o p u r i n u n d A z a t h i o p r i n a u f Menschliche C h r o m o s o m e n in vitro, Arzneimittel-Forsch., 21 (1971) 504 505 . 19 I{6HRBORN, G., T h e d o m i n a n t lethals: M e t h o d a n d c y t o g e n e t i c e x a m i n a t i o n of early cleavage stages, in F. VOGEL AND G. R/DHRBORN (Eds.), Chemical Mutagenesis in Mammals and Man, Springer, Berlin, 197 ° , pp. 148-155. 20 SCANNEL, J. t)., AND G. H. HITCHINGS, T h i o g u a n i n e in d e o x y r i b o n u c l e i c acid from t u m o r s of 6 - m e r c a p t o p u r i n e - t r e a t e d mice, Proc. Soc. Exptl. Biol. Med., 122 (1966) 627 629. 21 VAN ZYL, j., AND H. F. WISSMULLER, T h e clastogenic effect of a z a t h i o p r i n e on h u m a n c h r o m o s o m e s in vitro, Humangenetik, 21 (1974) 153-165. 22 ZEUTHEN, E., AND W. FRIEDRICH, C h r o m o s o m e n u n t e r s u c h u n g e n bei K i n d e r n y o n I m u r a n b e h a n d e l t e n Eltern, Humangenetik, 12 (1871) 74-76.
87
T H E MUTAGENICITY OF A Z A T H I O P R I N E IN MICE, DROSOPHILA M E L A N O G A S T E R AND NEUROSPORA CRASSA
J. M. CLARK School of Biological Sciences, F~inders University of South Australia, Bedford Park (South Australia, 5042) (Received October ist, 1974)
SUMMARY
The chemotherapeutic compound azathioprine was tested for possible mutagenicity in Swiss Albino mice, Drosophila melanogaster and Neurospora crassa. Utilizing the dominant-lethal assay it was found that acute oral doses of azathioprine (2 × 25 mg/kg body weight), induced dominant-lethal mutations in mouse spermatocytes. Chronic oral doses of azathioprine (2 × 25 mg/kg body weight/week for IO weeks) resulted in a greater rate of dominant-lethality. This increase was not permanent, and by week 4 of gamete sampling there was no significant increase in dominant-lethal mutations. Histological sections showed that chronic treatment of male mice with azathioprine caused pyknosis of spermatocyte nuclei and depletion of the spermatid population. Both acute and chronic doses of azathioprine caused a temporary reduction in sperm viability. Oral treatment of male Canton-S D. melanogaster with azathioprine caused an increase in dominant-lethality in broods assumed to correspond to spermatid and spermatocyte stages. Azathioprine also increased the rate of non-disjunction of the X and Y chromosomes, loss of the long arm of the Y chromosome, and loss of the X or Y chromosome in treated male R(r)2, v//B~Yy+D, melanogaster. Since sex-ratio deviation did not occur in progeny from treated rod-X (yv/B,yy+) male D. melanogaster, it was concluded that the observed sex-ratio deviation in the treated ring-X stock was the result of induced ring-X lethality. Azathioprine induced recessive-lethal mutations in the ad-3 region of a N. crassa heterokaryon. In the host-mediated assay using this same heterokaryon and male Swiss Albino mice as host, the mutagenic activity of azathioprine did not appear to be potentiated or detoxified by the host. The results show that azathioprine has a deleterious effect on reproduction in mice and probably induces mutational events in mice, D. melanogaster and N. crassa.
INTRODUCTION
Azathioprine (6-(i-methyl-4-nitro-5-imidazolyl) thiopurine, Imuran ®) is a drug which has been, and still is used extensively for the chemotherapy of a number of condiPresent Address: Institute cf Genetics, University of Glasgow, Glasgow (Scotland).
88
J . M . CI.AI{K
tions. For example, in Australian general hospitals, Io8 kg of azathioprine were consumed in over Io 6 tablets for the year I971-72 (ref. 5). This drug is used as an anti-inflammatory agent and a general imnmnosuppressor in organ transplantation and autoimmune diseases ; as an antineoplastic agent for the treatment of general malignancies and leukemia; and for the treatment of chronic hepatitis, glomerulo-nephritis, rheumatoid arthritis, gout, leprosy and psoriasis 1. The therapeutic dose is usually in the lower region of the range o.5 IOO mg/kg body weight/day 1. In view of the fact that azathioprine and its metabolites can cause reversible inhibition of DNA synthesis ~7and incorporation of nucleotide analogues into DNA '-'°,it is possible that treatment with azathiporine could cause both somatic and germ cell mutation. The available literature on the mutagenicity of azathioprine is conflicting. KROGHJENSEN 15 found an increase in chromosome aberrations in human bone-marrow cells treated i,~ vilro and i,t vivo. Similarly, HAMPEL ct al. la found a substantial increase in chromosome breakage after treatment with both azathioprine and a metabolite, 6-mercaptopurine. In contrast to these findings, GA.XNEReta[. ~2showed that azathioprine did not cause chromosome damage in bone-marrow cells. Chromosome aberrations in lvmphocytes from the same patients were attributed to the long life-span of these cells and the accumulation of cells with chromosomal abnormalities 12.The possibility of cytogenetic inertness of azathioprine was proposed by FRIEDRICH and ZEUTHEN 11,2'', who attributed chromosome damage during azathioprine treatment to uraemia rather than a direct action of the drug. EBERI.E7 also did not find evidence that azathioprine was clastogenic. It appears as if azathioprine only causes a significant increase in chromosome aberrations in vitro, and at levels exceeding normal therapeutic concentrationsl~, '-'~. As a result of the conflicting evidence on the mutagenie status of azathioprine, and since people of reproductive age are being treated with this conlpound, this study was undertaken to examine the potential genetic risks associated with its use. Currently available methods using mice, Drosophila melalzogaster and Ncurospora crassa make it possible to detect a wide spectrum of mutational and clastogenic events in these species, as well as reducing the problenls of sex, age and individual sensitivity which may be difficult to account for in clinical tests. MATERIALS AND METHODS
The azathioprine, originally from Burroughs-Wellcome, was a gift from Professor A. W. Murray, Flinders University, South Australia. The methods used have been described in greater detail by CI.ARK~. (±) Azathioprine i~ mice The effects of azathioprine in mice were observed using the dominant-lethal assay, sperm viability tests, and testis histological sections. Inbred, 8-week-old Swiss Albino mice were used for all mammalian systems. In the dominant-lethal assay, 8-week-old male mice were orally dosed with 2 × 25 mg/kg body weight azathioprine over 2 days (acute administration) or 2 x 25 mg/kg body weight/week for IO weeks (chronic administration). The chronic administration doses were given on the first and fourth day of the week. Each dose of azathioprine was suspended in o.5 ml physiological saline, pH 7.o and administered at IO:OO a.m. Control mice received saline only. After a recovery period of 24 h, the males were
M U T A G E N I C I T Y OF A Z A T H I O P R I N E
89
sequentially mated with groups of untreated virgin, 8-week-old females. Each male was caged with 3 virgin females/week for 6 weeks. Uterine contents of all females were examined 12 days after the mid-week of their caging and presumptive mating. For each brood, the number of pregnant females and the number of dead and live implants/fertilized female were calculated. Sperm viability in four males/brood was also determined by the supravital staining method of ELIASSONANn TREICHL9. Eosin Y (o. 5 % in o. 15 M phosphate buffer, pH 7.4) stained inviable sperm yellowish and viable sperm blue. Percentage sperm viability was calculated on the basis of 200 sperm/male. At the end of chronic treatment with azathioprine for IO weeks, the testes of IO control and IO treated males were removed and immediately weighed. Normal histological procedures were adopted for the examination of the testis. From both groups, 60 transverse sections of seminiferous tubules on coded slides were selected at random from different regions of the testes and photographed. The number of sectioned spermatogonia, spermatocytes and spermatids were counted in those tubules which were sectioned perpendicular to their axis. (2) A zathioprine in Drosophila A description of the stocks and marked chromosomes used can be found in LINDSLEY AND GRELLTM. Azathioprine was fed to the Drosophila by placing a drop of saline containing the suspension on the surface of the usual treacle-meal-agar medium. Ten starved 36-h old male flies were allowed to feed on 5 mg azathioprine per tube. Treated male Canton-S flies were mated sequentially with a brood interval of 3 days to 3-day-old virgin Canton-S females. The proportion of unhatched eggs from inseminated females was used as an indicator of dominant-lethality. Any tubes where all eggs failed to hatch were discarded. The ability of azathioprine to cause partial or total chromosome loss and nondisjunction was determined by treating R(r)2, vfiB ~Yy+ males and mating with 3-day brood intervals to 3-day-old virgin yw afemales. Exceptional progeny, indicating loss of the long arm of the Y chromosome, loss of the X or Y chromosome, non-disjunction or X-Y exchange were scored. The phenotypes of flies resulting from these events are described by BRINK3. The frequency of loss of Y chromosome segments was calculated on the basis of the number of male progeny. The rate of X or Y chromosome loss and nondisjunction were calculated on the basis of total number of progeny. Sex-ratios were recorded in the progeny of treated ring-X (R(~)2, vf/B~Yy+) and rod-X (yv/BsYy+) flies when mated to virgin yw ~females with 3-day brood intervals. All mosaic flies were tested for fertility.
(3) A zathioprine in Neurospora and the host-mediated assay Neurospora heterokaryon 12 (obtained from Dr. F. J. de Serres, Natl. Inst. of Environmental Health Sciences, Research Triangle Park, N,C. 27709. U.S.A.) was used for the host-mediated assay 8in male Swiss Albino mice. The in vitro dose of azathioprine was 12o mg in ioo ml of Hanks' Balanced Salt Solution treating 2. lO 7 conidia with stirring for 24 h at 37 °. Control conidia were kept under the same conditions but without azathioprine. Survival and forward mutation frequency at the ad-3 locus were determined as outlined by CLARK4. Forward mutation frequency at the ad-3 locus and survival of conidia in the intraperitoneal cavity of treated and control mice were also
9°
j . M . CI.ARK
d e t e r m i n e d . T h e i n i t i a l d o s e of a z a t h i o p r i n e (25 m g / k g b o d y w e i g h t ) w a s o r a l l y a d m i n i s t e r e d 3 h b e f o r e 2. IO 7 w a s h e d c o n i d i a w e r e i n j e c t e d in I m l of H a n k s ' B a l a n c e d S a l t S o l u t i o n i n t o t h e i n t r a p e r i t o n e a l c a v i t y , A s i m i l a r d o s e of a z a t h i o p r i n e w a s a d m i n i s t e r e d IO h a f t e r t h e c o n i d i a h a d b e e n i n j e c t e d . A f t e r 24 h, t h e c o n i d i a w e r e r e c o v e r e d a n d t h e mutation frequency was determined*. RESULTS
(±) M o u s e Dominant-lethal
a s s a y . T h e r e s u l t s for tile d o m i n a n t - l e t h a l a s s a y in m i c e a f t e r
a c u t e a n d c h r o n i c t r e a t m e n t w i t h a z a t h i o p r i n e a p p e a r in T a b l e s I a n d I I r e s p e c t i v e l y . I t is a p p a r e n t t h a t b o t h t r e a t m e n t s c a u s e d a r e d u c t i o n in t h e f e r t i l i t y i n d e x ( % m a t i n g s r e s u l t i n g in p r e g n a n c y ) . T h i s r e d u c t i o n , w h i c h w a s g r e a t e s t a f t e r t h e c h r o n i c t r e a t m e n t , c o u l d b e t h e r e s u l t of t w o f a c t o r s a r e d u c t i o n in l i b i d o , or a r e d u c t i o n in p h y s i o l o g i c a l f e r t i l i t y . A t t h i s s t a g e it is n o t p o s s i b l e t o d e c i d e w h i c h is m o r e i m p o r t a n t .
TABLE I R E S U L T S OF T H E D O M I N A N T - L E T H A L
ASSAY AFTER ACUTE TREATMENT
O F M A L E SV¢ISS A L B I N O M I C E
WITH AZATHIOPRINE
T, treated; C, control; 1, implants; LI, live inlplants; DI, dead implants. Fertility index is the percentage rate of pregnancy. Week •
2
3
"t
5
6
Number T of females C
3° 3°
3° 3°
3° 3°
3° 3°
3o 3o
3° 3o
Number pregnant
T C
18 24
I9 23
21 2o
20 22
20
2I
24
23
Fertility T index (o{~) C
60 80
63 77
7° 67
67 73
67 80
7o 77
i51 207
156 195
168 175
158 183
i68 206
174 195
Nunlber of [
T C
I/fenlale
T C
8.39 8.62
8.21 8.48
8.oo 8.75
7.go
8.40
8.2 9
8.32
8.58
8.48
T C T C
16 22 lO.6O lO.63
16 21 lO.26 lO.77
18 ~8 lO.71 lO.29
28 2o 17.72 to.93
42 23 25.00 1 ].16
33 21 18.97 1o.77
Number of DI °o DI
DI/ T femalea C
0.89--0.37 0.92 4_ o.35
o.84~o.39 o.91 -k o.32
0.86±0.34 o.9o 4_ o.33
r.4o:t 0.75 o.91±o.32
2.to:j 0.98 o.96~o.36
1.574-o.81 o.9o--o.35
LI/ female
7.5 o 7.5 °
7.37 7.57
7.I4 7.g5
6.5o 7.41
6.30 7.62
6.72 7..57
o.oo
o.o2
o.o2
3.23
12.33
4.94
z22DI, I
T C
a DI/female are means m s.d.
I n b o t h c a s e s a z a t h i o p r i n e c a u s e d a r e d u c t i o n b e l o w t h e c o n t r o l v a l u e in t h e n u m b e r of i m p l a n t s / f e m a l e . W i t h r e s p e c t t o t o t a l i m p l a n t s a n d live i m p l a n t s , t h e a c u t e a n d c h r o n i c c o n t r o l s w e r e h o m o g e n e o u s (Z5" - - o.o5 a n d o.13 r e s p e c t i v e l y ) . B o t h a c u t e a n d c h r o n i c t r e a t m e n t i n c r e a s e d t h e n u m b e r of d e a d i m p l a n t s / f e m a l e . A f t e r a c u t e
91
MUTAGENICITY OF AZATHIOPRINE TABLE II
RESULTS OF THE DOMINANT-LETHAL ASSAY AFTER CHRONIC TREATMENT OF MALE SWISS ALBINO MICE WITH AZATHIOPRINE T, t r e a t e d ; C, c o n t r o l ; I, i m p l a n t s ; LI, live i m p l a n t s ; D I, d e a d i m p l a n t s . F e r t i l i t y i n d e x is t h e p e r c e n t a g e r a t e of p r e g n a n c y . i
2
3
4
5
6
N u m b e r of T females C
Week
3° 30
3° 3°
3° 3°
3° 3°
3° 3°
3° 3°
Number T pregnantC
16 22
14 24
17 21
19 25
17 22
18 21
Fertility T index(%) C
53 73
47 80
57 7°
63 83
57 73
60 7°
126 189
114 212
139 177
161 221
146 192
155 175
Number of I
T C
I/female
T C
Number
T C
37 21
24 23
27 18
24 23
23 20
23 18
T
29.36 I I.I I
21.05 lO.84
19.42 lO.16
14.9o lO.4O
15.75 lO.41
14.83 lO.28
of DI % DI
C
DI/
T
femalea C LI/ female
)¢22 DI, I
T C
7.87 8.59
8.14 8.83
8.18 8.43
8.47 8.84
8.59 8.73
8.61 8.33
2.3Iii.o2 0.95 4-o.39
1.71-t-o.87 0.96 4- 0.40
1.59±o.81 0.86 4- 0.34
1.26io.59 o.92 ~:o.35
1.35-t-o.55 o.91 4- o.35
1.284-o.5o 0.86 4- o.31
5.56 7.64
6.43 7.87
6-59 7-57
7 .21 7-92
7.24 7.82
7.33 7.47
16.76
6.26
5.46
1.75
2.13
1.57
a D I / f e m a l e a r e m e a n s 4- s.d.
t r e a t m e n t the m a x i m u m sensitivity to the induction of dominant-lethal mutations occurred at week 5 (Z2~ = 12.33 ; t = 5.16; 38 degrees of freedom), which is assumed to represent the sampling of treated spermatocytes 1°. There was no significant increase in the induction of dominant-lethal mutations in post-meiotic stages. After completion of treatment with azathioprine for IO weeks, the m a x i m u m increase in dead implants/female was observed in week I 0C2~ = 16.76; t = 5.74; 28 degrees of freedom). B y week 4 of gamete sampling this increase had fallen and the difference from the control series was not significant. A sample correlation coefficient was calculated for the relationship between dead implants/female and the week of gamete sampling after chronic treatment. A linear regression found r = 0.899, a log transformation followed b y a linear regression found r = 0.902. The difference between the two p r o d u c t - m o m e n t correlation coefficients is small, and hence it is not possible to say whether the decrease in dead implants/female after cessation of chronic t r e a t m e n t is linear or exponential. F r o m the data, it is concluded t h a t azathioprine did not cause a permanent increase in the n u m b e r of dead implants/female. The value " n u m b e r of live implants/female" expresses both the a m o u n t of preimplantation egg loss and dead implantation. This value can be used to calculate the actual rate of dominant lethal mutation induction. E m b r y o s can die before or after implantation as a consequence of one or severallethal hits. The induced post-implantation dominant-lethality can be expressed as:
92
J.M. CLARK •
N u m b e r of living e m b r y o s / a l l i m p l a n t s in t r e a t e d group N u m b e r of living e m b r y o s / a l l i m p l a n t s in control group
a n d i n d u c e d pre- a n d p o s t - i m p l a n t a t i o n d o m i n a n t - l e t h a l i t y as: of living e m b r y o s / f e m a l e in t r e a t e d group
I--Number
N u m b e r of living e m b r y o s / f e m a l e in control group Hence the p r e - i m p l a n t a t i o n egg loss is found to be the difference between the induced pre- a n d p o s t - i m p l a n t a t i o n d o n l i n a n t - l e t h a l i t y and the i n d u c e d p o s t - i m p l a n t a t i o n d o m i n a n t - l e t h a l i t y 19. On the a s s u m p t i o n of a Poisson d i s t r i b u t i o n , the m u t a t i o n r a t e (p) in lethal hits per g a m e t e can be c a l c u l a t e d according to the m e t h o d described b y EDWARDS AND SEARLE s, where: # - - --log~ ( I - - i n d u c e d pre- a n d p o s t - i m p l a n t a t i o n d o m i n a n t lethality) The m a x i m u m p o s t - i m p l a n t a t i o n d o m i n a n t - l e t h a l i t y after acute t r e a t m e n t , occurring at week 5, was o.156. At the same stage, the i n d u c e d p r e - a n d p o s t - i n t p l a n t a t i o n domin a n t - l e t h a l i t y was o.173. Thus the i n d u c e d p r e - i m p l a n t a t i o n egg loss was 1.76°/o . The m a x i m u m true m u t a t i o n r a t e i n d u c e d b y acute t r e a t m e n t w i t h a z a t h i o p r i n e (at week 5) is o.19o lethal h i t s / g a m e t e . The m a x i m u m i n d u c e d p r e - i m p l a n t a t i o n egg loss after chronic t r e a t m e n t was 6.69% at week I. This gave the m a x i m u m true m u t a t i o n r a t e to be o.317 lethal h i t s / g a m e t e . The d a t a show t h a t c o n t i n u e d t r e a t m e n t with a z a t h i o p r i n e is more n m t a g e n i c in the d o m i n a n t - l e t h a l assay t h a n acute t r e a t m e n t . S p e r m z,iabili(v. The effects of acute a n d chronic t r e a t m e n t with a z a t h i o p r i n e on s p e r m v i a b i l i t y are shown in Table I I I . In both cases the s p e r m v i a b i l i t y r e t u r n s to within the control range b y week 5 (t values are found in the table). This r e d u c t i o n in s p e r m v i a b i l i t y could cause a r e d u c t i o n in the a p p a r e n t fertility of the animal. The p a t t e r n of i n d u c e d d o m i n a n t - l e t h a l s e n s i t i v i t y seen in Table I I suggests t h a t the initial decrease in s p e r m v i a b i l i t y is not r e l a t e d to the d o m i n a n t - l e t h a l m u t a t i o n s recovered. E f f e c t on s p e r m a t o g e n e s i s . A t the 5 % level of significance, chronic t r e a t m e n t with a z a t h i o p r i n e caused a r e d u c t i o n in testes weight from o. 1988 ~ o.o226 g for b o t h testes in the control to o x 7 5 4 ! o.o232 g in the t r e a t e d animals (t 2.29; I8 degrees of freedom). Comparison of b o d y - - t e s t e s weight i n d i c a t e d t h a t there was a general loss of weight from 27.36 ! 1.79 g in t h e control to 24.98 ! •.89 g in the t r e a t e d animals (significant at the 1% level; t 2.89; 18 degrees of freedom). The effect of a z a t h i o p r i n e on the m o r p h o l o g y of a section of seminiferous t u b u l e TABLE II[ THE EFFECT ALBINO
MICE
t6 ( P =
o.oi)
OF ACUTE
=
AND CHRONIC
DOSES
OF AZATHIOPRINE
ON SPERM
VIABILITY
IN FOUR
3.71 .
Acute treatment Control t(n-1) Chronic t r e a t m e n t Control t(n--1)
Percentage viability at week • 2 3
4
5
65.2
57-5
61.0
62.2
69.5
7 5 .2
74.7 3.52 53-2 73.0
75 .2 7.26 54.5 74.0 7.31
76.0 6.24 6I .2 71.7 3.49
76'5 5-55 65.2 75.7 5.14
77 .0 2"67 68.2 74 "a 1.95
76"2
7-75
6
0"47 70.2 74-7 1.62
S\VISS
MUTAGENICITY OF AZATHIOPRINE
93
Fig. ia. T y p i c a l histological section of a region of s e m i n i f e r o u s t u b u l e from a m a l e Swiss Albino m o u s e t r e a t e d w i t h chronic levels of azathioprine. N o t e p y k n o s i s of s p e r m a t o c y t e nuclei. Stained b y t h e F e u l g e n r e a c t i o n w i t h light green as c o u n t e r s t a i n , original m a g n i f i c a t i o n × iooo.
Fig. Ib. T y p i c a l histological section of a region of s e m i n i f e r o u s t u b u l e f r o m a n u n t r e a t e d m a l e Swiss Albino mouse. S t a i n e d b y t h e F e u l g e n reaction w i t h light green as c o u n t e r s t a i n , original m a g n i f i c a t i o n × iooo.
94
j.M. CLARK
TABLE IV THE PERCENTAGE OF THE THREE MAIN CELL TYPES IN 4 ° SEMINIFEROUS TUBULE T H R E E S~,VISS A L B I N O M I C E F O L L O W I N G C H R O N I C T R E A T M E N T W I T H A Z A T H I O P R I N E
SECTIONS
FROM
Values are means ± standard deviation.
Control
Treated
Number of cells
% Spermatogonia
% Spermatocytes
ql, Spermatids
IO32t 5823
15-6 ~h o.6 22.6 ~h 7.2
29.9 ± 2.2 45.8 q_ I3.5
54.5 7[ 3.0 31.6 ~: 2o.o
can be seen in Fig. I. T r e a t m e n t caused extreme pyknosis of p r i m a r y spermat,,mytes in m a n y tubules. Since differentiation into spermatozoa appeared unaffected, it is suggested t h a t this pyknosis resulted in a depletion of the spermatid population. In a sample of 4o seminiferous tubules the n u m b e r of cells present was a p p r o x i m a t e l y one half t h a t in the control tubules. This could account for some of the loss in weight. The percentages of the various cell types in a sample of 4o tubules are shown in Table IV. This a t t e m p t to q u a n t i t a t e the effect of azathioprine in the testis, d e m o n s t r a t e s a reduction in cell n u m b e r occurring at the spermatid stage and an increase in the n u m b e r of spermatocytes in the tubules. Many of these spermatocytes p r o b a b l y did not complete the meiotic divisions. The effect of the azathioprine was often highly localized to a section of a tubule. The localization of effects (evident front the large s t a n d a r d deviations) p r o b a b l y accounts for the fact t h a t the fertility of the mice was not reduced as m u c h as would be expected front the decrease in the n u m b e r of spermatids.
(2) Drosophila Dominant-lethal mutations. The effects on egg hatch of oral t r e a t m e n t of C a n t o n - S males with azathioprine are shown in Table V. At the 1% level of significance there was an increase in the proportion of u n h a t c h e d eggs in broods 2, 3 a n d 4. This increase in the proportion of u n h a t c h e d eggs was a t t r i b u t e d to genetic damage in the form of d o m i n a n t lethal m u t a t i o n s . Chromosome breakage, loss and non-disjunction. The effect of azathioprine oll chromosome breakage, loss a n d n o n - d i s j u n c t i o n in male D. melanogaster is seen in Table VI a n d Fig. 2. The m a x i m u m increases in the loss of the X or Y chromosome, loss of the long a r m of the Y chromosome (yL), a n d n o n - d i s j u n c t i o n were significant at the 1% level (Z1-+ 8.08 at brood 4, 8.58 at brood 3 a n d 8.52 at brood 4, respectively). There TABLE \: THE EFFECT OF AZATHIOPRINE TREATMENT OF MALE CANTON-.~ D. OF UNHATCHED EGGS (ll.h.), \VITH 3-DAY BROOD INTERVAL
melanogaster ON
Brood
Control
Treated
Zt ~
t. Total
3675
3473
~!~ u . h .
2. Total ~o u.h. 3. Total ~'o u . h .
4. Total o~ u.h. 5. Total °' o u . h .
13.83 ± 0.57
3824 14.8o }: 0.57 3438 I5.IO
-~: o . 6 I
2980 15.o3 ~ 0.65 2173 13-25 i
0.72
3.08
1 5 . 2 9 ~2 O . 6 1
3627 19.O8 ± 0.65 335° I9-I9
24.27 2o.o8
]c o.68
2993 19.14 ± o.71 2305 1 5 . 8 4 ~ 0"7.5
17-81 5-97
THE PROPORTION
95
MUTAGENICITY OF AZATHIOPRINE TABLE
VI
THE EFFECT OF AZATHIOPRINE TREATMENT OF RING-X ( R ( I ) 2 , R ( I ) 2 , v f / B S y y +) AND R o D - X (yv/-yv/ B s Y y +) MALE D. melanogaster ON PROGENY SEX-RATIO, LOSS OF X OR ~tr CHROMOSOMEN, LOSS OF THE LONG ARM OF THE Y CHROMOSOME ( y L ) , LOSS OF THE SHORT ARM OF THE Y CHROMOSOME ( y s ) , NON-DISJUNCTION AND EXCHANGE WHEN MATED TO WWa FEMALES WITH 3-DAY BROOD INTERVAL Numbers
in p a r e n t h e s e s
Brood
represent mosaic events.
•
2
3
4
Control
4327 3937 o.9o8 8 3 3 7
3541 3539 o.999 b IO
2417 2430 I.OO5b 12(2)
1473 1431 o.97Ia I4a
7629 6583 o.863 28
Ring-X Females Males Male/female sex-ratio L o s s of X o r Y L o s s of y L L o s s of Y s Non-disj unction X-Y Exchange
3
5(I) a
2
2
4
4(1)
1
4
7
4
--
I
7a
9
--
--
2402 229 o 0.954
4957 4789 0.950
Rod-X Females Males Male/female sex-ratio
3723 353 ° 0.948
2971 2868 0-965
3 6 9 5
3547 0.960
a p < o.oi. b p < 0.005.
ring-X 1.0 .o
- 0.9
0.5
04
> o
0.3
y
E
as~
c
g
o.2
Lc
0.1
IF-~
c Brood F i g . 2. T h e e f f e c t o f a z a t h i o p r i n e t r e a t m e n t o f y / R ( I ) 2 , v f / B S y y + m a l e D. melanogaster o n l o s s of the X or Y chromosome (-A--A-) ; loss of the short segment of the Y chromosome, ys (-O--O-), l o s s of t h e l o n g s e g m e n t o f t h e Y c h r o m o s o m e , y L ( - - © - - G - - ) , and non-disjunction of the X and Y c h r o m o s o m e ( - m - - m - ) , w h e n m a t e d t o yw a f e m a l e s . I n a d d i t i o n a r e i n c l u d e d t h e s e x - r a t i o s o f t h e p r o g e n y f r o m t h e a b o v e r i n g - X m a t i n g , a s w e l l a s t h o s e f r o m m a t i n g t r e a t e d r o d - X , y/yv/B s y y + males to yw a females. C refers to control values.
96
.I.M. {;LARK
was little increase in the m a x i m u m rate of loss of the short a r m of tile Y c h r o m o s o m e (Y~), (Zt ~ 2.26 at brood 3). The p e a k rate of non-disjunction in b r o o d 4 implies t h a t this brood represents s a m p l i n g of t r e a t e d s p e r m a t o c y t e s . Only 4 mosaic flies were o b s e r v e d a m o n g s t 95 e x c e p t i o n a l p r o g e n y in the t r e a t e d series. These mosaic i n d i v i d u a l s were s u b s e q u e n t l y found to 1)e fertile. Of ~4 p r o g e n y with p a r t i a l chromosome losses in the t r e a t e d series, 5 were fertile, 2 of which were mosaics. T h u s m a r k e r loss in 3 flies was not a c c o m p a n i e d b y loss of f e r t i l i t y factors. This would i m p l y t h a t a m u t a t i o n a l event involving a smaller segment of chrom(~sonae h a d occurred. The m a x i n m m ring-X m a l e / f e m a l e sex-ratio which occurred in brood 3 (see Table VI) differed significantly from t h a t of its control (ZI2 23.t)o). The rod--X m a l e / female sex-ratio was g r e a t e s t at brm)d 3 b u t did not differ significantly from its control (Zg~ - - o.23). Frnnl this it is inferred t h a t m u c h of tile sex-ratio shift o b s e r v e d in t h e p r o g e n y of t r e a t e d r i n g - X flies can be a t t r i b u t e d to events associated with the r i n g - X chromosome.
(3) Neuro@ora and the hosl-mediatect assay The results for tile h o s t - m e d i a t e d a s s a y a p p e a r in Table VI I. A z a t h i o p r i n e caused a reduction in the n u m b e r of survivors b o t h in vitro a n d in vivo. Using the tables described b y KASTENBAUM AND BOWMANI~ for testing the significance of m u t a t i o n frequencies, a z a t h i o p r i n e was seen to be m u t a g e n i c at the I °/o level of significance in the ad-2 region of N e u r o s p o r a conidia u n d e r b o t h the in vitro a n d in ",,ivo conditions. TABLI: POOLED
V[I RESULTS
AS INDICATOR
AND
OF
FOUR
MALE
REPLICATE
HOST-MEI)IATEI)
SV~rlSS ALBINO
MICE
N u m b e r of conidia
N u m b e r oj survivors
(. xo°)
(. ion)
18.05 I8.72
13.24 25.53
ASSAYS
USING *'\;~. crassa
HETEROKARYON
12
AS HOST
?o survivors
N u m b e r of mutants
Mutants/io ~ survivors
17.86 I3.87
99 74
lo 34
o. 5 {) 2.45
9.26 9.o~
7° 58
5° ~43
It* vitro Control Treated
I n vivo Control Treated
5.4 ° 25 .88
DISCUSSION
As an initial point, it should be n o t e d t h a t when the g r e a t e r m e t a b o l i c r a t e a n d small mass of a mouse are t a k e n into account, the a z a t h i o p r i n e dose was well with.in the range used for the t r e a t m e n t of humans. The d a t a p r e s e n t e d in this p a p e r suggest t h a t a z a t h i o p r i n e is m u t a g e n i c in mice, D. melanogasler a n d N. crassa. The d o m i n a n t - l e t h a l a s s a y in mice showed t h a t s p e r m a t o c y t e s were most sensitive to the i n d u c t i o n of domin a n t - l e t h a l m u t a t i o n s . The pre-meiotic stages did not a p p e a r to be sensitive, a n d perm a n e n t d a m a g e to the s t e m cells did not occur after chronic t r e a t m e n t . Selection a g a i n s t d a m a g e d s p e r m a t o c y t e s a n d s p e r m a t i d s p r o b a b l y resulted in u n d e r e s t i m a t e s of the m a x i m u m rates of d o m i n a n t - l e t h a l i t y . Tile a p p e a r a n c e of sortie s p e r m a t o c y t e
M U T A G E N I C I T Y OF A Z A T H I O P R I N E
97
nuclei in treated seminiferous tubules (see Fig. I) suggests that cell killing effects probably occur. It is not possible to say whether the pyknotic nuclei are related to the dominant-lethality observed in some implanted eggs, but it is likely that they represent one end of a spectrum of cytotoxic effects induced by azathioprine and probably do not differentiate into spermatids. The dominant-lethal assay in mice suggests that azathioprine is mutagenic. Continued administration resulted in greater dominant-lethality than acute adminstration. Since the total doses were not equivalent, the mutagenic effects of chronic and acute treatment are not strictly comparable .However, for initial screening, it was considered more appropriate to select a chronic level which represented continued administration of the acute dose--a situation often encountered in the therapeutic use of azathioprine. The m a x i m u m rate of induction of dominant-lethal mutations of o. 317 lethal hits/gamete was much less than that observed after, for example, treatment with the strong mutagen Trenimon. R/JHRBORN'S data TM show that Trenimon induced 0.978 lethal hits/gamete in mouse spermatids. Although the dose of azathioprine was greater and the mutagenic effects therefore not strictly comparable with that after Trenimon treatment, these figures illustrate that it is possible to induce much greater rates of dominant-lethality with selected compounds. On the basis of comparative induction of lethal hits in sampled gametes, it is concluded that azathioprine is a relatively weak mutagen. It should be noted that a high incidence of dominant-lethality does not prove the mutagenicity of azathioprine, since the effect produced m a y always result in lethality and never in mutation. Treatment of male D. melanogaster with azathioprine produced a different dominant-lethal sensitivity pattern to that observed in mice. Young spermatozoa and spermatids appear to be the most sensitive, whereas in mice the spermatocytes were the most sensitive. Such an effect m a y be due to the reproductive differences between mice and Drosophila. Inactivation of Drosophila sperm m a y lead to the laying of unfertilized eggs, and hence a greater proportion of unhatched eggs, but in mice the dominant-lethality is expressed as a fraction of fertilized eggs. It is possible that the mouse dominant-lethal sensitivity pattern m a y b e similar to that in Drosophila if preimplantation egg loss is considered. Alternative explanations for the difference in sensitivity pattern could lie in differences in the metabolic fate of azathioprine or in the mixing of germinal cell types. Azathioprine was shown to be capable of breaking chromosomes in Drosophila. Breakage was maximal in the spermatid stage. At this stage loss of the long arm of the Y chromosome occurred more frequently than loss of the short arm. Such an apparent difference in trequency of loss probably reflects a greater probability of breaks in the longer chromosome segment. The observation that azathioprine caused loss of Y chromosome markers in 3 out of 5 exceptional flies without loss of fertility, suggests that mutational events involving relatively small segments of chromatin can occur. Translocation of Y markers m a y result in an underestimate of loss, however, it is expected that such events are relatively infrequent. As expected, the rate of chromosome loss in Drosophila was maximal in spermatocytes, where a significant contribution would have been made by non-disjunction. The studies on sex-ratio shift in Drosophila after treatment with azathioprine demonstrate that ring-X chromosomes are in some way more sensitive to the establishment of lethal events than rod-X chromosomes. Although peak chromosome loss occurs
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in the same brood as m a x i m u m sex-ratio deviation, chromosome loss and tile occurrence of XO males account for only a fraction of the sex-ratio deviation. It has been suggested by BAKER2 that breakage of the ring-X chromosome is the type of primary lesion which causes both ctiromosome loss and dominant-lethality, these being expressed as XO males and sex-ratio shift. If lethality of the ring-X chromosome was responsible for the sex-ratio deviation in brood 3, then 12. 1% of the ring-X chromosomes would need to be lethal. When this lethality is interpreted as breakage it must be concluded t h a t the ring-X chromosome is more susceptible to the establishment of breaks than the Y chromosome, since the rate of breakage of the Y chromosome is o.45% when both y s and yL loss are considered (excluding the possibility of multiple hits). Sucll an interpretation supports the observations by BRINK a using heliotrine, and CLARK~using DDT, of the differing susceptibility of the ring-X and Y chromosomes to breaks and lethals. The data presented does not give any indication as to the mechanism of induction of ring-X lethality by azathioprine, however, breakage of the chromosome with the formation of a large dicentric and subsequent unbalanced gametes could account for much of the lethality. Also, breaks in the ring-X chromosome probably have a reduced chance of restitution to the original form. The host-mediated assay demonstrates t h a t the murine host did not detoxify or potentiate the mutagenic capabilities of azathioprine. The fact t h a t this compound caused recessive-lethal mutations in the a&2 region of Neurospora suggests that it is capable of producing interactions with genetic material resulting in m u t a t i o n at specific loci. These events nlay be the result of a n u m b e r of possibilities such as point mutation, deletion, or small rearrangements. Although the heterokaryon contained sufficient markers for a more detailed s t u d y and characterization of the ad-:,," locus mutants, this was not performed. The conclusion t h a t can be drawn from these separate assay systems is t h a t azathioprine is a mutagenic compound. Its effects include small events resulting in recessive-lethal mutations in Neurospora crassa and the induction of dominant-lethality in Drosophila melanogaster and mice. Although azathioprine has not been shown to cause point mutations in mammals, the mutagenic effects of this drug in mice and Drosophila are sufficient to say that it m a y have genetically deleterious effects on these organisms. The relevance of these data to man is uncertain. The fact t h a t azathioprine is nmtagcnic in such evolutionarv distant species such as mice, Drosophila and Neurospora suggests that it could be nmtagenic in man. Such a finding m a y indicate that caution is required in the use of this drug particularly in patients of reproductive age. ACKNO'WLEDGEMENTS
Tile a u t h o r is most grateful for the guidance given b y Dr. N. G. BRINK and Dr. D. E. A. CATCHESIDE during the course of this work ; and also wishes to t h a n k Dr. F. J. DE SERRES for N. crassa heterokaryon 12, and Professor A. W. MURRAY for the azathioprine. REFERENCES I ANO>-.,Australian Drug Compendiun~, 6th, ed., Seaforth, New South \Vales, I97 I. 2 BAKER,\¥. K., Induced loss of a ring and a telomeric chromosome in Drosophila mdanogaster, Genetics, 42 (I957) 735-748.
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3 BRINK, N. G., T h e m u t a g e n i c a c t i v i t y of t h e pyrrolizidine alkaloid heliotrine in Drosophila melanogaster, Mutation Res., 8 (1969) I 3 9 - I 4 6 . 4 CLARK, J. M., T h e m u t a g e n i c i t y of D D T in mice, Drosophila melanogaster a n d Neurospora crassa, Aust. J. Biol. Sci., 27 (I974) 427-44 o. 5 CRAFTER, K., Director, Medical Services, Flinders Univ. of S o u t h Australia, 1973 (personal communication). 6 DE SERRES, F. J., AND H. V. MALLING, M e a s u r e m e n t of recessive lethal d a m a g e over t h e entire g e n o m e a n d a t two specific loci in t h e ad-3 region of a t w o - c o m p o n e n t h e t e r o k a r y o n of Neurospora crassa, in A. HOLLAENDER (Ed.), Chemical Mutagens; Principles and Methods for their Detection, Vol. 2, P l e n u m , N e w York, 1971, pp. 311-342. 7 EBERLE, P., W. HUNSTEIN AND E. PERINGS, C h r o m o s o m e s in p a t i e n t s t r e a t e d w i t h I m u r a n , Humangenetik, 6 (1968) 69-73. 8 EDWARDS, R. G., AND A. G. SEARLE, Genetic r a d i o s e n s i t i v i t y of specific p o s t - d i c t a t e stages in m o u s e oocytes, Genet. Res., 4 (1963) 389-398. 9 ELIASSON, R., AND L. TREICHL, S u p r a v i t a l s t a i n i n g of h u m a n s p e r m a t o z o a , Fertil. Steril., 22 (1971 ) 134-137. io EPSTEIN, S. S., AND G. ROHRBORN, R e c o m m e n d e d p r o c e d u r e s for t e s t i n g genetic h a z a r d s f r o m chemicals, b a s e d on t h e i n d u c t i o n of d o m i n a n t lethal m u t a t i o n s in m a m m a l s , Nature, 230 (1971 ) 459-460. i i FRIEDRICH, U., AND E. ZEUTHEN, C h r o m o s o m e n a b n o r m i t R t e n u n d B e h a n d l u n g m i t I m u r a n (Azathioprin) n a c h N i e r e n t r a n s p l a n t a t i o n e n , Humangenetih, 8 (197 o) 289-294. i2 GANNER, E., J. OSMENT, P. DITTRICH AND H. HUBER, C h r o m o s o m e s in p a t i e n t s t r e a t e d w i t h a z a t h i o p r i n e , Humangenetik, 18 (1973) 231-236. 13 HAMPEL, I{. E., A. LACKNER, G. SCHULZ AND V. BUSSE, C h r o m o s o m e n m u t a t i o n e n d u t c h A z a t h i o p r i n bei m e n s c h l i c h e n L e u k o z y t e n in vitro, Z. Gastroenterol., 9 (1971) 47-51. 14 KASTENBAUM,M. A., AND I42. O. BOWMAN,Tables for d e t e r m i n i n g t h e s t a t i s t i c a l significance of m u t a t i o n frequencies, Mutation Res., 9 (197 o) 527-549. 15 KROGH-JENSEN, M., Effect of a z a t h i o p r i n e on t h e c h r o m o s o m e c o m p l e m e n t of h u m a n bone m a r r o w cells, Intern. J. Cancer, 5 (197 o) 147-15116 LINDSLEY, D. L., AND E. H. GRELL, Genetic v a r i a t i o n of Drosophila melanogaster, Carnegie Inst. Wash. Publ., 627 (1967). 17 MALAMUD, D., E. M. GONZALEZ, H. CHIU AND R. A. MALT, I n h i b i t i o n of cell proliferation b y a z a t h i o p r i n e , Cancer Res., 32 (1972) 1226-1229. 18 OBE, G., Die W i r k u n g y o n 6 - M e r c a p t o p u r i n u n d A z a t h i o p r i n a u f Menschliche C h r o m o s o m e n in vitro, Arzneimittel-Forsch., 21 (1971) 504 505 . 19 I{6HRBORN, G., T h e d o m i n a n t lethals: M e t h o d a n d c y t o g e n e t i c e x a m i n a t i o n of early cleavage stages, in F. VOGEL AND G. R/DHRBORN (Eds.), Chemical Mutagenesis in Mammals and Man, Springer, Berlin, 197 ° , pp. 148-155. 20 SCANNEL, J. t)., AND G. H. HITCHINGS, T h i o g u a n i n e in d e o x y r i b o n u c l e i c acid from t u m o r s of 6 - m e r c a p t o p u r i n e - t r e a t e d mice, Proc. Soc. Exptl. Biol. Med., 122 (1966) 627 629. 21 VAN ZYL, j., AND H. F. WISSMULLER, T h e clastogenic effect of a z a t h i o p r i n e on h u m a n c h r o m o s o m e s in vitro, Humangenetik, 21 (1974) 153-165. 22 ZEUTHEN, E., AND W. FRIEDRICH, C h r o m o s o m e n u n t e r s u c h u n g e n bei K i n d e r n y o n I m u r a n b e h a n d e l t e n Eltern, Humangenetik, 12 (1871) 74-76.