Aria pharmacol. et toxicol. 1979, 45. 399-402.
From the Department of Industrial Hygiene and Toxicology, Institute of Occupational Health, Haartmaninkatu 1, SF-00290 Helsinki 29, Finland
Embryotoxicity and Teratogenicity of Styrene and Styrene Oxide on Chick Embryos Enhanced by Trichloropropylene Oxide BY J. T. J . Kankaanpaa, K. Hemminki and H. Vainio (Received May 30, 1979; Accepted June 7, 1979)
Abstracr: The effects of TCPO (trichloropropylene oxide) on the embryotoxicity and teratogenicity of styrene and styrene oxide on chick embryos were investigated. The compounds were injected into the air space of the eggs in a total volume of 25 pl on the third day of embryogenesis. TCPO increased embryotoxicity and teratogenicity of styrene and styrene oxide. Our results present evidence that the epoxides possess embryotoxic and teratogenic properties in chick embryos. Key-words: Embryotoxicity embryos - epoxide.
-
teratogenicity
- styrene
Styrene (vinyl benzene) is widely used (world production 7 mill. tons 1977; Sleeth 1977) in the production of plastics and resins. The metabolism of styrene proceeds mainly via epoxide formation, and styrene oxide( 1,2-epoxyethyl benzene) is found as a reactive epoxide intermediate (cf. for references, Leibman 1975). TCPO (trichloropropylene oxide; 1.2-epoxy3,3,3-trichloropropane) acts on styrene metabolism by inhibiting the enzyme epoxide hydratase, which converts styrene oxide to the diol, styrene glycol (Oesch 1973). TCPO has been assumed t o enhance the toxicity of such compounds, which are detoxicated via epoxide formation, i.e. styrene. Both styrene and styrene oxide have been found to be mutagenic in bacteria (Vainio et a/. 1976) and in Drosophila (Donner er al. 1979). They also have been observed to be embryotoxic and to possess teratogenic potential in chick embryos (Vainio et 01. 1977). The purpose of this study is to investigate the effects of TCPO on the embryotoxicity and
- styrene oxide - trichloropropylene oxide
- chick
teratogenicity of styrene and styrene oxide, on chick embryos.
Materials and Methods Eggs (White Leghorn strains 'mittari' and SK 12) were obtained from Siipikarjanhoitajan Liitto ry. hatcherie, Hameenlinna, Finland. The eggs were incubated in an incubation chamber (Tu 40b Memmert-Oven, 854 Schwabach, F.R.G.) for 14 days from the start of their embryogenesis in a constant temperature of 37.5" (k0.5') and a relative humidity of 65-75 per cent. During the incubation the eggs were turned once a day. Styrene (purum grade; Fluka AG, Buchs SG, Switzerland), styrene oxide (97% purity; Fluka AG, Buchs SG, Switzerland) and TCPO (1,2-epoxy-3,3,3-trichloropropane; 98 9%; Aldrich-Europe, Beerse, Belgium) were dissolved in a polyoxyethylated vegetable oil, Emulphor in concentrations 10 pmol/egg, 0.8 pmol/egg andO.l pmol/ egg, respectively. Emulphor was used in 10 per cent water solution and all the solutions were made just before use. Then, in concentrations mentioned, each compound was injected into their air space of the eggs via a small hole. Styrene and styrene oxide were tested individually
J. T. J. KANKAANPAA ET A L .
xII
x
z
m
II
z
m 0
9
II 2
n m c
n
z
3
I
Noninlected controls
II z
c PO
2
... ... ... ... ... ... ... ... ... ... ... ... ..:
m 01
5
II
z
* .. .....
... ... ...
a
... I..
-1 I
z
... ... ... ... ... ... ... ... ...
-
.1Umol
Fig. 1. Effect of TCPO on embryotoxicity and teratogenicity of styrene and styrene oxide on chick embryos. The open columns describe the survival rates of chick embryos (%). The dotted columns present the percentages of malformed embryos per live embryos. The malformations include stunting, exteriorization of different organs (brain, viscera), eye defects (anophthalmias, microphthalmias) and (rare) limb and toe malformations. N=number of eggs (open columns), N=number of live embryos (dotted columns).
and in combination with TCPO. The solutions of two compounds were made immediately before injection. The total volume injected was 25 pl in each group. After injection the hole was sealed with paraffin wax. All injections were performed during the 3rd day of incubation. On the 14th day of incubation the embryos were prepared for macroscopic inspection.
Results and Discussion The test concentrations used were chosen to be lower than the LD50 dose levels determined earlier in this laboratory (40 pmol/egg for styrene and 1.5 pmol/egg for styrene oxide, Vainio et al. 1977). Styrene at a dose of 10 pmol/egg and styrene oxide at 0.8 pmol/egg brought about a mortality of 29% and 38%, respectively (fig. 1). In Emulphor-injected control embryos the mortality rate of 18% and in TCPO-treated (0.1 pmol/egg) embryos the respective value of 23% were found, while the untreated embryos showed a mortality rate of only 5.5% (fig. 1). The enhancement of embryotoxicity can be seen, when TCPO is added either together with styrene or styrene oxide. The mortality rate for styrene is 29% as compared to 72% for styrene+TCPO; for
styrene oxide the rate is 38% as compared to 62% for styrene oxide+TCPO. The dotted columns in fig. 1 describe the percentages of malformed chick embryos per live embryos. Both styrene and styrene oxide induce malformations. In styrene treated eggs the rate is 15% and in styrene oxide treated eggs a little higher, 20%. The malformation rates of styrene and styrene oxide administered with TCPO were 33% and 27%, respectively. In non-injected controls no malformations were detected. In injected controls 4.996, and in TCPO injected test group 11% of the embryos were malformed. Our unpublished experiments show that TCPO can have a higher teratogenic potential on chick embryos at larger doses. At the examined doses styrene and styrene oxide, however, are remarkably more teratogenic than TCPO. Macroscopically, 19 out of the total 29 malformed embryos exhibited stunting (embryos under 5.0 cm long with generalized oederna and deteriorated feather development) and either viscera or brain exteriorizations. In addition, 8 embryos had distinct eye defects (anophthalmias, microphthalmias). One embryo with two pairs of legs (fig.
TOXICITY O F STYRENE ON CHICK EMBRYOS
401
a.
b.
bT'I3Et:E
OXIDE
Fig. 2. a) The left embryo IS injected with styrene. The embryo has two pairs of hind limbs, the caudal pair of which IS stunted. A control embryo, on the right b) On the left, two embryos with anophthalmia, injected with styrene oxide. The embryo in the middle has additionally a profound oedema A control embryo, o n the right
402
J. T. J. KANKAANPAA ET AL.
2a) and one with two complete trunks and a common head with microphthalmic eyes were also found. The results obtained provide additional evidence that styrene oxide is more potent and reactive than its biological parent compound, styrene. Chick embryos have been observed to possess drug metabolizing activities which are required for metabolic activation of styrene into styrene oxide (Dutton 1975; Wishart & Dutton 1975). It is likely that, while we block the biotransformation of styrene by adding TCPO, the enhanced embryotoxicity seen is due to the styrene oxide. Also an effect, although not as clear, was seen in regard to teratogenicity, when styrene was injected with TCPO. Similarly, TCPO has been presumed to enhance diphenylhydantoin teratogenesis by increasing the diphenylhydantoin epoxide level, secondary to the inhibition of embryonic epoxide hydratase (Martz et 01. 1977). The results provide some evidence in support of the hypothesis that epoxide formation in embryos may be related to embryotoxicity and teratogenesis. Acknowledgements We thank Mrs. Elma Nieminen for the skillful technical assistance. Grant: ASF 241 A-C/77.
References Donner, M., M. Sorsa & H. Vainio: Recessive lethals induced by styrene and styrene oxide in Drosophila melanogaster. Mural. Res. 1979, 67, 373-376. Dutton, G. J.: Control of UDP-glucuronyltransferase activity. Biochem. Pharmacol. 1975, 24, 1835-1841. Leibman, K. C.: Metabolism and toxicity of styrene. Environ. health perspect. 1975, 11, 115-1 19. Martz, F., C. Failinger 111 & D. A. Blake: Phenytoin teratogenesis: Correlation between embryopathic effect and covalent binding of putative arene oxide metabolite in gestational tissue. J. Pharmarol. Exp. Therap. 1977, 203, 231-239. Oesch, F.: Mammalian epoxide hydrases: Inducible enzymes catalyzing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiorica 1973, 3 , 305-340. Sleeth, C. V.: Styrene monomer, Perrochemicals outlook - 13. Chem. eng. prog. Nov. 1977, 31-35. Vainio, H., K. Hemminki & E. Elovaara: Toxicity of styrene and styrene oxide on chick embryos. Toxicology 1977, 8, 319-325. Vainio, H., R. Paakkonen, K. Ronnholm, V. Raunio & 0. Pelkonen: A study of the mutagenic activity of styrene and styrene oxide. Scand. J. work Environ. Health 1976. 2, 147-151. Wishart, G. J. & G. J. Dutton: Precocious development of glucuronidating and hydroxylating enzymes in chick embryos treated with pituitary grafts. Biochem. J. 1975, 152, 325-331.
From the Department of Industrial Hygiene and Toxicology, Institute of Occupational Health, Haartmaninkatu 1, SF-00290 Helsinki 29, Finland
Embryotoxicity and Teratogenicity of Styrene and Styrene Oxide on Chick Embryos Enhanced by Trichloropropylene Oxide BY J. T. J . Kankaanpaa, K. Hemminki and H. Vainio (Received May 30, 1979; Accepted June 7, 1979)
Abstracr: The effects of TCPO (trichloropropylene oxide) on the embryotoxicity and teratogenicity of styrene and styrene oxide on chick embryos were investigated. The compounds were injected into the air space of the eggs in a total volume of 25 pl on the third day of embryogenesis. TCPO increased embryotoxicity and teratogenicity of styrene and styrene oxide. Our results present evidence that the epoxides possess embryotoxic and teratogenic properties in chick embryos. Key-words: Embryotoxicity embryos - epoxide.
-
teratogenicity
- styrene
Styrene (vinyl benzene) is widely used (world production 7 mill. tons 1977; Sleeth 1977) in the production of plastics and resins. The metabolism of styrene proceeds mainly via epoxide formation, and styrene oxide( 1,2-epoxyethyl benzene) is found as a reactive epoxide intermediate (cf. for references, Leibman 1975). TCPO (trichloropropylene oxide; 1.2-epoxy3,3,3-trichloropropane) acts on styrene metabolism by inhibiting the enzyme epoxide hydratase, which converts styrene oxide to the diol, styrene glycol (Oesch 1973). TCPO has been assumed t o enhance the toxicity of such compounds, which are detoxicated via epoxide formation, i.e. styrene. Both styrene and styrene oxide have been found to be mutagenic in bacteria (Vainio et a/. 1976) and in Drosophila (Donner er al. 1979). They also have been observed to be embryotoxic and to possess teratogenic potential in chick embryos (Vainio et 01. 1977). The purpose of this study is to investigate the effects of TCPO on the embryotoxicity and
- styrene oxide - trichloropropylene oxide
- chick
teratogenicity of styrene and styrene oxide, on chick embryos.
Materials and Methods Eggs (White Leghorn strains 'mittari' and SK 12) were obtained from Siipikarjanhoitajan Liitto ry. hatcherie, Hameenlinna, Finland. The eggs were incubated in an incubation chamber (Tu 40b Memmert-Oven, 854 Schwabach, F.R.G.) for 14 days from the start of their embryogenesis in a constant temperature of 37.5" (k0.5') and a relative humidity of 65-75 per cent. During the incubation the eggs were turned once a day. Styrene (purum grade; Fluka AG, Buchs SG, Switzerland), styrene oxide (97% purity; Fluka AG, Buchs SG, Switzerland) and TCPO (1,2-epoxy-3,3,3-trichloropropane; 98 9%; Aldrich-Europe, Beerse, Belgium) were dissolved in a polyoxyethylated vegetable oil, Emulphor in concentrations 10 pmol/egg, 0.8 pmol/egg andO.l pmol/ egg, respectively. Emulphor was used in 10 per cent water solution and all the solutions were made just before use. Then, in concentrations mentioned, each compound was injected into their air space of the eggs via a small hole. Styrene and styrene oxide were tested individually
J. T. J. KANKAANPAA ET A L .
xII
x
z
m
II
z
m 0
9
II 2
n m c
n
z
3
I
Noninlected controls
II z
c PO
2
... ... ... ... ... ... ... ... ... ... ... ... ..:
m 01
5
II
z
* .. .....
... ... ...
a
... I..
-1 I
z
... ... ... ... ... ... ... ... ...
-
.1Umol
Fig. 1. Effect of TCPO on embryotoxicity and teratogenicity of styrene and styrene oxide on chick embryos. The open columns describe the survival rates of chick embryos (%). The dotted columns present the percentages of malformed embryos per live embryos. The malformations include stunting, exteriorization of different organs (brain, viscera), eye defects (anophthalmias, microphthalmias) and (rare) limb and toe malformations. N=number of eggs (open columns), N=number of live embryos (dotted columns).
and in combination with TCPO. The solutions of two compounds were made immediately before injection. The total volume injected was 25 pl in each group. After injection the hole was sealed with paraffin wax. All injections were performed during the 3rd day of incubation. On the 14th day of incubation the embryos were prepared for macroscopic inspection.
Results and Discussion The test concentrations used were chosen to be lower than the LD50 dose levels determined earlier in this laboratory (40 pmol/egg for styrene and 1.5 pmol/egg for styrene oxide, Vainio et al. 1977). Styrene at a dose of 10 pmol/egg and styrene oxide at 0.8 pmol/egg brought about a mortality of 29% and 38%, respectively (fig. 1). In Emulphor-injected control embryos the mortality rate of 18% and in TCPO-treated (0.1 pmol/egg) embryos the respective value of 23% were found, while the untreated embryos showed a mortality rate of only 5.5% (fig. 1). The enhancement of embryotoxicity can be seen, when TCPO is added either together with styrene or styrene oxide. The mortality rate for styrene is 29% as compared to 72% for styrene+TCPO; for
styrene oxide the rate is 38% as compared to 62% for styrene oxide+TCPO. The dotted columns in fig. 1 describe the percentages of malformed chick embryos per live embryos. Both styrene and styrene oxide induce malformations. In styrene treated eggs the rate is 15% and in styrene oxide treated eggs a little higher, 20%. The malformation rates of styrene and styrene oxide administered with TCPO were 33% and 27%, respectively. In non-injected controls no malformations were detected. In injected controls 4.996, and in TCPO injected test group 11% of the embryos were malformed. Our unpublished experiments show that TCPO can have a higher teratogenic potential on chick embryos at larger doses. At the examined doses styrene and styrene oxide, however, are remarkably more teratogenic than TCPO. Macroscopically, 19 out of the total 29 malformed embryos exhibited stunting (embryos under 5.0 cm long with generalized oederna and deteriorated feather development) and either viscera or brain exteriorizations. In addition, 8 embryos had distinct eye defects (anophthalmias, microphthalmias). One embryo with two pairs of legs (fig.
TOXICITY O F STYRENE ON CHICK EMBRYOS
401
a.
b.
bT'I3Et:E
OXIDE
Fig. 2. a) The left embryo IS injected with styrene. The embryo has two pairs of hind limbs, the caudal pair of which IS stunted. A control embryo, on the right b) On the left, two embryos with anophthalmia, injected with styrene oxide. The embryo in the middle has additionally a profound oedema A control embryo, o n the right
402
J. T. J. KANKAANPAA ET AL.
2a) and one with two complete trunks and a common head with microphthalmic eyes were also found. The results obtained provide additional evidence that styrene oxide is more potent and reactive than its biological parent compound, styrene. Chick embryos have been observed to possess drug metabolizing activities which are required for metabolic activation of styrene into styrene oxide (Dutton 1975; Wishart & Dutton 1975). It is likely that, while we block the biotransformation of styrene by adding TCPO, the enhanced embryotoxicity seen is due to the styrene oxide. Also an effect, although not as clear, was seen in regard to teratogenicity, when styrene was injected with TCPO. Similarly, TCPO has been presumed to enhance diphenylhydantoin teratogenesis by increasing the diphenylhydantoin epoxide level, secondary to the inhibition of embryonic epoxide hydratase (Martz et 01. 1977). The results provide some evidence in support of the hypothesis that epoxide formation in embryos may be related to embryotoxicity and teratogenesis. Acknowledgements We thank Mrs. Elma Nieminen for the skillful technical assistance. Grant: ASF 241 A-C/77.
References Donner, M., M. Sorsa & H. Vainio: Recessive lethals induced by styrene and styrene oxide in Drosophila melanogaster. Mural. Res. 1979, 67, 373-376. Dutton, G. J.: Control of UDP-glucuronyltransferase activity. Biochem. Pharmacol. 1975, 24, 1835-1841. Leibman, K. C.: Metabolism and toxicity of styrene. Environ. health perspect. 1975, 11, 115-1 19. Martz, F., C. Failinger 111 & D. A. Blake: Phenytoin teratogenesis: Correlation between embryopathic effect and covalent binding of putative arene oxide metabolite in gestational tissue. J. Pharmarol. Exp. Therap. 1977, 203, 231-239. Oesch, F.: Mammalian epoxide hydrases: Inducible enzymes catalyzing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiorica 1973, 3 , 305-340. Sleeth, C. V.: Styrene monomer, Perrochemicals outlook - 13. Chem. eng. prog. Nov. 1977, 31-35. Vainio, H., K. Hemminki & E. Elovaara: Toxicity of styrene and styrene oxide on chick embryos. Toxicology 1977, 8, 319-325. Vainio, H., R. Paakkonen, K. Ronnholm, V. Raunio & 0. Pelkonen: A study of the mutagenic activity of styrene and styrene oxide. Scand. J. work Environ. Health 1976. 2, 147-151. Wishart, G. J. & G. J. Dutton: Precocious development of glucuronidating and hydroxylating enzymes in chick embryos treated with pituitary grafts. Biochem. J. 1975, 152, 325-331.
