389
Mutation Research, 40 (1976) 389--396 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication POTENTIAL MUTAGENICITY OF CADMIUM IN MAMMALIAN OOCYTES
TAKAMICHI SHIMADA, TOSHIAKI WATANABE and AKIRA ENDO
Department of Hygiene and Preventive Medicine,' Yamagata University School of Medicine, Zao-lida, Yamagata City (Japan) (Received January 30th, 1976} (Revision received April 4th, 1976) (Accepted April 27th, 1976}
Since the observation of toxic effects of cadmium in 1858 [11], the biological effects of cadmium in toxicology and environmental health sciences have been widely studied [6]. Recent studies have shown that cadmium is teratogenic in the mouse [10,13], rat [1,3] and hamster [5], and also possibly mutagenic in h u m a n leukocytes in vivo and in vitro [2,14,15]. So, in view of transmittance of mutations to the progeny, the search for mutagenic effects of cadmium on the germ cells will be of importance. Several investigators have inferred that the ovulation phase of female mammals is most sensitive to chemical agents [8,12], X-rays [17] or acute maternal hypoxia [9] for the induction of chromosome anomalies. We have, therefore, examined cytogenetically the effects of cadmium in vivo on the meiotic process of the ova of the mouse. The procedures used here for the treatment of cadmium and oocyte collection are as follows. Forty-eight hours after application of 5 i.u. of pregnant mare's serum (PMS), ddY mice received 5 i.u. of human chorionic gonadotrophin (HCG) intraperitoneally for stimulated ovulation. Females were given a single subcutaneous injection of 3 mg/kg or 6 mg/kg body weight of CdC12 3 h after the application of HCG and were dissected 12 h after the cadmium treatment. Cytogenetical preparations were made on unfertilized metaphase II oocytes recovered from the Fallopian tubes by the m e t h o d of Tarkowski [16]. These oocytes were treated with hyaluronidase (75 i.u. per ml of Hanks' solution) for 2 or 3 min, then with a hypotonic solution of 0.7% of sodium citrate for 10 min, dropped on microslides, fixed by adding two or three drops of methanol--acetic acid (3 : 1) and air dried. Slides were stained with Giemsa solution. Oocytes with adequately spread meiotic plates in metaphase II were examined in a search for chromosomal anomalies. The liver, kidney and ovary of each cadmium-treated mouse were digested in nitric and perchloric acids. The cadmium concentrations were then analysed by atomic absorption spectrophotometry. Tables I and II summarize toxic and cytogenic effects of cadmium on the fe-
a b c d
ON
25 25 25
Number of females examined
CADMIUM
ddY
MICE
0 7 c 19 c
Number of females with diffuse ovarian haemorrhage
FEMALE
C a d m i u n levels g i v e n as ~ g / g t i s s u e w e t w e i g h t w i t h S.E. Data from pooled ovaries of each group. p < 0.01 c o m p a r e d w i t h c o n t r o l b y F i s h e r ' s e x a c t t e s t . p < 0.02 compared with control by t test.
0•25 0/25 13/38
0 3 6
OF
Dead/ injected
TOXIC
CdCI2 dose (mg/kg)
I
EFFECTS
TABLE
5 5 2 ( 2 2 . 1 +_ 1 . 8 7 ) 437 (17.5 ± 1.70) 391 ('15.6 -+ 1 . 8 1 ) d
Total number of collected oocytes ( M e a n -+ S.E.) 1 0 5 ( 4 . 2 -+ 0 . 6 0 ) 96 ( 3 . 8 ± 0 . 6 0 ) 1 0 7 ( 4 . 3 +- 0 . 7 4 )
( M e a n +- S.E.)
ooeytes
Total number of degenerated
0 27.6 + 0.91 39.1 +- 1 . 6 4
Liver
8.62 ± 0.46 1 3 . 4 +- 0 . 8 4
0
Kidney
Cadmium concentrations a
0 2.5 5.0
Ovary b
Lo c.o O
391 T A B L E II CYTOGENETIC CdCI 2 dose (mg/kg)
0 3 6 a b c d e
EFFECTS
OF CADMIUM ON ddY FEMALE
Number of oocytes
Number of females Total examined
25 25 25
Numerical anomalies a
1 3 5 d
MICE
Blocked meiosis a
0 0 1
Total examined b
199 155 126
Blocked meiosis
Numerical anomalies n -- 1
n + 1
2n
Total
1 3 2
0 1 2
0 O 2
1 4 e 6 e
Females with one or more abnormal oocytes. Meiotic plates in hypoploidies with too much spreading were excluded from the scoring. p = 0.118. p = 0.095. p = 0 . 0 1 5 c o m p a r e d w i t h c o n t r o l s b y Fisher's e x a c t t e s t ( o n e t a i l e d ) .
male mice. The mean numbers of collected oocytes per female in the control group, the 3 mg/kg- and the 6 mg/kg~cadmium-treated groups were 22.1, 17.5 and 15.6 respectively. The difference between the control group and the cadmium-treated group (6-mg/kg) was statistically significant, which indicates the inhibitory effect of cadmium on ovulation. At autopsy, we also observed heavy and diffuse haemorrhage on ovaries of the cadmium-treated mice. However, no difference in the incidence of degenerated oocytes was observed among groups. These oocytes did n o t contribute to cytogenetic examination, because there were no chromosome configurations. Chromosome analysis was made on 75 females consisting in total of 480 oocytes from the cadmium-treated and the control mice. The reduction of analyzable metaphase II in cadmium-treated groups was due to fewer ovulated oocytes. There was a trend to increase in the females with chromsomally abnormal oocytes in cadmium-treated groups as compared with the control group. Chromosome anomalies of hypoploidy, hyperploidy {Fig. l a ) and diploidy (Fig. l b ) were found in ten oocytes from mice treated with cadmium. There was a statistically significant increase of chromosome anomalies in the cadmium group (6 mg/kg) as compared with the control group on the o o c y t e basis. One blocked metaphase I figure consisting of 20 bivalents (Fig. l c ) was observed in a cadmium-treated mouse. The chromosome anomalies observed in this study were mainly numerical, such as hypoploidy (n -- 1), hyperploidy (n + 1) or diploidy (2n). These anomalies originating in oocytes may eventually produce m o n o s o m y (2n -- 1), trisomy (2n + 1) or triploidy (3n) in subsequent progeny after fertilization. As for the blocked metaphase I chromosomes, it is difficult to interpret at this stage whether the o o c y t e with the blocked meiosis I may produce triploidy after fertilization, or only at the relatively prolonged meiotic stage of metaphase I and then followed by meiosis II. Contrary to the findings in human leukocyte culture studies [ 14], structural anomalies of chromosomes such as gaps, breaks or rearrangements were not observed in this experiment. This discrepancy may have arisen because in the present study the ova were exposed to the agent for a relatively short period just between diakinesis/metaphase I and metaphase II in the Graffian follicles and Fallopian tubes. So the cytogenetic effects ob-
392
served here might be caused by the action of cadmium on meiotic apparatus such as spindle fibres and kinetochores rather than on DNA synthesis which would lead to structural anomalies of chromosomes. In view of the relative narrowness of toxic and mutagenic dose ranges in this experiment, our results must be taken as tentative evidence that high doses of cadmium m a y influence meiotic chromosomes of the mouse ova. However, considering the present data along with the previously reported findings of chromosomal mutagenicity in h u m a n l y m p h o c y t e s [ 14] and of the carcinogenicity of cadmium in rat [7], we assume that cadmium has the potentiality of a mutagen in mammalian meiotic chromosomes.
A
Fig. l( a) .
393 As regards the mechanism of mutagenic action of cadmium, it is still speculative. It is well known that cadmium interacts with zinc in metalloenzymes. The preliminary determination of zinc in the liver of 6 female mice at intervals after treatment with cadmium at 6 mg/kg showed a wide fluctuation of its levels. During the first several hours after the treatment, the zinc decreased from its initial level of 27.7 -+ 0.81 pg/g to 22.4 • 0.79 pg/g, then recovered to the initial level within 12 h after injection. Thereafter the level continued to rise up to twice as high as the initial level until 72 h after the treatment (63.4 -+ 1.74 pg/ g). This result suggests that cadmium injection disturbed the dynamic equilibrium of zinc levels in organs. Moreover, the recent observation of the transient change of zinc distribution in the mitotic apparatus during cell division [4] is
Fig. l ( b ) .
394
Fig. 1. C h r o m o s o m e a n o m a l i e s in o o c y t e s f r o m m i c e t r e a t e d w i t h a single s u b c u t a n e o u s i n j e c t i o n o f cadm i u m c h l o r i d e . (a) H y p e r p l o i d m e t a p h a s e II o o c y t e w i t h n = 21 c h r o m o s o m e s . (b) D i p l o i d m e t a p h a s e II o o c y t e . (c) B l o c k e d m e t a p h a s e I o o c y t e c o n s i s t i n g o f 20 b i v a l e n t s .
suggestive of a significant role of zinc in the mitotic or meiotic mechanism. These findings imply that the interference of cadmium with zinc metalloenzymes which participate in the formation of the meiotic apparatus m a y be responsible for the development of aneuploidy and diploidy in the cadmiumtreated oocytes. We propose here that c h r o m o s o m e analysis of metaphase II oocytes may become one of the useful methods for screening of mutagenicity of environmental contaminants in mammalian germ cells in vivo, when sufficient control (negative and positive) data are accumulated and also comparative analysis is made with the effects upon spermatogenesis.
395
References 1 B a r r , M., J r . , T h e t e r a t o g e n i c i t y o f c a d m i u m c h l o r i d e in t w o s t o c k s o f W i s t a r r a t s , T e r a t o l o g y , 7 (1973) 237--242. 2 Bui, T-H., J. L i n d s t e n a n d G . F . N o r d b e r g , C h r o m o s o m e a n a l y s i s o f l y m p h o c y t e s f r o m c a d m i u m w o r k e r s a n d Itai-itai p a t i e n t s , E n v i r o n . Res., 9 ( 1 9 7 5 ) 1 8 7 - - 1 9 5 . 3 C h e r n o f f , N., T e r a t o g e n i c e f f e c t s o f c a d m i u m in r a t s , T e r a t o l o g y , 8 ( 1 9 7 3 ) 2 9 - - 3 2 . 4 F a l c h u k , K . H . , D.W. F a w c e t t a n d B.L. Vallee, R o l e o f z i n c in cell d i v i s i o n o f E u g l e n a gracillis, J. Cell Sci., 1 7 ( 1 9 7 5 ) 5 7 - - 7 8 . 5 F e r m , V . H . a n d S.J. C a r p e n t e r , T e r a t o g e n i c e f f e c t o f c a d m i u m a n d its i n h i b i t i o n b y z i n c , N a t u r e , 2 1 6 (1967) 1123. 6 F r i b e r g , L., M. P i s c a t o r , G . F . N o r d b e r g a n d T. K j e l l s t r S m , C a d m i u m in t h e E n v i r o n m e n t , C R C Press, Ohio, 1974. 7 G u n n , S.A., T.C. G o u l d a n d W . A . D . A n d e r s o n , S p e c i f i c r e s p o n s e o f m e s e n c h y m a l tissue t o c a n c e r i g e n e sis b y c a d m i u m , A r c h . P a t h . , 8 3 ( 1 9 6 7 ) 4 9 3 - - 4 9 9 . 8 H a n s m a n n , I., C h r o m o s o m e a b e r r a t i o n s in m e t a p h a s e II o o c y t e s . S t a g e s e n s i t i v i t y in t h e m o u s e oogenesis to amethopterin and cyclophosphamide, Mutation Res., 22 (1974) 175--191. 9 Ingalls, T . H . , T. S h i m a d a a n d M. Y a m a m o t o , H y p o x i a - i n d u c e d m u t a t i o n in M. a u r a t u s a n d t h e o r i g i n o f species, A r c h . E n v i r o n . H e a l t h , 3 1 ( 1 9 7 6 ) 1 5 3 - - 1 5 9 . 1 0 Ishizu, S., M. M i n a m i , A. S u z u k i a n d M. Y a m a d a , A n e x p e r i m e n t a l s t u d y o n t h e t e r a t o g e n i c e f f e c t o f c a d m i u m , J. T o k y o W o r n . M e d . Coll., 4 2 ( 1 9 7 2 ) 7 4 4 - - 7 5 2 (in J a p a n e s e ) . 11 P r o d a n , L., C a d m i u m p o i s o n i n g . I. T h e h i s t o r y o f c a d m i u m p o i s o n i n g a n d uses o f c a d m i u m , J. I n d u s t r . Hyg., 14 ( 1 9 3 2 ) 1 3 2 - - 1 5 5 . 1 2 R S h r b o r n , G. a n d I. H a n s m a n n , I n d u c e d c h r o m o s o m e a b e r r a t i o n s in u n f e r t i l i z e d o o e y t e s o f m i c e , H u m a n g e n e t i k , 13 ( 1 9 7 1 ) 1 8 4 - - 1 9 8 . 13 S c h r o e d e r , H . A . a n d M. M i t c h e n e r , T o x i c e f f e c t s o f t r a c e e l e m e n t s o n t h e r e p r o d u c t i o n o f m i c e a n d rats, Arch. Environ. Health, 23 (1971) 102--106. 1 4 S h i r a i s h i , Y., H. K u r a h a s h i a n d T . H . Y o s h i d a , C h r o m o s o m a l a b e r r a t i o n s in c u l t u r e d h u m a n l e u c o c y t e s induced by cadmium sulfide, Proc. Japan Acad., 48 (1972) 133--137. 1 5 S h i r a i s h i , Y., C y t o g e n e t i c s t u d i e s in 1 2 p a t i e n t s w i t h Itai-itai disease, H u m a n g e n e t i k , 2 7 ( 1 9 7 5 ) 3 1 - - 4 1 . 1 6 T a r k o w s k i , A . K . , A n a i r - d r y i n g m e t h o d f o r c h r o m o s o m e p r e p a r a t i o n s f r o m m o u s e eggs, C y t o g e n e s i s , 5 (1966) 394---400. 17 U c h i d a ° I.A. a n d C . P . V . Lee, R a d i a t i o n - i n d u c e d n o n d i s j u n c t i o n in m o u s e o o c y t e s , N a t u r e , 2 5 0 ( 1 9 7 4 ) 601--602.
Mutation Research, 40 (1976) 389--396 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication POTENTIAL MUTAGENICITY OF CADMIUM IN MAMMALIAN OOCYTES
TAKAMICHI SHIMADA, TOSHIAKI WATANABE and AKIRA ENDO
Department of Hygiene and Preventive Medicine,' Yamagata University School of Medicine, Zao-lida, Yamagata City (Japan) (Received January 30th, 1976} (Revision received April 4th, 1976) (Accepted April 27th, 1976}
Since the observation of toxic effects of cadmium in 1858 [11], the biological effects of cadmium in toxicology and environmental health sciences have been widely studied [6]. Recent studies have shown that cadmium is teratogenic in the mouse [10,13], rat [1,3] and hamster [5], and also possibly mutagenic in h u m a n leukocytes in vivo and in vitro [2,14,15]. So, in view of transmittance of mutations to the progeny, the search for mutagenic effects of cadmium on the germ cells will be of importance. Several investigators have inferred that the ovulation phase of female mammals is most sensitive to chemical agents [8,12], X-rays [17] or acute maternal hypoxia [9] for the induction of chromosome anomalies. We have, therefore, examined cytogenetically the effects of cadmium in vivo on the meiotic process of the ova of the mouse. The procedures used here for the treatment of cadmium and oocyte collection are as follows. Forty-eight hours after application of 5 i.u. of pregnant mare's serum (PMS), ddY mice received 5 i.u. of human chorionic gonadotrophin (HCG) intraperitoneally for stimulated ovulation. Females were given a single subcutaneous injection of 3 mg/kg or 6 mg/kg body weight of CdC12 3 h after the application of HCG and were dissected 12 h after the cadmium treatment. Cytogenetical preparations were made on unfertilized metaphase II oocytes recovered from the Fallopian tubes by the m e t h o d of Tarkowski [16]. These oocytes were treated with hyaluronidase (75 i.u. per ml of Hanks' solution) for 2 or 3 min, then with a hypotonic solution of 0.7% of sodium citrate for 10 min, dropped on microslides, fixed by adding two or three drops of methanol--acetic acid (3 : 1) and air dried. Slides were stained with Giemsa solution. Oocytes with adequately spread meiotic plates in metaphase II were examined in a search for chromosomal anomalies. The liver, kidney and ovary of each cadmium-treated mouse were digested in nitric and perchloric acids. The cadmium concentrations were then analysed by atomic absorption spectrophotometry. Tables I and II summarize toxic and cytogenic effects of cadmium on the fe-
a b c d
ON
25 25 25
Number of females examined
CADMIUM
ddY
MICE
0 7 c 19 c
Number of females with diffuse ovarian haemorrhage
FEMALE
C a d m i u n levels g i v e n as ~ g / g t i s s u e w e t w e i g h t w i t h S.E. Data from pooled ovaries of each group. p < 0.01 c o m p a r e d w i t h c o n t r o l b y F i s h e r ' s e x a c t t e s t . p < 0.02 compared with control by t test.
0•25 0/25 13/38
0 3 6
OF
Dead/ injected
TOXIC
CdCI2 dose (mg/kg)
I
EFFECTS
TABLE
5 5 2 ( 2 2 . 1 +_ 1 . 8 7 ) 437 (17.5 ± 1.70) 391 ('15.6 -+ 1 . 8 1 ) d
Total number of collected oocytes ( M e a n -+ S.E.) 1 0 5 ( 4 . 2 -+ 0 . 6 0 ) 96 ( 3 . 8 ± 0 . 6 0 ) 1 0 7 ( 4 . 3 +- 0 . 7 4 )
( M e a n +- S.E.)
ooeytes
Total number of degenerated
0 27.6 + 0.91 39.1 +- 1 . 6 4
Liver
8.62 ± 0.46 1 3 . 4 +- 0 . 8 4
0
Kidney
Cadmium concentrations a
0 2.5 5.0
Ovary b
Lo c.o O
391 T A B L E II CYTOGENETIC CdCI 2 dose (mg/kg)
0 3 6 a b c d e
EFFECTS
OF CADMIUM ON ddY FEMALE
Number of oocytes
Number of females Total examined
25 25 25
Numerical anomalies a
1 3 5 d
MICE
Blocked meiosis a
0 0 1
Total examined b
199 155 126
Blocked meiosis
Numerical anomalies n -- 1
n + 1
2n
Total
1 3 2
0 1 2
0 O 2
1 4 e 6 e
Females with one or more abnormal oocytes. Meiotic plates in hypoploidies with too much spreading were excluded from the scoring. p = 0.118. p = 0.095. p = 0 . 0 1 5 c o m p a r e d w i t h c o n t r o l s b y Fisher's e x a c t t e s t ( o n e t a i l e d ) .
male mice. The mean numbers of collected oocytes per female in the control group, the 3 mg/kg- and the 6 mg/kg~cadmium-treated groups were 22.1, 17.5 and 15.6 respectively. The difference between the control group and the cadmium-treated group (6-mg/kg) was statistically significant, which indicates the inhibitory effect of cadmium on ovulation. At autopsy, we also observed heavy and diffuse haemorrhage on ovaries of the cadmium-treated mice. However, no difference in the incidence of degenerated oocytes was observed among groups. These oocytes did n o t contribute to cytogenetic examination, because there were no chromosome configurations. Chromosome analysis was made on 75 females consisting in total of 480 oocytes from the cadmium-treated and the control mice. The reduction of analyzable metaphase II in cadmium-treated groups was due to fewer ovulated oocytes. There was a trend to increase in the females with chromsomally abnormal oocytes in cadmium-treated groups as compared with the control group. Chromosome anomalies of hypoploidy, hyperploidy {Fig. l a ) and diploidy (Fig. l b ) were found in ten oocytes from mice treated with cadmium. There was a statistically significant increase of chromosome anomalies in the cadmium group (6 mg/kg) as compared with the control group on the o o c y t e basis. One blocked metaphase I figure consisting of 20 bivalents (Fig. l c ) was observed in a cadmium-treated mouse. The chromosome anomalies observed in this study were mainly numerical, such as hypoploidy (n -- 1), hyperploidy (n + 1) or diploidy (2n). These anomalies originating in oocytes may eventually produce m o n o s o m y (2n -- 1), trisomy (2n + 1) or triploidy (3n) in subsequent progeny after fertilization. As for the blocked metaphase I chromosomes, it is difficult to interpret at this stage whether the o o c y t e with the blocked meiosis I may produce triploidy after fertilization, or only at the relatively prolonged meiotic stage of metaphase I and then followed by meiosis II. Contrary to the findings in human leukocyte culture studies [ 14], structural anomalies of chromosomes such as gaps, breaks or rearrangements were not observed in this experiment. This discrepancy may have arisen because in the present study the ova were exposed to the agent for a relatively short period just between diakinesis/metaphase I and metaphase II in the Graffian follicles and Fallopian tubes. So the cytogenetic effects ob-
392
served here might be caused by the action of cadmium on meiotic apparatus such as spindle fibres and kinetochores rather than on DNA synthesis which would lead to structural anomalies of chromosomes. In view of the relative narrowness of toxic and mutagenic dose ranges in this experiment, our results must be taken as tentative evidence that high doses of cadmium m a y influence meiotic chromosomes of the mouse ova. However, considering the present data along with the previously reported findings of chromosomal mutagenicity in h u m a n l y m p h o c y t e s [ 14] and of the carcinogenicity of cadmium in rat [7], we assume that cadmium has the potentiality of a mutagen in mammalian meiotic chromosomes.
A
Fig. l( a) .
393 As regards the mechanism of mutagenic action of cadmium, it is still speculative. It is well known that cadmium interacts with zinc in metalloenzymes. The preliminary determination of zinc in the liver of 6 female mice at intervals after treatment with cadmium at 6 mg/kg showed a wide fluctuation of its levels. During the first several hours after the treatment, the zinc decreased from its initial level of 27.7 -+ 0.81 pg/g to 22.4 • 0.79 pg/g, then recovered to the initial level within 12 h after injection. Thereafter the level continued to rise up to twice as high as the initial level until 72 h after the treatment (63.4 -+ 1.74 pg/ g). This result suggests that cadmium injection disturbed the dynamic equilibrium of zinc levels in organs. Moreover, the recent observation of the transient change of zinc distribution in the mitotic apparatus during cell division [4] is
Fig. l ( b ) .
394
Fig. 1. C h r o m o s o m e a n o m a l i e s in o o c y t e s f r o m m i c e t r e a t e d w i t h a single s u b c u t a n e o u s i n j e c t i o n o f cadm i u m c h l o r i d e . (a) H y p e r p l o i d m e t a p h a s e II o o c y t e w i t h n = 21 c h r o m o s o m e s . (b) D i p l o i d m e t a p h a s e II o o c y t e . (c) B l o c k e d m e t a p h a s e I o o c y t e c o n s i s t i n g o f 20 b i v a l e n t s .
suggestive of a significant role of zinc in the mitotic or meiotic mechanism. These findings imply that the interference of cadmium with zinc metalloenzymes which participate in the formation of the meiotic apparatus m a y be responsible for the development of aneuploidy and diploidy in the cadmiumtreated oocytes. We propose here that c h r o m o s o m e analysis of metaphase II oocytes may become one of the useful methods for screening of mutagenicity of environmental contaminants in mammalian germ cells in vivo, when sufficient control (negative and positive) data are accumulated and also comparative analysis is made with the effects upon spermatogenesis.
395
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