Arch Toxicol (1992) 6 6 : 4 4 3 - 4 4 5
Archives of
Toxicology 9 Springer-Verlag 1992
Short communication
Cellular toxicity of toluene on mouse gamete cells and preimplantation embryos Frank D. Yelian and W. Richard D u k e l o w Endocrine Research Center, Michigan State University, East Lansing, Michigan 48824, USA Received 7 November 1991/Accepted 25 February 1992
Abstract. Toluene is an aromatic hydrocarbon, which has been used in the paint, lacquer and glue industry. It has been detected in municipal water supplies. Previous mouse in vivo studies indicated that toluene administrated by gavage increased the embryonic mortality. The present in vitro study demonstrated that a concentration of toluene higher than 8.67 ~g/ml not only decreased sperm motility and inhibited fertilization, but also significantly increased preimplantation embryo degeneration. At lower levels no effects were observed and the adverse effect levels were approximately 780 fold higher than reported levels in municipal water supplies. Key words: Toluene - Spermatozoa - Oocytes - In vitro
fertilization - Preimplantation embryo
ally inhaled toluene in high doses throughout pregnancy (Goodwin 1988). Toluene has been shown to be teratogenic (cleft palate) in mice by Nawrot and Staples (1979). Similarly an inhalation study by Courtney et al. (1986) showed that toluene administered by inhalation at 2 0 0 400 ppm to CD-1 mice from days 6 to 16 of gestation was teratogenic but not fetotoxic. The limited studies on the teratogenic potential of toluene indicate that toluene could have some teratogenic and toxic effects on the mammalian fetus. The presence of toluene at low levels in municipal drinking water emphasizes the importance of determining if such an effect can occur. No in vitro toxicological studies on the mammalian gamete cells after exposure to toluene are available. The present study was designed to test the direct effects of toluene on mouse epididymal sperm motility, fertilizing capacity and preimplantation embryonic mortality in vitro.
Introduction Materials and m e t h o d s
Toluene has been detected in municipal water supplies at levels ranging from 0.1 l.tg/1 to 11 gg/1. The toluene metab01ites benzaldehyde and benzoic acid were also found in municipal water at concentrations up to 19 gg/1 (EPA 1980). Mouse and rat experiments show impaired growth of the mother and fetus and fetal skeletal anomalies after exposure to large doses of toluene (Hudak and Ungvary 1978 and Shigeta et al. 1982). Behavioral effects of toluene in mice exposed pre- and postnatally have also been described (Kostas and Hotchin 1981). Two children with multiple malformations were born to mothers who worked as shoemakers and were chronically exposed to toluene and trichlorethylene, used in a soling solution (Euler 1967). Additionally, there has been at least one report of congenital defects in children born to mothers who had intention-
Experimental animals. The animals used in this study were B6D2FI mice (Cosby et al. 1989). The mice were either purchased from the Jackson Laboratory (Bar Harbor, ME) or produced at the Endocrine Research Center of Michigan State University by crossing C57BL-6J and DBA-2J mice. B6D2F1 mice were sexed at 21 days of age, and maintained in separate cages. They were provided Breeder Blox| (Wayne Pet Food Division, Chicago, IL) and fresh water daily, ad libitum. The animal room light cycle was maintained on 12 h: 12 h, light:dark basis and temperature was maintained at 23-25* C.
Chemicals and culture media. Toluene (Sigma Chemical Company, St Louis, MO) was dissolved in tert-butanol (Sigma) and further diluted to concentrations of 86.7, 8.67, 0.867, and 0.0867 Ixg/ml in BMOC-3 medium (Gibco BRL, Grand Island, NY) (pH = 7.5 4-0.1; Osm = 290 _+ 10). The culture media were held at 1 - 4 ~ for a maximum of 3 weeks after opening the stock solution bottle. The medium was filtered with 0.22 I.tm sterile filter units (Milipore Products Division, Bedford, MA). DNA-specific fluorochrome Hoechst 33258 was dissolved in the BMOC-3 medium at a final concentration of l0 ~tg/ml. Sperm motility. Male B6D2F1 mice ( 3 - 6 months of age) were used.
Offprint requests to: W. R. Dukelow
Mice were sacrificed by cervical dislocation. The caudae epididymides
444 Table 1. Effect of toluene on mouse sperm motility in BMOC-3 medium during first 4 h incubation Group
% Motile sperm
Control A Control B 0.0867 p.glml (r) 0.867 [tg/ml (T) 8.67 ~tg/ml (T) 86.7 p.g/ml (T)
15-30 min (n = 8)
6 0 - 9 0 rain (n = 5)
4 h (n = 6)
74.2 +__4.6 74.1 ___3.9 71.4-+6.4 57.8-+6.7* 46.9-+6.8* 5.9-+5.7*
45.0 +__4.2 50.0___4.1 45.4 -+5.5 37.0___3.4* 27.9-+5.6* 4.4__.6.2*
28.0 _+2.0 31.1 --+7.9 25.8 ___5.3 18.7-+4.3" 17.3-+6.0" 2.0-+4.5*
Control A is BMOC-3 medium and Control B is BMOC-3 medium with 1% of t-butanol; T = toluene; Values are mean _ ___SEM, * p
Archives of
Toxicology 9 Springer-Verlag 1992
Short communication
Cellular toxicity of toluene on mouse gamete cells and preimplantation embryos Frank D. Yelian and W. Richard D u k e l o w Endocrine Research Center, Michigan State University, East Lansing, Michigan 48824, USA Received 7 November 1991/Accepted 25 February 1992
Abstract. Toluene is an aromatic hydrocarbon, which has been used in the paint, lacquer and glue industry. It has been detected in municipal water supplies. Previous mouse in vivo studies indicated that toluene administrated by gavage increased the embryonic mortality. The present in vitro study demonstrated that a concentration of toluene higher than 8.67 ~g/ml not only decreased sperm motility and inhibited fertilization, but also significantly increased preimplantation embryo degeneration. At lower levels no effects were observed and the adverse effect levels were approximately 780 fold higher than reported levels in municipal water supplies. Key words: Toluene - Spermatozoa - Oocytes - In vitro
fertilization - Preimplantation embryo
ally inhaled toluene in high doses throughout pregnancy (Goodwin 1988). Toluene has been shown to be teratogenic (cleft palate) in mice by Nawrot and Staples (1979). Similarly an inhalation study by Courtney et al. (1986) showed that toluene administered by inhalation at 2 0 0 400 ppm to CD-1 mice from days 6 to 16 of gestation was teratogenic but not fetotoxic. The limited studies on the teratogenic potential of toluene indicate that toluene could have some teratogenic and toxic effects on the mammalian fetus. The presence of toluene at low levels in municipal drinking water emphasizes the importance of determining if such an effect can occur. No in vitro toxicological studies on the mammalian gamete cells after exposure to toluene are available. The present study was designed to test the direct effects of toluene on mouse epididymal sperm motility, fertilizing capacity and preimplantation embryonic mortality in vitro.
Introduction Materials and m e t h o d s
Toluene has been detected in municipal water supplies at levels ranging from 0.1 l.tg/1 to 11 gg/1. The toluene metab01ites benzaldehyde and benzoic acid were also found in municipal water at concentrations up to 19 gg/1 (EPA 1980). Mouse and rat experiments show impaired growth of the mother and fetus and fetal skeletal anomalies after exposure to large doses of toluene (Hudak and Ungvary 1978 and Shigeta et al. 1982). Behavioral effects of toluene in mice exposed pre- and postnatally have also been described (Kostas and Hotchin 1981). Two children with multiple malformations were born to mothers who worked as shoemakers and were chronically exposed to toluene and trichlorethylene, used in a soling solution (Euler 1967). Additionally, there has been at least one report of congenital defects in children born to mothers who had intention-
Experimental animals. The animals used in this study were B6D2FI mice (Cosby et al. 1989). The mice were either purchased from the Jackson Laboratory (Bar Harbor, ME) or produced at the Endocrine Research Center of Michigan State University by crossing C57BL-6J and DBA-2J mice. B6D2F1 mice were sexed at 21 days of age, and maintained in separate cages. They were provided Breeder Blox| (Wayne Pet Food Division, Chicago, IL) and fresh water daily, ad libitum. The animal room light cycle was maintained on 12 h: 12 h, light:dark basis and temperature was maintained at 23-25* C.
Chemicals and culture media. Toluene (Sigma Chemical Company, St Louis, MO) was dissolved in tert-butanol (Sigma) and further diluted to concentrations of 86.7, 8.67, 0.867, and 0.0867 Ixg/ml in BMOC-3 medium (Gibco BRL, Grand Island, NY) (pH = 7.5 4-0.1; Osm = 290 _+ 10). The culture media were held at 1 - 4 ~ for a maximum of 3 weeks after opening the stock solution bottle. The medium was filtered with 0.22 I.tm sterile filter units (Milipore Products Division, Bedford, MA). DNA-specific fluorochrome Hoechst 33258 was dissolved in the BMOC-3 medium at a final concentration of l0 ~tg/ml. Sperm motility. Male B6D2F1 mice ( 3 - 6 months of age) were used.
Offprint requests to: W. R. Dukelow
Mice were sacrificed by cervical dislocation. The caudae epididymides
444 Table 1. Effect of toluene on mouse sperm motility in BMOC-3 medium during first 4 h incubation Group
% Motile sperm
Control A Control B 0.0867 p.glml (r) 0.867 [tg/ml (T) 8.67 ~tg/ml (T) 86.7 p.g/ml (T)
15-30 min (n = 8)
6 0 - 9 0 rain (n = 5)
4 h (n = 6)
74.2 +__4.6 74.1 ___3.9 71.4-+6.4 57.8-+6.7* 46.9-+6.8* 5.9-+5.7*
45.0 +__4.2 50.0___4.1 45.4 -+5.5 37.0___3.4* 27.9-+5.6* 4.4__.6.2*
28.0 _+2.0 31.1 --+7.9 25.8 ___5.3 18.7-+4.3" 17.3-+6.0" 2.0-+4.5*
Control A is BMOC-3 medium and Control B is BMOC-3 medium with 1% of t-butanol; T = toluene; Values are mean _ ___SEM, * p
