Toxicology, 5 (1975) 43--47 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
MUTAGENICITY T E S T S WITH CADMIUM IN THE MOUSE
N. GILLIAVOD and A. LI~ONARD Laboratory of Genetics, Department of Radiobiology C.E.N. --S.C.K., B-2400 Mol (Belgium)
(Received December 24th, 1974) (Accepted March 20th, 1975)
SUMMARY The testicle is k n o w n to be the critical organ in acute exposure of experimental mammals to cadmium. Such t r e a t m e n t results in t e m p o r a r y sterility and, very often, in the appearance of testicular interstitial cell tumors. The present experiments were p e r f o r m e d to determine w het her such deleterious effects on the male reproductive organs involved any genetic hazards for the surviving germ cells. The administration of 1.75 mg/kg cadmium chloride to male mice did n o t increase the d o m i n a n t lethals during the first three weeks after t r e a t m e n t and failed to induce translocation in the F 1 male off-spring. No c h r o m o s o m e rearrangement was observed in the treated males after i.p. injection of 0.5, 1.75 or 3.0 mg/kg cadmium chloride.
INTRODUCTION Although cadmium is n o t t h o u g h t to represent a hazard to man as importan t as lead or m e r c u r y it must nevertheless be considered a dangerous pollutant. The main sources of cadmium pollution are mines and metallurgy, chemical industries, scrap metal t r e a t m e n t , electroplating, superphosphate fertilizers and cadmium pesticides. Several cases of chronic cadmium poisoning have been r e p o r t e d in the literature [9--11,15,27] the Itai-itai disease which was endemic between 1940 and 1965 in the Zinzu River [10] being an e x t r e m e manifestation of such chronic cadmium poisoning. The most c o m m o n effect of cadmium exposure is renal tubular dysfunction, in m any cases indicated only by an increased excretion of proteins, while in more advanced cases there will also be disturbances in the metabolism of bone minerals [ 1 4 , 1 6 - - 1 8 , 2 2 , 2 3 ] . On the other hand [ 1 , 6 , 8 , 1 3 , 1 9 , 2 0 ] , the testicle has been d e m o n s t r a t e d to be the critical organ after acute exposure of experimental mammals, such t r e a t m e n t resulting in t e m p o r a r y sterility due to the destruction of spermatogonial cells. The present experiments were p e r f o r m e d to determine w het her such
43
d e l e t e r i o u s e f f e c t s on t h e m a l e r e p r o d u c t i v e organs involve a n y genetic h a z a r d s f o r t h e surviving g e r m cells. MATERIALS AND METHODS Male m i c e f r o m B A L B / c strain, aged 1 1 - - 1 3 w e e k s , w e r e injected i.p. w i t h 0.5, 1.75 or 3.0 m g / k g o f c a d m i u m chloride. O n e d a y a f t e r t r e a t m e n t each r e c i p i e n t o f 1.75 m g / k g was caged w i t h t h r e e virgin f e m a l e s f r o m t h e s a m e strain. A f t e r 7 and 14 d a y s t h e f e m a l e s w e r e r e p l a c e d b y fresh ones t o c o m p a r e t h e sensitivity o f s p e r m a t o z o a , m a t u r e a n d early s p e r m a t i d s . 10 d a y s later the f e m a l e s w e r e dissected a n d the n u m b e r o f c o r p o r a lutea, live a n d d e a d e m b r y o s c o u n t e d a n d c o m p a r e d w i t h t h o s e f r o m f e m a l e s m a t e d in t h e s a m e m a n n e r w i t h m a l e s w h i c h h a d received saline only. T h e t r e a t e d males w e r e killed t h r e e m o n t h s later a n d their testes e x a m i n e d b y m e a n s o f air-drying t e c h n i q u e o f Evans et al. [4] f o r t h e p r e s e n c e o f t r a n s l o c a t i o n c o n f i g u r a t i o n s [ 1 1 ] . F u r t h e r m o r e , t h e F 1 t r a n s l o c a t i o n test was p e r f o r m e d on 120 F 1 m a l e s sired d u r i n g t h e first t h r e e w e e k s a f t e r t r e a t m e n t b y B A L B / c m a l e m i c e given 1.75 m g / k g o f c a d m i u m chloride and m a t e d w i t h c o n t r o l females. In t h e s p e r m a t o c y t e test 100 cells w e r e a n a l y s e d p e r t r e a t e d m a l e ; in the F 1 t r a n s l o c a t i o n test, 25 p e r F] m a l e . RESULTS AND DISCUSSION T h e results o f t h e d o m i n a n t l e t h a l i t y test are s u m m a r i z e d in T a b l e I. T h e o n l y e f f e c t o b s e r v e d in the f e m a l e s m a t e d w i t h the t r e a t e d m a l e s was a slight b u t n o n - s i g n i f i c a n t decrease (t -- 0.6; 0.6 > P > 0.5) in t h e n u m b e r o f living e m b r y o s as well as a decrease in t h e rate o f p r e g n a n c y d u r i n g t h e t h i r d w e e k a f t e r t r e a t m e n t . E x a m i n a t i o n o f dividing s p e r m a t o c y t e s in t h e t r e a t e d ani-
TABLE I GENERAL RESULTS OF THE DISSECTIONS Treatment
Week Total of the females mating
% of Corpora preglutea per nant female females
Implantation per female
Live embryos per female
Dead embryos per female
Control
1st 2nd 3rd
67 67 77
65 68 57
8.95 9.15 9.22
8.11 8.52 8.09
6.13 6.13 6.09
1.97 2.39 2.00
Cadmium
1st 2nd 3rd
67 67 86
68 70 45
8.89 9.19 8.79
8.06 7.82 7.46
6.15 5.87 5.41
1.91 1.95 2.05
44
TABLE II RESULTS OF CYTOLOGICAL EXAMINATION Test
Treatment (mg/kg)
Spermatocyte test
0 0.5 1.75 3.OO 1.75
F1 translocation test
Animals examined
Cells analysed
Cells with translocation configuration
10 10 10 10
1000 1000 1000 1000
0 0 0 0
120
3000
0
mals or in the F1 male off-spring failed to reveal any c h r o m o s o m e rearrangem e n t such as reciprocal translocation (Table II). Very few experiments have been p e r f o r m e d up to now to determine the genetic hazards of cadmium to mammals. Cadmium chloride added to leukocy te cultures and to fibroblast cultures for 24, 38 or 72 h does n o t induce c h r o m o s o m e aberrations according to Pat t on and Allisson [ 2 1 ] , but Shiraishi et al. [25] observed several c h r o m o s o m e aberrations in cultured human leukocytes after t r e a t m e n t with cadmium sulfide at a final concentration of 6.2 • 10 - 2 pg/ml of culture fluid. These included chromatid and isochromatid breaks, translocations and dicentrics. Positive observations in vivo have been r e p o r t e d by Shiraishi and Yoshida [26] and D e k n u d t and L6onard [2] who observed severe c h r o m o s o m e aberrations such as dicentric and ring c h r o m o s o m e s in peripheral blood l eukocyt es of workers engaged in the m a n u f a c t u r e of cadmium. The fact t hat cadmium produces no genetic effects in male mouse germ cells is th er e f or e rather surprising since acute exposure to this metal has been shown to kill spermatogonia [ 1,6,8,13,19 ] and to induce testicular interstitial cell tumors in mice and rats [ 7 , 2 4 ] . Similar observations have been r ep o r ted with ot her chemicals such as m i t o m y c i n C which is c y t o t o x i c to spermatogonia (resulting in t e m p o r a r y sterility of treated males [ 1 2 ] ) but does n o t p roduce reciprocal translocations in premeiotic male germ cells [5] while having only a slight d o m i n a n t lethal effect in spermatocytes [ 3 ] . This observation demonstrates once more that the action of chemicals on mammalian reproductive organs differs markedly from t hat of ionizing radiation. ACKNOWLEDGEMENTS This work has been p e r f o r m e d with grant from the " F o n d s National de la Recherche Scientifique Medieale".
45
REFERENCES 1 M. Allanson and R. Deanesly, Observation on cadmium damage and repair in rat testes and the effects on the pituitary gonadotrophs, J. Endocrinol., 24 (1962) 453. 2 Gh. DeKnudt and A. L~onard, Cytogenetic investigations on leucocytes of workers occupationally exposed to cadmium, Environ. Physiol., (in press). 3 U.H. Ehling, Comparison of radiation and chemically induced dominant lethal mutations in male mice, Mutation Res., 11 (1971) 35. 4 E.P. Evans, G. Breckon and C.E. Ford, An air-drying method for meiotic preparations from mammalian testes, Cytogenetics, 3 (1964) 289. 5 N. Gilliavod and A. L6onard, Test for mutagenic effects of chemicals in mice, I. Effects of mitomycin C on spermatogonia, Mutation Res., 13 (1971) 274. 6 S.A. Gunn, T.C. Gould and W.A.D. Anderson, The selective injurious response of testicular and epidimyal blood vessel to cadmium and its prevention by zinc, Amer. J. Pathol., 42 (1963a) 685. 7 S.A. Gunn, T.C. Gould and W.A.D. Anderson, Cadmium induced interstitial cell tumors in rats and mice and their prevention by zinc, J. Natl. Cancer Inst., 31 (1963b) 745. 8 A.B. Kar and R.P. Das, Testicular changes in rats after treatment with cadmium chloride, Acta Biol. Med. Ger., 5 (1960) 153. 9 L. Friberg, Health hazards in the manufacture of alkaline accumulators with special reference to chronic cadmium poisoning, Acta Med. Scand., 138 (1950) Suppl. 240. 10 M. Kohno, H. Sugihara, A. Kanai, Y. Someya, M. Tomotaki, M. Tsuruta, H. Atoba, T. Takahoshi and N. Hagino, Surveys on so-called Itai-itai disease, Juzen Igakukai Zasshi, 57 (1955) 2071. 11 A. L~onard, Observations on meiotic chromosomes of the male mouse as a test of the potential mutagenicity of chemical in mammals, in A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 3, Plenum, New York, 1973, pp. 21--56. 12 A. L6onard and N. Gilliavod, Testicular changes in mice after treatment with mitomycin C, Toxicology, 1 (1973) 217. 13 E.S. Meck, Cellular changes induced by cadmium in mouse testis and liver, Brit. J. Exp. Pathol., 40 (1959) 503. 14 I. Murata, Chronic entero-osteo-nephropathy cadmium, Nippon Ishikai Zasshi, 65 (1971) 15. 15 P. Nicaud, A. Lafitte and A. Gross, Les troubles de l'intoxication chronique par le cadmium, Arch. Mal. Prof., 4 (1942) 192. 16 K. Nomiyama and Y. Sugata, Urinary low-molecular-weight proteins in Itai-Itai disease, Environ. Res., 6 (1973) 373. 17 G.F. Nordberg, Cadmium metabolism and toxicity, Environ. Phys. Biochem., 2 (1972)7. 18 G.F. Nordberg and M. Piscator, Influence of long-term cadmium exposure on urinary excretion of protein and cadmium in mice, Environ. Physiol. Biochem., 2 (1972) 37. 19 J. Parizek, The destructive effect of cadmium ion on testicular tissue and its prevention by zinc, J. Endocrinol., 15 (1957) 56. 20 J. Parizek and Z. Zahor, Effect of cadmium salts on testicular tissue, Nature, 177 (1956) 1036. 21 G.R. Patton and A.C. Allison, Chromosome damage in human cell cultures induced by metal salts, Mutation Res., 16 (1972) 332. 22 M. Piscator, Proteinuria in chronic cadmium poisoning, IV. Gel filtration and ion exchange chromatography of urinary proteins from cadmium workers, Arch. Environ. HeaJth, 12 (1955) 345. 23 Research Committee, Report on surveys on so-called Itai-Itai disease, Research Committee on Itai-Itai disease, Kanazawa, 1967.
46
24 F.J.C. Roe, C.E. Dukes and K.H. Cameron, Cadmium neoplasia: testicular atrophy and leydig cell hyperplasia and neoplasia in rats and mice following the subcutaneous injection of cadmium salts, Brit. J. Cancer, 18 (1964) 674. 25 Y. Shiraishi, H. Kurahashi and T.W. Yoshida, Chromosomal aberrations in cultured human leucocytes induced by cadmium sulfide, Proc. Jap. Acad., 48 (1972) 133, 26 Y. Shiraishi and T.H. Yoshida, Chromosomal abnormalities in cultured leucocyte cells from Itai Itai disease patients, Proc. Jap. Acad., 48 (1972) 248. 27 G.A. Stephens, Cadmium poisoning, J. Industr. Hyg., 2 (1920) 129.
47
MUTAGENICITY T E S T S WITH CADMIUM IN THE MOUSE
N. GILLIAVOD and A. LI~ONARD Laboratory of Genetics, Department of Radiobiology C.E.N. --S.C.K., B-2400 Mol (Belgium)
(Received December 24th, 1974) (Accepted March 20th, 1975)
SUMMARY The testicle is k n o w n to be the critical organ in acute exposure of experimental mammals to cadmium. Such t r e a t m e n t results in t e m p o r a r y sterility and, very often, in the appearance of testicular interstitial cell tumors. The present experiments were p e r f o r m e d to determine w het her such deleterious effects on the male reproductive organs involved any genetic hazards for the surviving germ cells. The administration of 1.75 mg/kg cadmium chloride to male mice did n o t increase the d o m i n a n t lethals during the first three weeks after t r e a t m e n t and failed to induce translocation in the F 1 male off-spring. No c h r o m o s o m e rearrangement was observed in the treated males after i.p. injection of 0.5, 1.75 or 3.0 mg/kg cadmium chloride.
INTRODUCTION Although cadmium is n o t t h o u g h t to represent a hazard to man as importan t as lead or m e r c u r y it must nevertheless be considered a dangerous pollutant. The main sources of cadmium pollution are mines and metallurgy, chemical industries, scrap metal t r e a t m e n t , electroplating, superphosphate fertilizers and cadmium pesticides. Several cases of chronic cadmium poisoning have been r e p o r t e d in the literature [9--11,15,27] the Itai-itai disease which was endemic between 1940 and 1965 in the Zinzu River [10] being an e x t r e m e manifestation of such chronic cadmium poisoning. The most c o m m o n effect of cadmium exposure is renal tubular dysfunction, in m any cases indicated only by an increased excretion of proteins, while in more advanced cases there will also be disturbances in the metabolism of bone minerals [ 1 4 , 1 6 - - 1 8 , 2 2 , 2 3 ] . On the other hand [ 1 , 6 , 8 , 1 3 , 1 9 , 2 0 ] , the testicle has been d e m o n s t r a t e d to be the critical organ after acute exposure of experimental mammals, such t r e a t m e n t resulting in t e m p o r a r y sterility due to the destruction of spermatogonial cells. The present experiments were p e r f o r m e d to determine w het her such
43
d e l e t e r i o u s e f f e c t s on t h e m a l e r e p r o d u c t i v e organs involve a n y genetic h a z a r d s f o r t h e surviving g e r m cells. MATERIALS AND METHODS Male m i c e f r o m B A L B / c strain, aged 1 1 - - 1 3 w e e k s , w e r e injected i.p. w i t h 0.5, 1.75 or 3.0 m g / k g o f c a d m i u m chloride. O n e d a y a f t e r t r e a t m e n t each r e c i p i e n t o f 1.75 m g / k g was caged w i t h t h r e e virgin f e m a l e s f r o m t h e s a m e strain. A f t e r 7 and 14 d a y s t h e f e m a l e s w e r e r e p l a c e d b y fresh ones t o c o m p a r e t h e sensitivity o f s p e r m a t o z o a , m a t u r e a n d early s p e r m a t i d s . 10 d a y s later the f e m a l e s w e r e dissected a n d the n u m b e r o f c o r p o r a lutea, live a n d d e a d e m b r y o s c o u n t e d a n d c o m p a r e d w i t h t h o s e f r o m f e m a l e s m a t e d in t h e s a m e m a n n e r w i t h m a l e s w h i c h h a d received saline only. T h e t r e a t e d males w e r e killed t h r e e m o n t h s later a n d their testes e x a m i n e d b y m e a n s o f air-drying t e c h n i q u e o f Evans et al. [4] f o r t h e p r e s e n c e o f t r a n s l o c a t i o n c o n f i g u r a t i o n s [ 1 1 ] . F u r t h e r m o r e , t h e F 1 t r a n s l o c a t i o n test was p e r f o r m e d on 120 F 1 m a l e s sired d u r i n g t h e first t h r e e w e e k s a f t e r t r e a t m e n t b y B A L B / c m a l e m i c e given 1.75 m g / k g o f c a d m i u m chloride and m a t e d w i t h c o n t r o l females. In t h e s p e r m a t o c y t e test 100 cells w e r e a n a l y s e d p e r t r e a t e d m a l e ; in the F 1 t r a n s l o c a t i o n test, 25 p e r F] m a l e . RESULTS AND DISCUSSION T h e results o f t h e d o m i n a n t l e t h a l i t y test are s u m m a r i z e d in T a b l e I. T h e o n l y e f f e c t o b s e r v e d in the f e m a l e s m a t e d w i t h the t r e a t e d m a l e s was a slight b u t n o n - s i g n i f i c a n t decrease (t -- 0.6; 0.6 > P > 0.5) in t h e n u m b e r o f living e m b r y o s as well as a decrease in t h e rate o f p r e g n a n c y d u r i n g t h e t h i r d w e e k a f t e r t r e a t m e n t . E x a m i n a t i o n o f dividing s p e r m a t o c y t e s in t h e t r e a t e d ani-
TABLE I GENERAL RESULTS OF THE DISSECTIONS Treatment
Week Total of the females mating
% of Corpora preglutea per nant female females
Implantation per female
Live embryos per female
Dead embryos per female
Control
1st 2nd 3rd
67 67 77
65 68 57
8.95 9.15 9.22
8.11 8.52 8.09
6.13 6.13 6.09
1.97 2.39 2.00
Cadmium
1st 2nd 3rd
67 67 86
68 70 45
8.89 9.19 8.79
8.06 7.82 7.46
6.15 5.87 5.41
1.91 1.95 2.05
44
TABLE II RESULTS OF CYTOLOGICAL EXAMINATION Test
Treatment (mg/kg)
Spermatocyte test
0 0.5 1.75 3.OO 1.75
F1 translocation test
Animals examined
Cells analysed
Cells with translocation configuration
10 10 10 10
1000 1000 1000 1000
0 0 0 0
120
3000
0
mals or in the F1 male off-spring failed to reveal any c h r o m o s o m e rearrangem e n t such as reciprocal translocation (Table II). Very few experiments have been p e r f o r m e d up to now to determine the genetic hazards of cadmium to mammals. Cadmium chloride added to leukocy te cultures and to fibroblast cultures for 24, 38 or 72 h does n o t induce c h r o m o s o m e aberrations according to Pat t on and Allisson [ 2 1 ] , but Shiraishi et al. [25] observed several c h r o m o s o m e aberrations in cultured human leukocytes after t r e a t m e n t with cadmium sulfide at a final concentration of 6.2 • 10 - 2 pg/ml of culture fluid. These included chromatid and isochromatid breaks, translocations and dicentrics. Positive observations in vivo have been r e p o r t e d by Shiraishi and Yoshida [26] and D e k n u d t and L6onard [2] who observed severe c h r o m o s o m e aberrations such as dicentric and ring c h r o m o s o m e s in peripheral blood l eukocyt es of workers engaged in the m a n u f a c t u r e of cadmium. The fact t hat cadmium produces no genetic effects in male mouse germ cells is th er e f or e rather surprising since acute exposure to this metal has been shown to kill spermatogonia [ 1,6,8,13,19 ] and to induce testicular interstitial cell tumors in mice and rats [ 7 , 2 4 ] . Similar observations have been r ep o r ted with ot her chemicals such as m i t o m y c i n C which is c y t o t o x i c to spermatogonia (resulting in t e m p o r a r y sterility of treated males [ 1 2 ] ) but does n o t p roduce reciprocal translocations in premeiotic male germ cells [5] while having only a slight d o m i n a n t lethal effect in spermatocytes [ 3 ] . This observation demonstrates once more that the action of chemicals on mammalian reproductive organs differs markedly from t hat of ionizing radiation. ACKNOWLEDGEMENTS This work has been p e r f o r m e d with grant from the " F o n d s National de la Recherche Scientifique Medieale".
45
REFERENCES 1 M. Allanson and R. Deanesly, Observation on cadmium damage and repair in rat testes and the effects on the pituitary gonadotrophs, J. Endocrinol., 24 (1962) 453. 2 Gh. DeKnudt and A. L~onard, Cytogenetic investigations on leucocytes of workers occupationally exposed to cadmium, Environ. Physiol., (in press). 3 U.H. Ehling, Comparison of radiation and chemically induced dominant lethal mutations in male mice, Mutation Res., 11 (1971) 35. 4 E.P. Evans, G. Breckon and C.E. Ford, An air-drying method for meiotic preparations from mammalian testes, Cytogenetics, 3 (1964) 289. 5 N. Gilliavod and A. L6onard, Test for mutagenic effects of chemicals in mice, I. Effects of mitomycin C on spermatogonia, Mutation Res., 13 (1971) 274. 6 S.A. Gunn, T.C. Gould and W.A.D. Anderson, The selective injurious response of testicular and epidimyal blood vessel to cadmium and its prevention by zinc, Amer. J. Pathol., 42 (1963a) 685. 7 S.A. Gunn, T.C. Gould and W.A.D. Anderson, Cadmium induced interstitial cell tumors in rats and mice and their prevention by zinc, J. Natl. Cancer Inst., 31 (1963b) 745. 8 A.B. Kar and R.P. Das, Testicular changes in rats after treatment with cadmium chloride, Acta Biol. Med. Ger., 5 (1960) 153. 9 L. Friberg, Health hazards in the manufacture of alkaline accumulators with special reference to chronic cadmium poisoning, Acta Med. Scand., 138 (1950) Suppl. 240. 10 M. Kohno, H. Sugihara, A. Kanai, Y. Someya, M. Tomotaki, M. Tsuruta, H. Atoba, T. Takahoshi and N. Hagino, Surveys on so-called Itai-itai disease, Juzen Igakukai Zasshi, 57 (1955) 2071. 11 A. L~onard, Observations on meiotic chromosomes of the male mouse as a test of the potential mutagenicity of chemical in mammals, in A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 3, Plenum, New York, 1973, pp. 21--56. 12 A. L6onard and N. Gilliavod, Testicular changes in mice after treatment with mitomycin C, Toxicology, 1 (1973) 217. 13 E.S. Meck, Cellular changes induced by cadmium in mouse testis and liver, Brit. J. Exp. Pathol., 40 (1959) 503. 14 I. Murata, Chronic entero-osteo-nephropathy cadmium, Nippon Ishikai Zasshi, 65 (1971) 15. 15 P. Nicaud, A. Lafitte and A. Gross, Les troubles de l'intoxication chronique par le cadmium, Arch. Mal. Prof., 4 (1942) 192. 16 K. Nomiyama and Y. Sugata, Urinary low-molecular-weight proteins in Itai-Itai disease, Environ. Res., 6 (1973) 373. 17 G.F. Nordberg, Cadmium metabolism and toxicity, Environ. Phys. Biochem., 2 (1972)7. 18 G.F. Nordberg and M. Piscator, Influence of long-term cadmium exposure on urinary excretion of protein and cadmium in mice, Environ. Physiol. Biochem., 2 (1972) 37. 19 J. Parizek, The destructive effect of cadmium ion on testicular tissue and its prevention by zinc, J. Endocrinol., 15 (1957) 56. 20 J. Parizek and Z. Zahor, Effect of cadmium salts on testicular tissue, Nature, 177 (1956) 1036. 21 G.R. Patton and A.C. Allison, Chromosome damage in human cell cultures induced by metal salts, Mutation Res., 16 (1972) 332. 22 M. Piscator, Proteinuria in chronic cadmium poisoning, IV. Gel filtration and ion exchange chromatography of urinary proteins from cadmium workers, Arch. Environ. HeaJth, 12 (1955) 345. 23 Research Committee, Report on surveys on so-called Itai-Itai disease, Research Committee on Itai-Itai disease, Kanazawa, 1967.
46
24 F.J.C. Roe, C.E. Dukes and K.H. Cameron, Cadmium neoplasia: testicular atrophy and leydig cell hyperplasia and neoplasia in rats and mice following the subcutaneous injection of cadmium salts, Brit. J. Cancer, 18 (1964) 674. 25 Y. Shiraishi, H. Kurahashi and T.W. Yoshida, Chromosomal aberrations in cultured human leucocytes induced by cadmium sulfide, Proc. Jap. Acad., 48 (1972) 133, 26 Y. Shiraishi and T.H. Yoshida, Chromosomal abnormalities in cultured leucocyte cells from Itai Itai disease patients, Proc. Jap. Acad., 48 (1972) 248. 27 G.A. Stephens, Cadmium poisoning, J. Industr. Hyg., 2 (1920) 129.
47
