Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
In vitro embryotoxicity of petroleum creosote monitored via mouse preimplantation embryo culture Poorni Iyer , J.E. Martin & T. Rick Irvin To cite this article: Poorni Iyer , J.E. Martin & T. Rick Irvin (1992) In vitro embryotoxicity of petroleum creosote monitored via mouse preimplantation embryo culture, Journal of Toxicology and Environmental Health, 37:2, 231-245, DOI: 10.1080/15287399209531667 To link to this article: http://dx.doi.org/10.1080/15287399209531667
Published online: 20 Oct 2009.
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Date: 07 November 2015, At: 02:15
IN VITRO EMBRYOTOXICITY OF PETROLEUM CREOSOTE MONITORED VIA MOUSE PREIMPLANTATION EMBRYO CULTURE Poorni Iyer, J. E. Martin, T. Rick Irvin
Downloaded by [University of Florida] at 02:15 07 November 2015
Laboratory of Toxicology, Department of Veterinary Anatomy, College of Veterinary Medicine, Texas A & M University, College Station, Texas A mouse preimplantation embryo culture system was utilized to characterize the in vitro embryotoxicity of petroleum creosote (PC), a complex mixture of aliphatic and polycyclic aromatic hydrocarbons. ICR mouse embryos, collected on d 3.5 of gestation (blastocyst stage), were exposed for 7 h to varying concentrations of petroleum creosote in serum-supplemented culture medium. Parallel embryo cultures were exposed to PC in medium supplemented with rodent hepatic S9 microsomal fractions to monitor the role of bioactivation in PC-induced embryotoxicity. Embryos were subsequently cultured in control medium for 72 h and observed for viability as well as specific, time-dependent developmental end points—hatching and attachment to the culture dish at 48 h, and trophoblastic outgrowth with a distinct inner cell mass at 72 h. Embryonic viability varied in inverse proportion to PC concentration. Petroleum creosote caused embryolethal effects at concentrations of 33 μg/ml of culture medium and 54 μg/ml. Embryotoxicity was not observed at 22 μg/ml. Culture supplementation with rodent hepatic S9 fractions did not modify, either qualitatively or quantitatively, the embryotoxicity of PC in vitro. These findings implicate PC as a prenatal toxicant and support environmental and human health concerns regarding PC exposure from PC-containing chemical waste sites.
INTRODUCTION Petroleum creosote [PC], a distillation product of coal tar, is used principally as a wood preservative and consists of complex mixtures of polycyclic aromatic hydrocarbons [PAHs] (American Wood Preservers' Institute, 1977). The components of PC include known mutagens and carcinogens such as benzo[a]pyrene, fluoranthene, benzanthracene, and phenanthrene (EPA, 1984). Previous studies have reported commercial PC preparations exhibit carcinogenic effects subsequent to skin application in albino mice (Sail and Shear, 1940). Urine samples from Wistar rats exposed to PC also demonstrated mutagenicity, as indicated by an increase in number of revertant colonies, when evaluated in Salmonella Present address for P. Iyer is Institute for Environmental Studies, Louisiana State University, Baton Rouge, LA 70803; and for J. E. Martin, Department of Veterinary Anatomy, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803. Requests for reprints should be sent to T. R. Irvin, Institute for Environmental Studies, Louisiana State University, Baton Rouge, LA 70803. 231 Journal of Toxicology and Environmental Health, 37:231-245, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
Downloaded by [University of Florida] at 02:15 07 November 2015
232
P. IYER ET AL.
typhimurium strains TA98, 100, 1537, and 1538 (Bos et al., 1983a, 1984, 1985). Acute toxicity ascribed to PC has also been observed in livestock accidentally exposed to creosote (Hanlon, 1938). Public health concerns regarding human risks have focused on occupational as well as general environmental exposure to PAHs in creosote (Bos et al., 1983b). 1-Hydroxypyrene, a major metabolite of the PAH pyrene, has been identified in the urine of workers exposed to PC in wood processing and wood preservation industries Gongeneelen et al., 1985). Reports of PC contamination in areas adjacent to abandoned wood processing sites have intensified concern regarding human exposure to PCderived polycyclic aromatic hydrocarbons. Surveys of hazardous waste sites in the southern and southeastern United States indicate over 15% contains PC or PC mixtures (EPA, 1984); PC has been found in 31 of the 1177 sites on the National Priorities List of the EPA (Agency for Toxic Substances and Disease Registry, 1990). The most common routes of exposure around hazardous waste sites are likely to be through the skin, and drinking water contaminated with creosote (Agency for Toxic Substances and Disease Registry, 1990). Groundwater contamination from PC seepage has resulted in closure of a number of municipal wells (Bedient et al., 1984; Hickok et al., 1982). Groundwater contamination from creosote wastewaters and sludge stored in unlined surface impoundments at a wood treatment facility has been reported in Pensacola, Fla. (Goerlitz et al., 1985). Similar contamination problems have been reported in Conroe, Tex. (Borden, 1986) and St. Louis Park, Minn. (Hickok et al., 1982). Creosote may be released to soils at treatment facilities as a result of bleeding of the product from treated timber in stock yard and storage areas and through disposal of the mixture at hazardous waste sites. Rain water may also wash the soluble components directly from the surface of treated timber and into the soil (Henningsson, 1983). While the carcinogenic and mutagenic properties of PC have been investigated, the prenatal effects of this chemical mixture have not been studied. Some individual constituents of PC are known to exhibit embryotoxic properties. Maternally administered individual members of the PAH family have been reported to impair early fetal growth (Pereira et al., 1982). Studies by Pedersen et al. (1978) indicate benzo[a]pyrene, a consistently reported component of PC, is toxic to cultured rodent embryos in the presence of enzymes competent to bioactivate this compound to chemically reactive derivatives. Another PAH, fluoranthene, whose presence has been connected with the observed volatile mutagenicity of creosote (Bos et al., 1987), has been documented to exert embryotoxic effects (Irvin and Martin, 1988). Naphthalene, a bicyclic aromatic hydrocarbon reported in water supplies adjacent to creosotecontaminated waste sites (Bedient et al., 1984), has been demonstrated to traverse the placenta (Anziulewicz, 1959) and exert embryotoxic effects in vitro subsequent to metabolic activation (Iyer et al., 1991). Results of fetal
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EMBRYOTOXICITY OF CREOSOTE
233
translocation and metabolism of PAHs from coal fly ash administered intratracheally to pregnant rats support initiation of cell transformation in the developing fetus (Srivastava et al., 1986). In addition, increasing concern regarding embryotoxicity due to synergism among components of PAH mixtures within PC has been supported by a report of potent developmental toxicity following oral administration of high-boiling point coal liquids to pregnant rats (Hackett et al., 1984). As per the most recent document on the toxicological profile of creosote, no studies were located regarding the developmental and reproductive effects in humans following oral exposure (Agency for Toxic Substances and Disease Registry, 1990). In this study, we have utilized a mouse preimplantation embryo culture system to characterize in qualitative terms the in vitro embryotoxicity of PC. Further, the role of biotransformation in PC-mediated embryotoxicity was evaluated by supplementing culture medium with rodent hepatic microsomal preparations previously demonstrated to be competent to metabolically activate individual PAH creosote constituents (Martin et al., 1989). MATERIALS AND METHODS Chemicals Culture medium NCTC 109, fetal bovine serum, penicillinstreptomycin, and Hanks balanced salt solution [HBSS] powder were obtained from Gibco Laboratories (Chagrin Falls, Ohio). Dimethyl sulfoxide [DMSO], beta-nicotinamide adenine dinucleotide phosphate (reduced form), and glucose 6-phosphate were obtained from Sigma Chemical Company (St. Louis, Mo.). Rat hepatic S9 (a postmitochondrial supernatant—9000 x g fraction), isolated from Aroclor 1254-induced animals, was purchased from Molecular Toxicology Inc. (College Park, Md.). Petroleum creosote [PC] stock solution (CX1984) was obtained from Matheson, Coleman & Bell (Norwood, Ohio). Chemical characterization of the same stock solution has been studied at various fractions of the solution employing GC/MS (Okaygun, 1988). Animal Breeding and Embryo Collection ICR mice from Harlan Sprague-Dawley (Houston, Tex.) were maintained in a 12-h light-dark cycled (lights on between 7:00 a.m. and 7:00 p.m.) environment. All mice were fed Teklad rodent feed (Harlan Sprague-Dawley, Houston, Tex.) and had access to water ad libitum. Animals were housed in polypropylene cages (four per cage) lined with wood bedding material. Females were placed with males overnight and successful mating was determined by the presence of a vaginal copulation plug the next morning. The day of vaginal plug detection was considered gestation d 0. Plug-positive animals were euthanized with chloroform on d 3 of gestation, and whole mouse embryos were collected by
234
P. IYER ET AL.
flushing uterine horns with HBSS as originally described by Hogan et al. (1986). All embryos were examined, and only those in the blastocyst stage of development were used in the study.
Downloaded by [University of Florida] at 02:15 07 November 2015
Culture Methods Embryos were randomly distributed into 11 treatment groups and cultured in NCTC 109 medium supplemented with 10% fetal bovine serum and a 1% penicillin-streptomycin mixture as described earlier by Spielmann et al. (1985). Embryos assigned to chemical-treated groups (each containing 15 embryos) were exposed to PC (dissolved in DMSO) in serum-supplemented medium for 1 h in a 37°C incubator maintaining a humidified atmosphere of 5% CO2 in air. Embryos were exposed to different percentages of the stock solution of PC. Three exposure levels employed—22 /*g creosote/ml of medium, 33 /*g/ml, and 54 jug/ml—are reported in this study. Levels lower than 22 ftg/ml and higher than 54 /*g/ ml manifested a dose-dependent pattern and are not mentioned here so as not to be redundant. Parallel exposures at each concentration were performed, in which mouse embryos were exposed to PC in medium supplemented with rodent hepatic S9 and cofactors as previously described (Irvin and Irgolic, 1988). The exposure time of 1 h was decided on, as the activity of the S9 mixture diminishes after 2 h (A. Spindle, personal communication). Control groups consisted of embryos exposed to the solvent vehicle, DMSO, either with or without S9 supplementation, and an untreated group. Embryos were subsequently transferred to PC-free medium after rinsing and cultured for 72 h under incubator conditions as already described. Microscopic (150 x) evaluations were performed at 24-h intervals monitoring the following parameters: viability at 24 h, hatching and attachment to the culture dish at 48 h, trophoblastic outgrowth with development of a distinct inner cell mass (ICM) at 72 h. Previous reports have documented that development of mouse blastocysts in vitro employing this culture procedure is comparable to developmental progress observed in vivo (Conda and Hsu, 1980; Spielmann et al., 1985; Mertes and Irvin, 1987).
Statistics Using the Z test statistic for a binomial parameter, the proportion of embryos in each group that reached the specific development end point, at the time of observation, was compared with that of control groups at each exposure level. The sample size used for each group met with the requirements suggested by Fleiss (1980), for comparison of proportions for a binomial parameter. The significance level chosen was p < .05 as recommended by Ott (1984).
235
EMBRYOTOX1CITY OF CREOSOTE
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RESULTS Figure 1 summarizes the concentration-dependent embryotoxicity of PC to mouse embryos, cultured in the presence and absence of rodent hepatic microsomal fractions, 24 h after PC exposure and subsequent culture. Mouse embryos evidenced no toxic effects after PC exposure at the 22 [xg of PC/ml of medium concentration, both with and without microsomal culture supplementation. At 33 jug/ml, however, embryonic viability decreased by 43% and 26%, respectively, in groups cultured in medium with or without S9. At 54 ^g/ml, no viable embryos were detected irrespective of microsomal medium supplementation. The embryotoxic effects of PC on mouse embryos after 24 h of culture subsequent to a 1-h exposure to PC are illustrated in Figure 2. Embryos exposed to PC at 22 Mg/ml (Figs. 2a and b) developed to the point of exhibiting a distinct blastocoele with organization of the ICM and layering of mural trophoblastic cells on the zona pellucida. Supplemen-
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Exposure levels of petroleum creosote in media FIGURE 1. Effect of petroleum creosote exposure on embryo viability 24 h after culture. Fifteen embryos were assessed for each exposure level. Each data bar represents percentages with standard deviations. Asterisk indicates significant at .05 level.
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FIGURE 2. (a) Expanded blastocyst 24 h after exposure to petroleum creosote (22 ftg/ml) in the absence of the S9 fraction (250X). (b) Expanded blastocyst 24 h after exposure to petroleum creosote (22 jig/ml) in the presence of the S9 fraction (250 x).
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EMBRYOTOXICITY OF CREOSOTE
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FIGURE 2. {Continued) (c) Mouse embryo displaying degenerative features 24 h after exposure to petroleum creosote (54 ^g/ml) in the absence of the S9 fraction (150 x). (d) Mouse embryo displaying degenerative features 24 h after exposure to petroleum creosote (54 jjg/ml) in the presence of the S9 fraction (150 x).
P. IYER ET AL.
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238
tation of the cell culture medium with S9 did not alter the toxicity of the 22 /*g/ml concentration PC. In contrast, embryos cultured after exposure to PC at 54 ng/m\ (Fig. 2c and d) displayed degenerative features such as shrinkage of the embryonic mass from the zona pellucida and reduction in apparent embryonic volume, in the range of 40-60%. Embryos exposed to PC at the intermediate exposure level, 33 /xg/ml displayed a time dependency in the manifestation of PC-induced effects. Figures 3 and 4 summarize the embryotoxic effects of PC as a function of concentration 48 and 72 h, respectively, after exposure. At 48 and 72 h, the developmental progress of embryos exposed to PC at 22 /*g/ml was not significantly affected. Embryos exposed to 33 /ig/ml PC, however, exhibited decreasing developmental potential with increasing time in culture. Greater than 90% did not attach to the culture dish surface at 48 h, an index for developmental progress at this time point. At 72 h, a
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ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
In vitro embryotoxicity of petroleum creosote monitored via mouse preimplantation embryo culture Poorni Iyer , J.E. Martin & T. Rick Irvin To cite this article: Poorni Iyer , J.E. Martin & T. Rick Irvin (1992) In vitro embryotoxicity of petroleum creosote monitored via mouse preimplantation embryo culture, Journal of Toxicology and Environmental Health, 37:2, 231-245, DOI: 10.1080/15287399209531667 To link to this article: http://dx.doi.org/10.1080/15287399209531667
Published online: 20 Oct 2009.
Submit your article to this journal
Article views: 2
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [University of Florida]
Date: 07 November 2015, At: 02:15
IN VITRO EMBRYOTOXICITY OF PETROLEUM CREOSOTE MONITORED VIA MOUSE PREIMPLANTATION EMBRYO CULTURE Poorni Iyer, J. E. Martin, T. Rick Irvin
Downloaded by [University of Florida] at 02:15 07 November 2015
Laboratory of Toxicology, Department of Veterinary Anatomy, College of Veterinary Medicine, Texas A & M University, College Station, Texas A mouse preimplantation embryo culture system was utilized to characterize the in vitro embryotoxicity of petroleum creosote (PC), a complex mixture of aliphatic and polycyclic aromatic hydrocarbons. ICR mouse embryos, collected on d 3.5 of gestation (blastocyst stage), were exposed for 7 h to varying concentrations of petroleum creosote in serum-supplemented culture medium. Parallel embryo cultures were exposed to PC in medium supplemented with rodent hepatic S9 microsomal fractions to monitor the role of bioactivation in PC-induced embryotoxicity. Embryos were subsequently cultured in control medium for 72 h and observed for viability as well as specific, time-dependent developmental end points—hatching and attachment to the culture dish at 48 h, and trophoblastic outgrowth with a distinct inner cell mass at 72 h. Embryonic viability varied in inverse proportion to PC concentration. Petroleum creosote caused embryolethal effects at concentrations of 33 μg/ml of culture medium and 54 μg/ml. Embryotoxicity was not observed at 22 μg/ml. Culture supplementation with rodent hepatic S9 fractions did not modify, either qualitatively or quantitatively, the embryotoxicity of PC in vitro. These findings implicate PC as a prenatal toxicant and support environmental and human health concerns regarding PC exposure from PC-containing chemical waste sites.
INTRODUCTION Petroleum creosote [PC], a distillation product of coal tar, is used principally as a wood preservative and consists of complex mixtures of polycyclic aromatic hydrocarbons [PAHs] (American Wood Preservers' Institute, 1977). The components of PC include known mutagens and carcinogens such as benzo[a]pyrene, fluoranthene, benzanthracene, and phenanthrene (EPA, 1984). Previous studies have reported commercial PC preparations exhibit carcinogenic effects subsequent to skin application in albino mice (Sail and Shear, 1940). Urine samples from Wistar rats exposed to PC also demonstrated mutagenicity, as indicated by an increase in number of revertant colonies, when evaluated in Salmonella Present address for P. Iyer is Institute for Environmental Studies, Louisiana State University, Baton Rouge, LA 70803; and for J. E. Martin, Department of Veterinary Anatomy, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803. Requests for reprints should be sent to T. R. Irvin, Institute for Environmental Studies, Louisiana State University, Baton Rouge, LA 70803. 231 Journal of Toxicology and Environmental Health, 37:231-245, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
Downloaded by [University of Florida] at 02:15 07 November 2015
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P. IYER ET AL.
typhimurium strains TA98, 100, 1537, and 1538 (Bos et al., 1983a, 1984, 1985). Acute toxicity ascribed to PC has also been observed in livestock accidentally exposed to creosote (Hanlon, 1938). Public health concerns regarding human risks have focused on occupational as well as general environmental exposure to PAHs in creosote (Bos et al., 1983b). 1-Hydroxypyrene, a major metabolite of the PAH pyrene, has been identified in the urine of workers exposed to PC in wood processing and wood preservation industries Gongeneelen et al., 1985). Reports of PC contamination in areas adjacent to abandoned wood processing sites have intensified concern regarding human exposure to PCderived polycyclic aromatic hydrocarbons. Surveys of hazardous waste sites in the southern and southeastern United States indicate over 15% contains PC or PC mixtures (EPA, 1984); PC has been found in 31 of the 1177 sites on the National Priorities List of the EPA (Agency for Toxic Substances and Disease Registry, 1990). The most common routes of exposure around hazardous waste sites are likely to be through the skin, and drinking water contaminated with creosote (Agency for Toxic Substances and Disease Registry, 1990). Groundwater contamination from PC seepage has resulted in closure of a number of municipal wells (Bedient et al., 1984; Hickok et al., 1982). Groundwater contamination from creosote wastewaters and sludge stored in unlined surface impoundments at a wood treatment facility has been reported in Pensacola, Fla. (Goerlitz et al., 1985). Similar contamination problems have been reported in Conroe, Tex. (Borden, 1986) and St. Louis Park, Minn. (Hickok et al., 1982). Creosote may be released to soils at treatment facilities as a result of bleeding of the product from treated timber in stock yard and storage areas and through disposal of the mixture at hazardous waste sites. Rain water may also wash the soluble components directly from the surface of treated timber and into the soil (Henningsson, 1983). While the carcinogenic and mutagenic properties of PC have been investigated, the prenatal effects of this chemical mixture have not been studied. Some individual constituents of PC are known to exhibit embryotoxic properties. Maternally administered individual members of the PAH family have been reported to impair early fetal growth (Pereira et al., 1982). Studies by Pedersen et al. (1978) indicate benzo[a]pyrene, a consistently reported component of PC, is toxic to cultured rodent embryos in the presence of enzymes competent to bioactivate this compound to chemically reactive derivatives. Another PAH, fluoranthene, whose presence has been connected with the observed volatile mutagenicity of creosote (Bos et al., 1987), has been documented to exert embryotoxic effects (Irvin and Martin, 1988). Naphthalene, a bicyclic aromatic hydrocarbon reported in water supplies adjacent to creosotecontaminated waste sites (Bedient et al., 1984), has been demonstrated to traverse the placenta (Anziulewicz, 1959) and exert embryotoxic effects in vitro subsequent to metabolic activation (Iyer et al., 1991). Results of fetal
Downloaded by [University of Florida] at 02:15 07 November 2015
EMBRYOTOXICITY OF CREOSOTE
233
translocation and metabolism of PAHs from coal fly ash administered intratracheally to pregnant rats support initiation of cell transformation in the developing fetus (Srivastava et al., 1986). In addition, increasing concern regarding embryotoxicity due to synergism among components of PAH mixtures within PC has been supported by a report of potent developmental toxicity following oral administration of high-boiling point coal liquids to pregnant rats (Hackett et al., 1984). As per the most recent document on the toxicological profile of creosote, no studies were located regarding the developmental and reproductive effects in humans following oral exposure (Agency for Toxic Substances and Disease Registry, 1990). In this study, we have utilized a mouse preimplantation embryo culture system to characterize in qualitative terms the in vitro embryotoxicity of PC. Further, the role of biotransformation in PC-mediated embryotoxicity was evaluated by supplementing culture medium with rodent hepatic microsomal preparations previously demonstrated to be competent to metabolically activate individual PAH creosote constituents (Martin et al., 1989). MATERIALS AND METHODS Chemicals Culture medium NCTC 109, fetal bovine serum, penicillinstreptomycin, and Hanks balanced salt solution [HBSS] powder were obtained from Gibco Laboratories (Chagrin Falls, Ohio). Dimethyl sulfoxide [DMSO], beta-nicotinamide adenine dinucleotide phosphate (reduced form), and glucose 6-phosphate were obtained from Sigma Chemical Company (St. Louis, Mo.). Rat hepatic S9 (a postmitochondrial supernatant—9000 x g fraction), isolated from Aroclor 1254-induced animals, was purchased from Molecular Toxicology Inc. (College Park, Md.). Petroleum creosote [PC] stock solution (CX1984) was obtained from Matheson, Coleman & Bell (Norwood, Ohio). Chemical characterization of the same stock solution has been studied at various fractions of the solution employing GC/MS (Okaygun, 1988). Animal Breeding and Embryo Collection ICR mice from Harlan Sprague-Dawley (Houston, Tex.) were maintained in a 12-h light-dark cycled (lights on between 7:00 a.m. and 7:00 p.m.) environment. All mice were fed Teklad rodent feed (Harlan Sprague-Dawley, Houston, Tex.) and had access to water ad libitum. Animals were housed in polypropylene cages (four per cage) lined with wood bedding material. Females were placed with males overnight and successful mating was determined by the presence of a vaginal copulation plug the next morning. The day of vaginal plug detection was considered gestation d 0. Plug-positive animals were euthanized with chloroform on d 3 of gestation, and whole mouse embryos were collected by
234
P. IYER ET AL.
flushing uterine horns with HBSS as originally described by Hogan et al. (1986). All embryos were examined, and only those in the blastocyst stage of development were used in the study.
Downloaded by [University of Florida] at 02:15 07 November 2015
Culture Methods Embryos were randomly distributed into 11 treatment groups and cultured in NCTC 109 medium supplemented with 10% fetal bovine serum and a 1% penicillin-streptomycin mixture as described earlier by Spielmann et al. (1985). Embryos assigned to chemical-treated groups (each containing 15 embryos) were exposed to PC (dissolved in DMSO) in serum-supplemented medium for 1 h in a 37°C incubator maintaining a humidified atmosphere of 5% CO2 in air. Embryos were exposed to different percentages of the stock solution of PC. Three exposure levels employed—22 /*g creosote/ml of medium, 33 /*g/ml, and 54 jug/ml—are reported in this study. Levels lower than 22 ftg/ml and higher than 54 /*g/ ml manifested a dose-dependent pattern and are not mentioned here so as not to be redundant. Parallel exposures at each concentration were performed, in which mouse embryos were exposed to PC in medium supplemented with rodent hepatic S9 and cofactors as previously described (Irvin and Irgolic, 1988). The exposure time of 1 h was decided on, as the activity of the S9 mixture diminishes after 2 h (A. Spindle, personal communication). Control groups consisted of embryos exposed to the solvent vehicle, DMSO, either with or without S9 supplementation, and an untreated group. Embryos were subsequently transferred to PC-free medium after rinsing and cultured for 72 h under incubator conditions as already described. Microscopic (150 x) evaluations were performed at 24-h intervals monitoring the following parameters: viability at 24 h, hatching and attachment to the culture dish at 48 h, trophoblastic outgrowth with development of a distinct inner cell mass (ICM) at 72 h. Previous reports have documented that development of mouse blastocysts in vitro employing this culture procedure is comparable to developmental progress observed in vivo (Conda and Hsu, 1980; Spielmann et al., 1985; Mertes and Irvin, 1987).
Statistics Using the Z test statistic for a binomial parameter, the proportion of embryos in each group that reached the specific development end point, at the time of observation, was compared with that of control groups at each exposure level. The sample size used for each group met with the requirements suggested by Fleiss (1980), for comparison of proportions for a binomial parameter. The significance level chosen was p < .05 as recommended by Ott (1984).
235
EMBRYOTOX1CITY OF CREOSOTE
Downloaded by [University of Florida] at 02:15 07 November 2015
RESULTS Figure 1 summarizes the concentration-dependent embryotoxicity of PC to mouse embryos, cultured in the presence and absence of rodent hepatic microsomal fractions, 24 h after PC exposure and subsequent culture. Mouse embryos evidenced no toxic effects after PC exposure at the 22 [xg of PC/ml of medium concentration, both with and without microsomal culture supplementation. At 33 jug/ml, however, embryonic viability decreased by 43% and 26%, respectively, in groups cultured in medium with or without S9. At 54 ^g/ml, no viable embryos were detected irrespective of microsomal medium supplementation. The embryotoxic effects of PC on mouse embryos after 24 h of culture subsequent to a 1-h exposure to PC are illustrated in Figure 2. Embryos exposed to PC at 22 Mg/ml (Figs. 2a and b) developed to the point of exhibiting a distinct blastocoele with organization of the ICM and layering of mural trophoblastic cells on the zona pellucida. Supplemen-
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Exposure levels of petroleum creosote in media FIGURE 1. Effect of petroleum creosote exposure on embryo viability 24 h after culture. Fifteen embryos were assessed for each exposure level. Each data bar represents percentages with standard deviations. Asterisk indicates significant at .05 level.
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P. IYER ET AL.
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FIGURE 2. (a) Expanded blastocyst 24 h after exposure to petroleum creosote (22 ftg/ml) in the absence of the S9 fraction (250X). (b) Expanded blastocyst 24 h after exposure to petroleum creosote (22 jig/ml) in the presence of the S9 fraction (250 x).
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EMBRYOTOXICITY OF CREOSOTE
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FIGURE 2. {Continued) (c) Mouse embryo displaying degenerative features 24 h after exposure to petroleum creosote (54 ^g/ml) in the absence of the S9 fraction (150 x). (d) Mouse embryo displaying degenerative features 24 h after exposure to petroleum creosote (54 jjg/ml) in the presence of the S9 fraction (150 x).
P. IYER ET AL.
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238
tation of the cell culture medium with S9 did not alter the toxicity of the 22 /*g/ml concentration PC. In contrast, embryos cultured after exposure to PC at 54 ng/m\ (Fig. 2c and d) displayed degenerative features such as shrinkage of the embryonic mass from the zona pellucida and reduction in apparent embryonic volume, in the range of 40-60%. Embryos exposed to PC at the intermediate exposure level, 33 /xg/ml displayed a time dependency in the manifestation of PC-induced effects. Figures 3 and 4 summarize the embryotoxic effects of PC as a function of concentration 48 and 72 h, respectively, after exposure. At 48 and 72 h, the developmental progress of embryos exposed to PC at 22 /*g/ml was not significantly affected. Embryos exposed to 33 /ig/ml PC, however, exhibited decreasing developmental potential with increasing time in culture. Greater than 90% did not attach to the culture dish surface at 48 h, an index for developmental progress at this time point. At 72 h, a
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