No abstract

The toxicokinetics of 2,2,4,4-tetrabromodiphenyl ether (PBDE-47) was studied in the Japanese Medaka (Oryzias latipes) after a single oral exposure followed by termination at specific time points. The effects of repeated oral exposure to PBDE-47 on reproductive performance was assessed using a pair breeding experimental design with fathead minnows (Pimephales promelas) given daily PBDE-47 exposures for 25 days, during which fecundity was measured as an indicator of reproductive performance. Medaka and fathead minnows were orally exposed to PBDE-47 by bioencapsulation in brine shrimp, Artemia sp. In the medaka studies, measurable levels of PBDE-47 were detected in the carcass within 0.25 h with peak levels occurring at 8 h. The body levels of PBDE-47 slowly declined and were still 25% of peak levels at 624 h after dosing. Assimilation of the bioencapsulated dose was at least 80% and may well approach 100%. The PBDE-47 concentration-time profile was fitted to a one-compartment clearance-volume toxicokinetic model and the model-predicted value for elimination half-life was determined to be 281 h and the first-order absorption rate constant was Ka = 0.26 hr(-1). In the fathead minnow study, egg laying in the PBDE-treated breeding pairs stopped after 10 days. The condition factor of PBDE-treated males was significantly reduced (P <0.011) compared with control males, whereas no significant difference was observed in females. Histological examination revealed a greater than 50% reduction in mature sperm in PBDE-47 exposed minnows compared to controls. Collectively, these results suggest PBDE-47 is selectively toxic to sexually mature male fathead minnows.

The increased availability and use of DNA microarrays has allowed the characterization of gene expression patterns associated with exposure to different toxicants. An important question is whether toxicant induced changes in gene expression in fish are sufficiently diverse to allow for identification of specific modes of action and/or specific contaminants. In theory, each class of toxicant may generate a gene expression profile unique to its mode of toxic action. In this study, isogenic (cloned) rainbow trout Oncorhynchus mykiss were exposed to sublethal levels of a series of model toxicants with varying modes of action, including ethynylestradiol (xeno-estrogen), 2,2,4,4'-tetrabromodiphenyl ether (BDE-47, thyroid active), diquat (oxidant stressor), chromium VI, and benzo[a]pyrene (BaP) for a period of 1-3 weeks. An additional experiment measured trenbolone (anabolic steroid; model androgen) induced gene expression changes in sexually mature female trout. Following exposure, fish were euthanized, livers removed and RNA extracted. Fluorescently labeled cDNA were generated and hybridized against a commercially available Atlantic Salmon/Trout array (GRASP project, University of Victoria) spotted with 16,000 cDNA's. The slides were scanned to measure abundance of a given transcript in each sample relative to controls. Data were analyzed via Genespring (Silicon Genetics) to identify a list of up- and downregulated genes, as well as to determine gene clustering patterns that can be used as "expression signatures". The results indicate each toxicant exposure caused between 64 and 222 genes to be significantly altered in expression. Most genes exhibiting altered expression responded to only one of the toxicants and relatively few were co-expressed in multiple treatments. For example, BaP and Diquat, both of which exert toxicity via oxidative stress, upregulated 28 of the same genes, of over 100 genes altered by either treatment. Other genes associated with steroidogenesis, p450 and estrogen responsive genes appear to be useful for selectively identifying toxicant mode of action in fish, suggesting a link between gene expression profile and mode of toxicity. Our array results showed good agreement with quantitative real time polymerase chain reaction (qRT PCR), which demonstrates that the arrays are an accurate measure of gene expression. The specificity of the gene expression profile in response to a model toxicant, the link between genes with altered expression and mode of toxic action, and the consistency between array and qRT PCR results all suggest that cDNA microarrays have the potential to screen environmental contaminants for biomarkers and mode of toxic action.

No abstract

The synthetic estrogen 17 alpha-ethynylestradiol (EE2) is a commonly used oral contraceptive that has been increasingly detected in sewage effluents. This study determined whether EE2 exposure adversely affected reproduction in sexually maturing male rainbow trout (Oncorhynchus mykiss). We exposed male trout to graded water concentrations of EE2 (10, 100, and 1,000 ng/ L) for 62 d leading up to the time of spawning. Semen and blood plasma samples were removed from each fish. Semen was used to fertilize groups of eggs from one nonexposed female. As a measure of fertility, eggs were incubated for 28 d after fertilization to determine the proportion that attained the eyed stage of embryonic development. Additional endpoints also measured included sperm motility, spermatocrit, gonadosomatic and hepatosomatic indices, testis histology, and circulating plasma levels of the sex steroids 17 alpha, 20 beta-dihydroxyprogesterone (17,20-DHP) and 11-ketotestosterone (11-KT). Exposure to 1,000 ng/L of EE2 caused complete mortality of the treatment group by day 57. Exposure to lower EE2 water concentrations (10 and 100 ng/L) caused an increase in sperm density, while a significant reduction in testis mass was observed only in the 100-ng/L exposure group. Most significantly, semen harvested from fish exposed to 10 and 100 ng/L EE2 caused an approximately 50% reduction in the number of eggs attaining the eyed stage of embryonic development. Plasma levels of 17,20-DHP in exposed fish were roughly twice the level of the controls, while levels of 11-KT were significantly reduced in fish exposed to 100 ng/L EE2. These results suggest that sexually maturing male rainbow trout are susceptible to detrimental reproductive effects of short-term exposures to environmentally relevant levels of EE2.

The synthetic estrogen 17 alpha-ethynylestradiol (EE2) is a commonly used oral contraceptive that has been increasingly detected in sewage effluents. This study determined whether EE2 exposure adversely affected reproduction in sexually maturing male rainbow trout (Oncorhynchus mykiss). We exposed male trout to graded water concentrations of EE2 (10, 100, and 1,000 ng/ L) for 62 d leading up to the time of spawning. Semen and blood plasma samples were removed from each fish. Semen was used to fertilize groups of eggs from one nonexposed female. As a measure of fertility, eggs were incubated for 28 d after fertilization to determine the proportion that attained the eyed stage of embryonic development. Additional endpoints also measured included sperm motility, spermatocrit, gonadosomatic and hepatosomatic indices, testis histology, and circulating plasma levels of the sex steroids 17 alpha, 20 beta-dihydroxyprogesterone (17,20-DHP) and 11-ketotestosterone (11-KT). Exposure to 1,000 ng/L of EE2 caused complete mortality of the treatment group by day 57. Exposure to lower EE2 water concentrations (10 and 100 ng/L) caused an increase in sperm density, while a significant reduction in testis mass was observed only in the 100-ng/L exposure group. Most significantly, semen harvested from fish exposed to 10 and 100 ng/L EE2 caused an approximately 50% reduction in the number of eggs attaining the eyed stage of embryonic development. Plasma levels of 17,20-DHP in exposed fish were roughly twice the level of the controls, while levels of 11-KT were significantly reduced in fish exposed to 100 ng/L EE2. These results suggest that sexually maturing male rainbow trout are susceptible to detrimental reproductive effects of short-term exposures to environmentally relevant levels of EE2.

Among proposed uses for microarrays in environmental toxiciology is the identification of key contributors to toxicity within a mixture. However, it remains uncertain whether the transcriptomic profiles resulting from exposure to a mixture have patterns of altered gene expression that contain identifiable contributions from each toxicant component. We exposed isogenic rainbow trout Onchorynchus mykiss, to sublethal levels of ethynylestradiol, 2,2,4,4-tetrabromodiphenyl ether, and chromium VI or to a mixture of all three toxicants Fluorescently labeled complementary DNA (cDNA) were generated and hybridized against a commercially available Salmonid array spotted with 16,000 cDNAs. Data were analyzed using analysis of variance (p<0.05) with a Benjamani-Hochberg multiple test correction (Genespring [Agilent] software package) to identify up and downregulated genes. Gene clustering patterns that can be used as "expression signatures" were determined using hierarchical cluster analysis. The gene ontology terms associated with significantly altered genes were also used to identify functional groups that were associated with toxicant exposure. Cross-ontological analytics approach was used to assign functional annotations to genes with "unknown" function. Our analysis indicates that transcriptomic profiles resulting from the mixture exposure resemble those of the individual contaminant exposures, but are not a simple additive list. However, patterns of altered genes representative of each component of the mixture are clearly discernible, and the functional classes of genes altered represent the individual components of the mixture. These findings indicate that the use of microarrays to identify transcriptomic profiles may aid in the identification of key stressors within a chemical mixture, ultimately improving environmental assessment.

17alpha-Ethynylestradiol (EE2) is a synthetic estrogen identified in sewage effluents. To understand better the absorption kinetics of EE2 and the induction of vitellogenin (VTG) and estrogen receptor alpha (ERalpha) mRNA, we subjected male rainbow trout (Onchorynchus mykiss) to continuous water exposures of 125 ng/L of EE2 for up to 61 d. Trout were either repetitively sampled for blood plasma or serially killed at selected time intervals. Vitellogenin, ERalpha mRNA, and EE2 were measured using enzyme-linked immunosorbent assay and using quantitative polymerase chain reaction and gas chromatography-mass spectrometry, respectively. In separate experiments, trout were exposed to EE2 for 7 d, and hepatic gene expression was assessed using a low- and high-density cDNA microarray. The EE2 was rapidly absorbed by the trout, with an apparent equilibrium at 16 h in plasma and liver. The ERalpha mRNA levels also increased rapidly, reaching near-peak levels by 48 h. In contrast, plasma levels of VTG continuously increased for 19 d. After 61 d, tissues with the highest levels of VTG were the liver, kidney, and testes. Microarray-based gene expression studies provided unexpected results. In some cases, known estrogen-responsive genes (e.g., ERalpha) were unresponsive, whereas many of the genes that have no apparent link to estrogen function or EE2 toxicity were significantly altered in expression. Of the two microarray approaches tested in the present study, the high-density array appeared to be superior because of the improved quality of the hybridization signal and the robustness of the response in terms of the number of genes identified as being EE2 responsive.