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Acute cytogenetic effect of sterigmatocystin on rat bone-marrow cells in vivo
Mutation Research, 139 (1984) 203-206 Elsevier
203
MRLett 0541
Acute cytogenetic effect of sterigmatocystin on rat bone-marrow cells in vivo Norifumi
Ueda, Kimiko Fujie, Keiko Gotoh-Mimura, Taketoshi Sugiyama
Subal C. Chattopadhyay
and
Department of Pathology, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650 (Japan)
(Accepted 4 January 1984)
Summary The acute cytogenetic effects of sterigmatocystin (SC) on rat bone-marrow cells in vivo were studied. The incidence of chromosome aberrations in bone-marrow cells increased and reached the maximum level at 12 h after an intraperitoneal injection of 10-1 mM(31.2 mg)/kg body weight of SC and decreased gradually to the original level after 96 h. A dose-response relationship was observed between the dose of 10-6 and 1 mM. The chromosome aberrations consisted o f chromatid breaks and gaps, the distribution of breaks and gaps within chromosomes (Nos. 1 and 2) was similar to that induced by a carcinogen, 7,12-dimethylbenz[a]anthracene.
Sterigmatocystin (SC), a compound structurally related to aflatoxin, is well known as a toxic metabolite of Aspergillus nidulans and an undescribed strain o f Bipolaris (Hatsuda and Kuyama, 1954; Purchase and van der Watt, 1970). Recently, it has been suggested that SC is produced by Aspergillus flavus and Aspergillus parasiticus and aflatoxin by Aspergillus glaucus. Several researchers have reported that SC induces skin papilloma and carcinoma (Purchase and van der Watt, 1970), fibrosarcoma (Dickens et
al., 1966), hepatocellular carcinoma (Purchase and van der Watt, 1970; van der Watt, 1974) and cholangioma (Dickens, 1966) in rats. Terao (1973) studied the acute toxicity of SC and found that it was no greater than that of aflatoxin B1. Mycobacteria-induced SC has, however, a wide spectrum of action in the Aspergillus species. Sterigmatocystin is one of many carcinogens which should be avoided by man. In the present paper, we describe the cytogenetic toxicity of SC, administered intraperitoneally, on bone-marrow cells in rats.
Mailing address: Dr. Norifumi Ueda, Department of Pathology, Kobe University School of Medicine, Kobe 650 (Japan).
Materials and methods
Abbreviations: SC, sterigmatocystin;DMBA, dimethylbenz[a]anthracene.
0165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.
Female Long-Evans rats, 26-30 days old and weighing 50-80 g, were used. They were fed a commercial ration of NMF (Oriental Ferment Co.,
204
Tokyo) and water ad libitum. Several doses (mM/kg body weight) of SC, in the form of yellow crystals, presented by Dr. Tajima (National Institute of Genetics, Mishima), were carefully suspended in 2 ml of a 0.1°70 PF-68 solution dissolved in physiological saline using Polytron PT-10 (Kinematica, Luzern, Steinhofhalde) and injected intraperitoneally. At various times after treatment, the animals were sacrificed to study the aberrant metaphase cells of the femur bonemarrow cells. Colchicine (0.3 mg dissolved in 0.3 ml of saline) was administered 1 h before sacrifice. Chromosome specimens were prepared as has already been described (Sugiyama, 1971). There were 5 treatment groups, each consisting of 5 rats. The chromosome aberrations defining aberrant metaphase cells consisted o f a break with chromatid discontinuity, and an exchange between the chromosomes. 100 metaphase cells were examined for each animal and all aberrations were recorded photographically. Distribution of the breaks and gaps along the No. 1 and No. 2 chromosomes was examined by previously described methods (Sugiyama, 1971). Results and discussion
The dose-response relationship was studied in ceils sampled 6 h after the intraperitoneal administration of various amounts of SC. The animals injected with 10 -3 mM(0.312 mg)/kg body weight of SC had 6°7o aberrations which is statistically significant from the percentage obtained with 0 mM, 10 - 6 , 10 - 5 and 10 - 4 mM. The frequency of the aberrant metaphase cells in 10 -2 mM was 10°70, 33°70 in 10-1 mM and 72°70 in 1 mM. The dose-response was clearly observed as shown in Fig. 1. The first increase in percentage of aberrant metaphase cells was observed 1 h after intraperitoneal injection of SC. The aberrant cells increased progressively, reaching their maximum level after 12 h, followed by a gradual decrease to the 5°7o level after 96 h. The variation of the interchromosomal distribution in the aberrant metaphase cells induced by SC indicated that the No. 1 and No. 2 chromosomes were involved at all times (Table I).
% 60
5O .o_
~4o 30
~ 2o lO
~23 6
1'2
1'8
214
3'6
4'8
hrs
Fig. 1. Variations in metaphase cells with chromosome breaks and gaps (%) in the bone marrow after intraperitoneal injection of 10-1 mM(31.2 mg)/kg of sterigmatoeystin, contained in saline with PF-68 (e). Each value is the mean and standard deviation of the results of five animals.
% 7O 60
._o
5O '~
40
~ 30 E ~ 2O 10
'
// 1'0-6 I'0 -5 10 .4 1'0-3 li0 -2 1'0-I
i
I
mM
dose
Fig. 2. Dose-response relationship examined 6 h after intraperitoneal administration of sterigmatocystin. Each value indicates the mean and standard deviation of the results of 5 animals.
The distribution of the breaks and gaps in the No. 1 and No. 2 chromosomes is shown in Fig. 3. Both distributions showed a pattern similar to those induced by 7,12-dimethylbenz[a]anthracene (DMBA) or other chemical carcinogens (Sugiyama, 1981).
205
percentage of aberrant metaphase cells 12 h after administration of SC to be 51.6°70, and this high percentage indicates the high carcinogenic capacity of SC, although SC is not a benz[a]anthracene derivative. SC is a potent hepatic carcinogen, and several research workers (Purchase and van der Watt, 1970; van der Watt, 1974) have reported developing hepatic carcinogenesis after using this compound. Terao (1973) reported its acute toxic effect on liver cells of rats. He observed toxic hepatitis within a few hours of oral administration of a large amount of SC, and single cell necrosis following administration of a small amount. It is possible that the chromosome-damaging action of SC was also evoked in hepatic cells. The activating mechanism for SC is considered to be similar to that for aflatoxin (Hatsuda and Kuyama, 1954). The epoxide formation is the result of activation of aflatoxin or SC, just as with 3-methylcholanthrene and benz[a]anthracene. The activated products of SC react with the nuclear DNA in the cells. Engelbrecht and Altenkirk (1972) reported that SC prevented the incorporation of [3H]thymidine and [3H]uridine into DNA and RNA during their synthesis in cultured monkey kidney epithelial cells. The distribution of the breaks and gaps along the No. 1 chromosome is similar to that induced by DMBA. The distribution along the No. 2 chromosome represents only one peak at 55°70,
2o
i 1° ¢
50
0
• -
100
o
~20.
g & 10
0
50 100 relative d i s t a n c e from the centromere
Fig. 3. Plot of distribution of chromosome aberrations along the No. 1 and No. 2 chromosomes. Each distribution indicates the frequency of involvement of each chromosomal region per 100 aberrations induced 6 h after an injection of 10- ~ mM(31.2 m g ) / k g of sterigmatocystin.
SC is a mycotoxin and the mutagenicity of SC has been reported by Ames et al. (1973). In the present studies, the chromosome-breaking effects induced by the SC in rat bone-marrow cells are considered to be remarkably strong. Sugiyama (1973) reported a correlation between chromosome-damaging capacity and carcinogenicity of benz[a]anthracene derivatives, and suggested that chromosome damage played an important role in carcinogenesis. We found the
TABLE 1 I N T E R C H R O M O S O M A L DISTRIBUTION OF C H R O M O S O M E A B E R R A T I O N S I N D U C E D BY S T E R I G M A T O C Y S T I N (31.2 mg/kg) lh
2h
3h
6h
12h
18h
24h
48h
72h
No. 1
29.4
21.8
14.1
20.1
16.1
20.5
12.8
21.2
7.9 (%)
No. 2
23.5
47.2
27.5
26.5
21.0
36.4
33.0
21.2
18.4 (070)
The other telocentric chromosome
39.2
29.1
38.7
42.7
50.3
40.2
42.6
50.0
36.8 (07o)
The other submetacentric chromosome
7.8
1.8
16.9
9.8
10.6
3.0
11.7
3.8
18.4 (%)
Metacentric chromosome
0
0
2.8
0.9
2.0
0
0
1.9
2.6 (°7o)
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0 (070)
Total
206 a l t h o u g h 3 susceptible regions, 30, 55 a n d 80°7o were r e c o g n i z e d in D M B A - i n d u c e d d i s t r i b u t i o n ( S u g i y a m a , 1971; S u g i y a m a et al., 1975, 1981). W e s t u d i e d the d i s t r i b u t i o n a l o n g N o . 1 a n d N o . 2 c h r o m o s o m e s i n d u c e d b y v a r i o u s chemical carcinogens, a n d f o u n d one susceptible region, 4007o o f the long a r m , o n the No. 1 c h r o m o s o m e , a n d t h r e e regions o n the N o . 2 c h r o m o s o m e were always o b s e r v e d in d i f f e r e n t b u t s t r u c t u r a l l y r e l a t e d chemicals, such as D M B A , 7,8,12-trimethylbenz[a]anthracene, urethane and m i t o m y c i n C ( S u g i y a m a et al., 1975, 1981). T h e d i s t r i b u t i o n on the N o . 1 a n d N o . 2 c h r o m o s o m e s i n d u c e d b y SC s h o w e d similar p a t t e r n s , b u t diff e r e d slightly f r o m the a b o v e results. T h e c h r o m o s o m e - d a m a g i n g a c t i o n o f SC to b o n e m a r r o w cells does not, t h e r e f o r e , seem to be identical with t h o s e chemicals at the c h r o m o s o m a l levels. S u g i y a m a (1981) suggested these susceptible regions were late replicating regions, a n d t h a t the exact time after a d m i n i s t r a t i o n o f the c o m p o u n d s was r e q u i r e d for m e a s u r e m e n t . I n the present s t u d y , we m e a s u r e d o n l y 6 h a f t e r the intrap e r i t o n e a l i n j e c t i o n . It is feasible t h a t m e a s u r e m e n t s h o u l d be c a r r i e d o u t s o o n e r a f t e r the inject i o n o f SC. To establish this fact, f u r t h e r studies are necessary. T h e c h r o m o s o m e - d a m a g i n g a c t i o n o f SC lasts for a long t i m e in rats, as can be seen f r o m Fig. 1. This fact indicates t h a t the effect o f SC continues in the t a r g e t o r g a n s , a l t h o u g h the e x p e r i m e n t was c a r r i e d o u t via the i n t r a p e r i t o n e a l route, a n d a l t h o u g h SC is n o n - s o l u b l e in w a t e r . M a n s h o u l d n o t be e x p o s e d to SC, b e c a u s e o f the c o n s i d e r a b l e genetic risks a n d its d e l a y e d action.
Acknowledgements W e wish to t h a n k D r . Y a t a r o T a j i m a for his gift o f s t e r i g m a t o c y s t i n . This w o r k was s u p p o r t e d b y a
g r a n t - i n - a i d for C a n c e r Research f r o m the M i n i s t r y o f E d u c a t i o n , Science a n d C u l t u r e , a n d f r o m the M i n i s t r y o f H e a l t h a n d W e l f a r e .
References Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee (1973) Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. (U.S.A.), 70, 2281-2285. Dickens, F., H.E.H. Jones and H.B. Waynforth (1966) Oral, subcutaneous and intratraeheal administration of carcinogenic lactones and related substances: The intratracheal administration of cigarette tar in the rat, Br. J. Cancer, 20, 134-144. Engelbrecht, J.C., and B. Altenkirk (1972) Comparison of some biological effects of sterigmatocystin and aflatoxin analogues on primary cell culture, J. Natl. Cancer Inst., 48, 1647-1655. Hatsuda, Y., and S. Kuyama (1954) Studies on the metabolic products of Aspergillus versicolor. 1. Cultivation of Aspergillus versicolor, isolation and purification of metabolic products (in Japanese), J. Agr. Chem. Soc. (Japan), 28, 989-992. Purchase, I.F.H., and J.J. van der Watt (1970) Carcinogenicity of sterigmatocystin, Food Cosmet. Toxicol., 8, 289-295. Sugiyama, T. (1971) Specific vulnerability of the largest telocentric chromosome of rat bone marrow cells to 7,12-dimethylbenz[a]anthracene, J. Natl. Cancer Inst., 47, 1267-1275. Sugiyama, T. (1973) Chromosome aberration and carcinogenesis by various benz[a]anthracene derivatives, Gann, 64, 637-639. Sugiyama, T., K. Gotoh and H. Uenaka (1975) Acut~ cytogenetic effect of 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF2, a food preservative) on rat bone marrow cells in vivo, Mutation Res., 31, 241-246. Sugiyama, T., N. Ueda, S. Maeda, N. Shiraishi, K. GotoMimura, S. Murao and S.C. Chattopadhyay (1981) Chemical carcinogenesis in the rat: Common mode of action of car• cinogens at the chromosomal level, J. Natl. Cancer Inst., 67, 831-839. Terao, K. (1973) Massive and single cell necrosis in the rat liver induced by aflatoxin B~ and sterigmatocystin, Acta Pathol. Jap., 23, 647-653. Van der Watt, J.J. (1974) Sterigmatocystin, in: I.F.H. Purchase (Ed.), Mycotoxins, Elsevier, Amsterdam, pp. 369-382.
203
MRLett 0541
Acute cytogenetic effect of sterigmatocystin on rat bone-marrow cells in vivo Norifumi
Ueda, Kimiko Fujie, Keiko Gotoh-Mimura, Taketoshi Sugiyama
Subal C. Chattopadhyay
and
Department of Pathology, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650 (Japan)
(Accepted 4 January 1984)
Summary The acute cytogenetic effects of sterigmatocystin (SC) on rat bone-marrow cells in vivo were studied. The incidence of chromosome aberrations in bone-marrow cells increased and reached the maximum level at 12 h after an intraperitoneal injection of 10-1 mM(31.2 mg)/kg body weight of SC and decreased gradually to the original level after 96 h. A dose-response relationship was observed between the dose of 10-6 and 1 mM. The chromosome aberrations consisted o f chromatid breaks and gaps, the distribution of breaks and gaps within chromosomes (Nos. 1 and 2) was similar to that induced by a carcinogen, 7,12-dimethylbenz[a]anthracene.
Sterigmatocystin (SC), a compound structurally related to aflatoxin, is well known as a toxic metabolite of Aspergillus nidulans and an undescribed strain o f Bipolaris (Hatsuda and Kuyama, 1954; Purchase and van der Watt, 1970). Recently, it has been suggested that SC is produced by Aspergillus flavus and Aspergillus parasiticus and aflatoxin by Aspergillus glaucus. Several researchers have reported that SC induces skin papilloma and carcinoma (Purchase and van der Watt, 1970), fibrosarcoma (Dickens et
al., 1966), hepatocellular carcinoma (Purchase and van der Watt, 1970; van der Watt, 1974) and cholangioma (Dickens, 1966) in rats. Terao (1973) studied the acute toxicity of SC and found that it was no greater than that of aflatoxin B1. Mycobacteria-induced SC has, however, a wide spectrum of action in the Aspergillus species. Sterigmatocystin is one of many carcinogens which should be avoided by man. In the present paper, we describe the cytogenetic toxicity of SC, administered intraperitoneally, on bone-marrow cells in rats.
Mailing address: Dr. Norifumi Ueda, Department of Pathology, Kobe University School of Medicine, Kobe 650 (Japan).
Materials and methods
Abbreviations: SC, sterigmatocystin;DMBA, dimethylbenz[a]anthracene.
0165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.
Female Long-Evans rats, 26-30 days old and weighing 50-80 g, were used. They were fed a commercial ration of NMF (Oriental Ferment Co.,
204
Tokyo) and water ad libitum. Several doses (mM/kg body weight) of SC, in the form of yellow crystals, presented by Dr. Tajima (National Institute of Genetics, Mishima), were carefully suspended in 2 ml of a 0.1°70 PF-68 solution dissolved in physiological saline using Polytron PT-10 (Kinematica, Luzern, Steinhofhalde) and injected intraperitoneally. At various times after treatment, the animals were sacrificed to study the aberrant metaphase cells of the femur bonemarrow cells. Colchicine (0.3 mg dissolved in 0.3 ml of saline) was administered 1 h before sacrifice. Chromosome specimens were prepared as has already been described (Sugiyama, 1971). There were 5 treatment groups, each consisting of 5 rats. The chromosome aberrations defining aberrant metaphase cells consisted o f a break with chromatid discontinuity, and an exchange between the chromosomes. 100 metaphase cells were examined for each animal and all aberrations were recorded photographically. Distribution of the breaks and gaps along the No. 1 and No. 2 chromosomes was examined by previously described methods (Sugiyama, 1971). Results and discussion
The dose-response relationship was studied in ceils sampled 6 h after the intraperitoneal administration of various amounts of SC. The animals injected with 10 -3 mM(0.312 mg)/kg body weight of SC had 6°7o aberrations which is statistically significant from the percentage obtained with 0 mM, 10 - 6 , 10 - 5 and 10 - 4 mM. The frequency of the aberrant metaphase cells in 10 -2 mM was 10°70, 33°70 in 10-1 mM and 72°70 in 1 mM. The dose-response was clearly observed as shown in Fig. 1. The first increase in percentage of aberrant metaphase cells was observed 1 h after intraperitoneal injection of SC. The aberrant cells increased progressively, reaching their maximum level after 12 h, followed by a gradual decrease to the 5°7o level after 96 h. The variation of the interchromosomal distribution in the aberrant metaphase cells induced by SC indicated that the No. 1 and No. 2 chromosomes were involved at all times (Table I).
% 60
5O .o_
~4o 30
~ 2o lO
~23 6
1'2
1'8
214
3'6
4'8
hrs
Fig. 1. Variations in metaphase cells with chromosome breaks and gaps (%) in the bone marrow after intraperitoneal injection of 10-1 mM(31.2 mg)/kg of sterigmatoeystin, contained in saline with PF-68 (e). Each value is the mean and standard deviation of the results of five animals.
% 7O 60
._o
5O '~
40
~ 30 E ~ 2O 10
'
// 1'0-6 I'0 -5 10 .4 1'0-3 li0 -2 1'0-I
i
I
mM
dose
Fig. 2. Dose-response relationship examined 6 h after intraperitoneal administration of sterigmatocystin. Each value indicates the mean and standard deviation of the results of 5 animals.
The distribution of the breaks and gaps in the No. 1 and No. 2 chromosomes is shown in Fig. 3. Both distributions showed a pattern similar to those induced by 7,12-dimethylbenz[a]anthracene (DMBA) or other chemical carcinogens (Sugiyama, 1981).
205
percentage of aberrant metaphase cells 12 h after administration of SC to be 51.6°70, and this high percentage indicates the high carcinogenic capacity of SC, although SC is not a benz[a]anthracene derivative. SC is a potent hepatic carcinogen, and several research workers (Purchase and van der Watt, 1970; van der Watt, 1974) have reported developing hepatic carcinogenesis after using this compound. Terao (1973) reported its acute toxic effect on liver cells of rats. He observed toxic hepatitis within a few hours of oral administration of a large amount of SC, and single cell necrosis following administration of a small amount. It is possible that the chromosome-damaging action of SC was also evoked in hepatic cells. The activating mechanism for SC is considered to be similar to that for aflatoxin (Hatsuda and Kuyama, 1954). The epoxide formation is the result of activation of aflatoxin or SC, just as with 3-methylcholanthrene and benz[a]anthracene. The activated products of SC react with the nuclear DNA in the cells. Engelbrecht and Altenkirk (1972) reported that SC prevented the incorporation of [3H]thymidine and [3H]uridine into DNA and RNA during their synthesis in cultured monkey kidney epithelial cells. The distribution of the breaks and gaps along the No. 1 chromosome is similar to that induced by DMBA. The distribution along the No. 2 chromosome represents only one peak at 55°70,
2o
i 1° ¢
50
0
• -
100
o
~20.
g & 10
0
50 100 relative d i s t a n c e from the centromere
Fig. 3. Plot of distribution of chromosome aberrations along the No. 1 and No. 2 chromosomes. Each distribution indicates the frequency of involvement of each chromosomal region per 100 aberrations induced 6 h after an injection of 10- ~ mM(31.2 m g ) / k g of sterigmatocystin.
SC is a mycotoxin and the mutagenicity of SC has been reported by Ames et al. (1973). In the present studies, the chromosome-breaking effects induced by the SC in rat bone-marrow cells are considered to be remarkably strong. Sugiyama (1973) reported a correlation between chromosome-damaging capacity and carcinogenicity of benz[a]anthracene derivatives, and suggested that chromosome damage played an important role in carcinogenesis. We found the
TABLE 1 I N T E R C H R O M O S O M A L DISTRIBUTION OF C H R O M O S O M E A B E R R A T I O N S I N D U C E D BY S T E R I G M A T O C Y S T I N (31.2 mg/kg) lh
2h
3h
6h
12h
18h
24h
48h
72h
No. 1
29.4
21.8
14.1
20.1
16.1
20.5
12.8
21.2
7.9 (%)
No. 2
23.5
47.2
27.5
26.5
21.0
36.4
33.0
21.2
18.4 (070)
The other telocentric chromosome
39.2
29.1
38.7
42.7
50.3
40.2
42.6
50.0
36.8 (07o)
The other submetacentric chromosome
7.8
1.8
16.9
9.8
10.6
3.0
11.7
3.8
18.4 (%)
Metacentric chromosome
0
0
2.8
0.9
2.0
0
0
1.9
2.6 (°7o)
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0 (070)
Total
206 a l t h o u g h 3 susceptible regions, 30, 55 a n d 80°7o were r e c o g n i z e d in D M B A - i n d u c e d d i s t r i b u t i o n ( S u g i y a m a , 1971; S u g i y a m a et al., 1975, 1981). W e s t u d i e d the d i s t r i b u t i o n a l o n g N o . 1 a n d N o . 2 c h r o m o s o m e s i n d u c e d b y v a r i o u s chemical carcinogens, a n d f o u n d one susceptible region, 4007o o f the long a r m , o n the No. 1 c h r o m o s o m e , a n d t h r e e regions o n the N o . 2 c h r o m o s o m e were always o b s e r v e d in d i f f e r e n t b u t s t r u c t u r a l l y r e l a t e d chemicals, such as D M B A , 7,8,12-trimethylbenz[a]anthracene, urethane and m i t o m y c i n C ( S u g i y a m a et al., 1975, 1981). T h e d i s t r i b u t i o n on the N o . 1 a n d N o . 2 c h r o m o s o m e s i n d u c e d b y SC s h o w e d similar p a t t e r n s , b u t diff e r e d slightly f r o m the a b o v e results. T h e c h r o m o s o m e - d a m a g i n g a c t i o n o f SC to b o n e m a r r o w cells does not, t h e r e f o r e , seem to be identical with t h o s e chemicals at the c h r o m o s o m a l levels. S u g i y a m a (1981) suggested these susceptible regions were late replicating regions, a n d t h a t the exact time after a d m i n i s t r a t i o n o f the c o m p o u n d s was r e q u i r e d for m e a s u r e m e n t . I n the present s t u d y , we m e a s u r e d o n l y 6 h a f t e r the intrap e r i t o n e a l i n j e c t i o n . It is feasible t h a t m e a s u r e m e n t s h o u l d be c a r r i e d o u t s o o n e r a f t e r the inject i o n o f SC. To establish this fact, f u r t h e r studies are necessary. T h e c h r o m o s o m e - d a m a g i n g a c t i o n o f SC lasts for a long t i m e in rats, as can be seen f r o m Fig. 1. This fact indicates t h a t the effect o f SC continues in the t a r g e t o r g a n s , a l t h o u g h the e x p e r i m e n t was c a r r i e d o u t via the i n t r a p e r i t o n e a l route, a n d a l t h o u g h SC is n o n - s o l u b l e in w a t e r . M a n s h o u l d n o t be e x p o s e d to SC, b e c a u s e o f the c o n s i d e r a b l e genetic risks a n d its d e l a y e d action.
Acknowledgements W e wish to t h a n k D r . Y a t a r o T a j i m a for his gift o f s t e r i g m a t o c y s t i n . This w o r k was s u p p o r t e d b y a
g r a n t - i n - a i d for C a n c e r Research f r o m the M i n i s t r y o f E d u c a t i o n , Science a n d C u l t u r e , a n d f r o m the M i n i s t r y o f H e a l t h a n d W e l f a r e .
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