Grantee Research Project Results
Final Report: Mechanistic Approach to Screening Chemicals and Mixtures for Endocrine Activity Using an Invertebrate Model
EPA Grant Number: R831300Title: Mechanistic Approach to Screening Chemicals and Mixtures for Endocrine Activity Using an Invertebrate Model
Investigators: LeBlanc, Gerald A.
Institution: North Carolina State University
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 2003 through August 31, 2006 (Extended to May 31, 2007)
Project Amount: $391,598
RFA: Development of High-Throughput Screening Approaches for Prioritizing Chemicals for the Endocrine Disruptors Screening Program (2003) RFA Text | Recipients Lists
Research Category: Endocrine Disruptors , Environmental Justice , Human Health , Safer Chemicals
Objective:
The overall objective of this proposed research program was to generate a sound foundation for the development of mechanism-based high-throughput screening tests for evaluating endocrine-disrupting properties of chemicals in invertebrates. The currently proposed screening and testing scheme for endocrine-active compounds contains a tier 2 test aimed at establishing the No Observed Effect Level (NOEL) for such chemicals with an invertebrate species. However, no tier 1 screen with an invertebrate is proposed as a preamble to the tier 2 test. Thus, no mechanistic information will exist to support any judgment as to whether effects observed during the tier 2 test are truly due to endocrine toxicity.
This research utilized a model invertebrate in which hormones that are functionally analogous to vertebrate estrogens, androgens, and thyroid hormones are known, and the physiological endpoints they regulate have been well characterized. Specifically, ecdysteroid-dependent and terpenoid-dependent endpoints were evaluated for use in a high-throughput format with the crustacean daphnid Daphnia magna.
Specific aims were as follows.
- Identify relevant endpoints of endocrine activity that could be formatted into a high-throughput screening assay to assess endocrine activity of chemicals towards invertebrates.
- Investigate receptor cross-talk and interactive effects of endocrine toxicants as a consequence of receptor cross-talk.
- Evaluate the relationships between effects observed in the high-throughput mechanistic screens and true endocrine toxicity.
Summary/Accomplishments (Outputs/Outcomes):
Identify Relevant Endpoints of Endocrine Activity That Could be Formatted Into a High-Throughput Screening Assay To Assess Endocrine Activity of Chemicals Towards Invertebrates
Efforts were initially devoted to developing and refining an integrated screening paradigm with which: (1) maternal daphnids are exposed to the chemical and developmental abnormalities of offspring are scored as an indicator of altered ecdysteroid signaling; and (2) aberrant sex determination among offspring is evaluated as an indicator of terpenoid signaling activity (Figure 1). We had previously demonstrated that these developmental processes are regulated by the respective hormone signaling pathways (Mu and LeBlanc, 2002; Olmstead and LeBlanc, 2002; Olmstead and LeBlanc, 2003).
Figure 1. Screening Assay Used To Evaluate Chemicals for Terpenoid and Antiecdysteroid Activity Using the Crustacean D. magna
Terpenoid Signal Disruption
We initially evaluated endpoints that could be used to detect terpenoid activity using 18 chemicals (Table 1). Positive responses were detected with methyl farnesoate, the putative endogenous terpenoid hormone, and juvenile hormone III, the insect homolog to methyl farnesoate. Significant activity also was detected with pyriproxyfen, fenoxycarb, and methoprene. These compounds are known juvenile hormone mimics in insects. Finally, weak terpenoid activity was detected with the cyclodiene insecticide dieldrin. Assays performed repeatedly with compounds that gave either strong positive, weak positive, or negative responses were 100% consistent indicating that the assay is not prone to false positive or negative responses. Five candidate chemicals were evaluated for antiterpenoid activity and none registered positive (Figure 2). Four chemicals (all trans-retinoic acid, methoprene, kinoprene, bisphenol A) also were evaluated for their ability to potentiate the activity of the endogenous hormone methyl farnesoate (Figures 3, 4). All registered positive. Results demonstrate that this in vivo assay can be used to effectively screen chemicals for terpenoid-modulating activity.
Table 1. Terpenoid Agonist Activity as Measured by the Ability of the Test Chemicals To Stimulate the Production of Male Offspring
Figure 2. Screening of Five Chemicals for Antiterpenoid Activity. Assays were performed in the presence of 100 ng/l pyriproxyfen (PP) as the terpenoid and the ability of the test chemicals to elicit terpenoid activity (in the absence of pyriproxyfen) or antagonize the terpenoid activity associated with pyriproxifen was evaluated. Antiterpenoid activity was evaluated with: A, all trans-retinoic acid (RA); B, 20-hydroxyecdysone (20E); C, fenarimol (FEN); D, tert-amylphenol (TAP); E, cis-chlordane. White bars: percentage male offspring in male-containing broods. Error bars represent the SEM. Gray bars: percentage broods (third brood from the 10 maternal daphnids) that contained male offspring. An asterisk indicates that the test chemical significantly (p < 0.05) altered the terpenoid activity as compared to that measured with pyriproxyfen alone.
Figure 3. Potentiation of Terpenoid Activity Associated With Methyl Farnesoate (MF) by All trans-Retinoic Acid. White bars: percentage male offspring in male-containing broods. Error bars represent the SEM. Gray bars: percentage broods that contained male offspring. An asterisk indicates that the test chemical significantly (p ≤ 0.05) elevated terpenoid activity over that measured with methyl farnesoate alone.
Figure 4. Potentiation of Terpenoid Activity Associated With Methyl Farnesoate (MF) by: A, Methoprene (MP); B, Kinoprene (KP); and, C, Bisphenol A (BPA). White bars: percentage male offspring in male-containing broods. Error bars represent the SEM. Gray bars: percentage broods that contained male offspring. An asterisk indicates that the test chemical significantly (p ≤ 0.05) altered the terpenoid activity as compared to that measured with methyl farnesoate alone.
We occasionally observed a bilateral gynandromorph (daphnids that are morphologically male on one side of the body and female on the other) in our experiments. We hypothesized that gynandromorphs may be a more sensitive indicator of endocrine disruption than overt male sex determination. We therefore undertook an investigation of the causes of gynandromorphism and discovered that gynandromorphism is initiated by the sex-determining hormone methyl farnesoate when levels of the hormone are intermediate between low levels that stimulate the production of broods containing all-female offspring and high levels that stimulate the production of broods of all-male offspring (Figure 5). The incidence of hormonally induced gynandromorphism was low (0.14% at the maximum stimulatory hormone concentrations) but was significantly increased (46-fold) when the animals were hormone-treated at 30°C. Some environmental chemicals also can stimulate the gynandromorphic phenotype as we demonstrated with the insecticide pyriproxyfen (Figure 6). Gynandromorphism occurs due to inadequate signaling of male-sex determination since: (a) gynandromorphs did not occur in a population that was producing only female offspring; and (b) conditions that stimulated gynandromorphism also reduced the incidence of male offspring. We suggest that male sex determination occurs as a result of methyl farnesoate signaling just after the first embryonic cleavage and bilateral gynandromorphism occurs as a consequence of signaling to only one of the daughter cells. These results identify an endocrine pathway which, if perturbed by environmental factors, can result in aberrant sex determination. Since bilateral gynandromorphs occur at the EC50 for male sex determination, and since the incidence of gynandromorphs is very low, we concluded that this endpoint posed no advantage over male sex determination in a high-throughput screening assay. However, a significant incidence of gynandromorphs in field populations would be highly indicative of endocrine disruption that might not be detected by the occurrence of males which are normally found in field populations.
Figure 5. Female (panel A), male (panel B), and gynandromorphic (panel C) D. magna. Differentiating sex characteristics include the pair of minute first antennae (FA) of the females and the elongated FA of the males. Gynandromorphic individuals possessed an elongated FA partnered with a diminutive antenna. The female-like diminutive FA is obscured by the male-like elongated FA in the micrograph. The bivalved-like carapace of the female has two uniform, symmetrical edges (CE). Both CEs of the male are asymmetrical and are edged by setae. The gynandromorphic daphnid has one female-like symmetrical CE and one male-like asymmetrical CE.
Figure 6. Incidence of Gynandromorphic Offspring Produced Among Maternal Daphnids Exposed to Various Concentrations of Pyriproxyfen at 20oC (panel A) and 30oC (panel B). Panel A: Results from exposure to ranges of pyriproxyfen concentrations are presented to provide sufficient numbers of offspring per treatment to detect low incidences of gynandromorphism. Between 881 and 3,605 neonates were examined at each treatment level. Panel B: Maternal daphnids (5 per treatment level) were treated with the indicated concentration of insecticide and elevated temperature for 24 hours during ovarian oocyte maturation. Between 143 and 204 neonates were examined at each treatment level.
Ecdysteroid Signaling Disruption
We identified specific developmental abnormalities in neonatal daphnids that are indicative of prenatal exposure to chemical with antiecdysteroidal properties (Figure 7). Next, we evaluated the use of these biomarkers in the screening format described above to detect chemicals that elicit antiecdysteroidal activity. This evaluation was undertaken using both chemicals with known antiecdysteroidal activity and with water samples from an extensive surface water sampling throughout North Carolina. The pure chemicals found to have antiecdysteroid-like activity included testosterone, triclosan, sodium nitrate, 4-nonylphenol, fenarimol, and piperonyl butoxide. Antiecdysteroidal activity could be attributed to antagonistic activity towards the ecdysteroid receptor (testosterone), inhibition of ecdysteroid synthesis (fenarimol, piperonyl butoxide), and possible interaction with downstream transcription factors (sodium nitrate).
Figure 7. Developmental Abnormalities Among Neonatal Daphnids Resulting From Exposure to the Antiecdysteroidal Fungicide Fenarimol. a: Incidence of developmental abnormalities resulting from maternal exposure to concentrations of fenarimol. The open circle represents the incidence of developmental abnormalities among offspring produced by 10 control daphnids. b: normal (control) neonatal daphnid. c–f: developmental abnormalities resulting from fenarimol exposure. Daphnids depicted in micrographs c-f are the same age as the control neonate depicted in b.
Among the surface water samples, antiecdysteroid-like activity was detected in Six Runs Creek in eastern North Carolina. Six locations along the Six Runs Creek served as the study site. This tributary of the Cape Fear River provides drainage to an area replete with concentrated swine feeding operations. Water was sampled from the six locations (two upstream, two midstream, and two downstream) in spring and again in autumn. Water samples were returned to the laboratory, and either used in the screening assay or were extracted for analyses of testosterone and 17β-estradiol concentrations by radioimmunoassay (RIA) or liquid chromatography/mass spectrometry (LC/MS). Daphnids exposed to water from the two upstream sites sampled in spring produced offspring with an increased incidence of developmental abnormalities indicative of disrupted ecdysteroid signaling (Table 2). Daphnids placed in midstream and downstream samples elicited a negative response in the screening assay. Testosterone and 17β-estradiol was detected in the upstream samples in the range of ~2–20 ng/L. Lab studies revealed that testosterone interferes with embryo development with a threshold concentration of greater than 30 ng/L but less than 300 ng/L (Figure 8A). Thus, testosterone levels measured in the surface waters approached levels that cause developmental abnormalities in crustaceans. 17β-estradiol had no such effect (Figure 8B). Water samples taken in autumn contained significantly lower testosterone and 17β-estradiol concentrations than in the spring (< 1 ng/L) and daphnids reared in the samples produced no abnormal offspring. These results suggest that testosterone levels in surface waters in this region may seasonally attain sufficiently high levels to interfere with ecdysteroid signaling in Crustacea. Comparison of RIA and LC/MS analyses of water samples for the hormones demonstrated that either method is acceptable for the analyses of surface water samples.
Table 2. Embryo Abnormalities Among Neonatal Daphnids Maternally Exposed to Water Samples From Six Runs Creek Along With Measures of 17β-Estradiol and Testosterone in the Sampled Water
Figure 8. Embryo Abnormalities Resulting From Maternal Exposure to Testosterone (A) or 17 β-Estradiol (B)
In the course of these experiments, we discovered that exposure of maternal daphnids to some chemicals caused them to develop red coloration due to increased hemoglobin production (Figure 9). We viewed this as a potential endpoint of endocrine disruption that may be conducive to a high-throughput assay. We demonstrated that hemoglobin accumulation and male offspring production are co-elevated by the terpenoid signaling compounds (Figure 10). Potency of various compounds to induce both hemoglobin and male offspring was highly correlated, suggesting that both processes are regulated by the same terpenoid signaling pathway (Figure 10). Six clones of the D. pulex/pulicaria species complex that were previously characterized as unable to produce male offspring and five clones that were capable of producing males were evaluated for both hemoglobin induction and male offspring production in response to methyl farnesoate. Four of the five male-producing clones produced both hemoglobin and male offspring in response to the hormone (Table 3). Five of the six obligate parthenogenetic clones produced neither hemoglobin nor males in response to the hormone. These results provide additional evidence that both physiological processes are regulated by the same hormone signaling pathway. Furthermore, results indicate that the non-male-producing clones are largely defective in some methyl farnesoate signaling component downstream from methyl farnesoate synthesis but upstream from the genes regulated by the hormone. A likely candidate for the site of the defect is the methyl farnesoate receptor. Based upon results of this investigation, increased hemoglobin production, detected either as increased coloration, spectrophotometric hemoglobin measurements, or quantification of hemoglobin mRNA levels by quantitative RT-PCR is now routinely used in our screening of chemicals for endocrine activity.
Figure 9. Induction of Hemoglobin in Daphnids (D. magna) Exposed to 0 or 3 nM Pyriproxyfen. A. Control and exposed daphnids. B. Electrophoretically separated pigmented protein from individual control and exposed daphnids. C. Hemoglobin protein levels (μg/mg protein) in daphnids by absorbance at 415 nm (dark bars) and relative hb2 mRNA levels, normalized to levels of actin transcripts, measured by real-time RT PCR (light bars). Hemoglobin protein levels represent the mean and standard error of four samples, each prepared from 17–20 individuals. Hemoglobin mRNA levels represent the mean and standard error of two samples, each prepared from 30 individuals. An asterisk denotes a significant (p ≤ 0.05) difference between control and pyriproxyfen-treated daphnids (Student’s t test).
Figure 10. Relationship Between the Concentrations of Methyl Farnesoate and Analogs That Elevated Hemoglobin to 70% of the Maximum Level (X Axis) and Concentrations That Caused a 50% Incidence of Male Broods of Offspring (Y Axis) in D. magna. The 70% of maximal induction was used as the descriptor of relative potency for hemoglobin levels because this value could be interpolated from all of the concentration-response relationships established. Correlation coefficient (r2) = 0.999.
Table 3. Responsiveness of Male Producing (MP) and Non-Male Producing (NP) Clones of Daphnids to Methyl Farnesoate (MF). Daphnids were exposed to either 0 or 220 nM methyl farnesoate and evaluated for both male offspring and hemoglobin production. Data are presented as mean ± SE (n = 5–10).
An example evaluation involved the herbicide atrazine. We found during a screening of chemicals that the herbicide atrazine elevated hemoglobin levels in daphnids and hypothesized that atrazine induced hemoglobin in daphnids through the hormonal regulatory pathway. This hypothesis was tested by modeling the combined effects of atrazine and the terpenoid hormone mimic pyriproxyfen on hemoglobin mRNA levels assuming the same mechanism of action (concentration addition model) and, alternatively, assuming different mechanisms of action (response addition model). Model predictions were then compared to experimental assessments of the combined action of these two chemicals on hemoglobin mRNA levels. Changes in hemoglobin expression were evaluated using real-time RT PCR with primers specific to each of three D. magna hemoglobin genes (dhb1, dhb2, and dhb3). Both atrazine and pyriproxyfen significantly elevated levels of the hb2 gene product while having little effect on hb1 and hb3 gene products (Figure 11). Induction of dhb2 by combinations of atrazine and pyriproxyfen did not conform to the concentration addition predictions (Figure 12). Rather, dhb2 induction by these binary combinations was highly consistent with response addition model predictions. These results indicate that atrazine does not induce hemoglobin through the terpenoid hormone signaling pathway. Results from this study demonstrate that mixtures modeling can be used to assess a chemical’s mechanism of action and that atrazine likely stimulates hemoglobin accumulation through an alternative pathway, likely to be the oxygen-sensing pathway. Thus, caution must be exercised when using this endpoint as an indicator of endocrine disruption and can best be used to support indications of endocrine activity as judged by male sex determination.
Figure 11. Time Course of Hemoglobin Induction From Atrazine Exposure. Dhb1 (A), dhb2 (B), and dhb3 (C) mRNA levels among control (○) and 1.0 mg/L atrazine-treated (■) daphnids were measured by real-time RT PCR. Hemoglobin mRNA levels were normalized to β-actin levels in the same sample and are relative to the time zero controls. Each data point represents the mean (± standard error) from two replicate samples. Asterisks denote significant (p < 0.05) increases in hemoglobin mRNA relative to levels at 0 hours (ANOVA, Tukey HSD test).
Figure 12. Dhb2 mRNA Levels Following Exposure to Mixtures of Atrazine and Pyriproxyfen Compared to Concentration Addition (Dashed Line) and Response Addition (Dotted Line) Model Predictions. Dhb2 mRNA levels in daphnids were measured by real-time RT PCR at four mixtures of atrazine and pyriproxyfen. Data points represent the mean (± standard deviation) from three replicate samples.
Investigate Receptor Cross-Talk and Interactive Effects of Endocrine Toxicants as a Consequence of Receptor Cross-Talk
Within this objective, we set out to clone the two major hormone receptors in daphnids, the ecdysone receptor (EcR) and the retinoid X-receptor (RXR). Evidence indicates that RXR dimerizes with EcR to produce an ecdysteroid-activated transcription factor. RXR also appears to function independently to regulate gene transcription. Our preliminary data indicated that RXR may be transcriptionally activated by the hormone methyl farnesoate. Efforts to clone these receptors were successful, though more challenging than anticipated. A full length EcR cDNA (2,619 base pairs) was cloned from daphnids which coded for a 599-amino acid protein with all of the hallmark characteristics of the EcR of decapod crustaceans and insects (Figure 13). Following completion of this effort, the completely sequenced genome of the related species Daphnia pulex was released (http://daphnia.cgb.indiana.edu). We searched this genome and discovered the existence of two EcR genes, designated EcR-A and EcR-B. Shortly thereafter, Kato, et al. (2007) reported the existence of two EcR cDNAs in D. magna. From these recent results with D. pulex and D. magna, we concluded that we had cloned EcR-A.
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E E L I N R L V Y F Q E E F D Q P S E E
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D L R K I S T S G I H E S D A D A K F K
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Figure 13. Nucleotide and Deduced Amino Acid Sequences of the D. magna EcR
RXR was cloned and characterized with respect to phylogeny, developmental expression, and hormonal regulation. The full length daphnid RXR cDNA was cloned by initial PCR amplification of a cDNA fragment from the highly conserved DNA-binding domain followed by extension of the fragment using rapid amplification of cDNA ends (RACE) PCR. The full length cDNA was 1,888 base pairs in length and coded for a 400-amino acid protein that exhibited the five-domain structure of a nuclear receptor superfamily member (Figure 14). The RXR protein shared significant identity with other NR2B group members. Phylogenetic analyses of the ligand-binding domain of the receptor revealed that daphnid RXR clustered with RXR from decapod crustaceans on a branch of the phylogenetic tree that was distinct from RXRs known to bind retinoic acids and juvenile hormones (Figure 15). Daphnid RXR mRNA levels were greatest in embryos that were early in development and progressively declined through the initial five stages of embryo development. Adult females expressed higher levels of RXR mRNA than did males, and exposure of females to the terpenoid mimic pyriproxyfen reduced RXR mRNA to levels approaching levels in males. RXR mRNA levels in males were refractory to pyriproxyfen. These results show that branchiopod crustaceans dynamically express RXR, which should be evaluated as a candidate receptor for the terpenoid hormone methyl farnesoate.
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V Q E E R Q R N K E K G E M D M D A T S
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G G Q G D M P I D R V L E A E K R V E C
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K D E P Q V N S A T A A L G N I C A A T
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R E K V Y A T L E E Y T R T N Y A D E P
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L E H L F F F K L I G D T P I E S F L L
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E M L E A P A E T -
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Figure 14. Nucleotide and Deduced Amino Acid Sequences of the D. magna RXR cDNA (GenBank Accession Number DQ530508)
Figure 15. Phylogenetic Tree for the Ligand-Binding Domain of RXR. Numbers represent the bootstrap values (%) for 1,000 simulations. Accession numbers are in parentheses.
Our next effort was to express EcR and RXR in cells equipped with a reporter gene that would be expressed in response to ligand-activated EcR and RXR. This would allow for the high-throughput screening of individual chemicals and chemical mixtures for interaction with these receptors. Co-transfection experiments would allow for assessment of cross-talk between receptor-ligand complexes. We have successfully constructed a reporter system consisting of a chimeric Gal4/RXR gene that is constitutively expressed in cells transfected with a luciferase reporter gene. The Gal4 component of the chimeric receptor protein contains the DNA-binding domain allowing for the receptor to bind the Gal4 response element in the luciferase promoter. The RXR component of the chimeric receptor protein contains the ligand-binding domain that binds ligands leading to transcriptional activation of the reporter. This reporter construct was first assembled using the human RXR as a test of the system. The human RXR/Gal4 construct activated the reporter gene in response to 9 cis-retinoic acid and tributyl tin, two known ligands to this receptor. This success indicates that the reporter with the daphnid RXR should function as expected. Construction of the daphnid reporter is near completion. Validation of this reporter system, construction of the EcR reporter, and cross-talk experimentation will continue as alternative sources of funds become available.
Evaluate the Relationships Between Effects Observed in the High-Throughput Mechanistic Screens and True Endocrine Toxicity
Tier 1, high-throughput screening assays have utility in identifying chemicals that have the potential to elicit endocrine toxicity. Characterization of such toxicity is then relegated to tier 2 testing during which the endocrine-related toxicity is characterized and a threshold level (> NOEL < LOEL) for such effects are established. Endocrine toxicity to crustaceans would likely cause disturbances in embryo development, altered sex of offspring, changes in growth rates, changes in molt frequencies, and altered fecundity. All of these effects would likely present as alterations in the number of viable offspring produced (fecundity). Fecundity is the ultimate endpoint of ecological significance. Toxicity resulting from acute exposure (48-hour EC50) and from chronic toxicity (21-day chronic value (CV) based upon reduced fecundity) were derived either experimentally or from published values (https://cfpub.epa.gov/ecotox/) and used to calculate acute:chronic ratios (ACR). ACR values of > 10 were considered indicative of chronic (i.e., endocrine) toxicity (LeBlanc, 2004). ACR values were then compared to results from tier 1-type screening for endocrine activity as described above. Four of five compounds that clearly had no effect on fecundity (dieldrin, tributyltin, atrazine, chlordane, pentachlorophenol) scored negative in the screening assay (Table 4). The single false positive, dieldrin, provided a very weak positive response in the screening assay. This weak response may be indicative of a false positive response, or the weak terpenoid activity detected may be insufficient to have a significant bearing on fecundity. Most importantly, those chemicals that were clearly reproductive toxicants (fenarimol, methoprene, bisphenol A, pyriproxyfen) all scored positive in the screening assay. Two compounds with questionable reproductive toxicity (3-nonylphenol and triclosan) also scored positive in the screening assay. These results suggest that the screening approach is effective in detecting chemicals with endocrine toxicity, though it may be susceptible to false positive responses. Further evaluation with a greater number and variety of compounds is necessary to confirm this preliminary assessment.
Table 4. Results of Tier 1-Type Screening and Tier 2-Type Testing of Environmental Chemicals Using D. magna. Acute toxicity (EC50) was established over 48 hours of exposure. Chronic values were based upon reductions in fecundity over 21 days of exposure.
Conclusions Drawn From This Research
- Male sex determination can be used as an endpoint in tier 1-type screening assays with branchiopod crustaceans (e.g., Daphnia, Ceriodaphnia) to evaluate chemicals for terpenoid hormone activity.
- The occurrence of bilateral gynandromorphs among field populations of branchiopod crustaceans can be indicative of disruption of the terpenoid signaling pathway by environmental chemicals.
- Increased production of hemoglobin by daphnids, as determined visually, spectrophotometrically, or by real time RT PCR, can be used as a biomarker of exposure to terpenoid-active compounds. However, caution must be exercised in its use since hypoxia can produce false positive responses.
- Developmental abnormalities among neonatal daphnids can be used to screen chemicals for antiecdysteroidal activity.
- The cloned receptors, EcR and RXR, can be used in a reporter gene assay to assess the ability of individual chemicals to activate these receptors and to screen chemical mixtures for combined endocrine toxicity involving receptor cross-talk.
- The mechanistic screens identified in this research program are highly effective in identifying chemicals that have endocrine disrupting activity and should be targeted for tier 2 testing.
- The insecticide pyriproxyfen has profound endocrine toxicity towards D. magna. The tier 1-type screen indicated that this compound interferes with normal sex determination of offspring. Reproductive toxicity testing revealed that this compound interferes with normal reproduction with a threshold concentration of 0.048 μg/L and an ACR of 8,333.
References:
Kato Y, Kobayashi K, et al. Cloning and characterization of the ecdysone receptor and ultraspiracle protein from the water flea Daphna magna. Journal of Endocrine 2007;193:183-194.
LeBlanc GA. Basics of environmental toxicology. In: Hodson E, ed. A Textbook of Modern Toxicology. Hoboken, NJ: John Wiley & Sons Publishing, Inc., 2004, pp. 463-478.
Mu X, LeBlanc GA. Environmental antiecdysteroids alter embryo development in the crustacean Daphnia magna. The Journal of Experimental Zoology 2002;292:287-292.
Olmstead AW, LeBlanc GA. The juvenoid hormone methyl farnesoate is a sex determinant in the crustacean Daphnia magna. The Journal of Experimental Zoology 2002;293:736-739.
Olmstead AW, LeBlanc GA. Insecticidal juvenile hormone analogs stimulate the production of male offspring in the crustacean Daphnia magna. Environmental Health Perspectives 2003;111:919-924.
Journal Articles on this Report : 8 Displayed | Download in RIS Format
Other project views: | All 22 publications | 9 publications in selected types | All 9 journal articles |
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Gorr TA, Rider CV, Wang HY, Olmstead AW, LeBlanc GA. A candidate juvenoid hormone receptor cis-element in the Daphnia magna hb2 hemoglobin gene promoter. Molecular and Cellular Endocrinology 2006;247(1-2):91-102. |
R831300 (2005) R831300 (2006) R831300 (Final) R829358 (Final) R832739 (2008) |
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LeBlanc GA. Crustacean endocrine toxicology: a review. Ecotoxicology 2007;16(1):61-81. |
R831300 (Final) R826129 (Final) R832739 (2007) R832739 (Final) |
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Mu XY, Rider CV, Hwang GS, Hoy H, LeBlanc GA. Covert signal disruption: anti-ecdysteroidal activity of bisphenol A involves cross talk between signaling pathways. Environmental Toxicology and Chemistry 2005;24(1):146-152. |
R831300 (2004) R831300 (Final) R829358 (2004) R829358 (Final) R832739 (2008) |
Exit Exit |
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Olmstead AW, LeBlanc GA. The environmental-endocrine basis of gynandromorphism (intersex) in a crustacean. International Journal of Biological Sciences 2006;3(2):77-84. |
R831300 (Final) R832739 (2006) R832739 (2007) R832739 (2008) R832739 (Final) |
Exit Exit |
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Rider CV, Gorr TA, Olmstead AW, Wasilak BA, LeBlanc GA. Stress signaling: coregulation of hemoglobin and male sex determination through a terpenoid signaling pathway in a crustacean. Journal of Experimental Biology 2005;208(Pt 1):15-23. |
R831300 (2004) R831300 (2006) R831300 (Final) R829358 (Final) R832739 (2008) |
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Rider CV, LeBlanc GA. Atrazine stimulates hemoglobin accumulation in Daphnia magna: is it hormonal or hypoxic? Toxicological Sciences 2006;93(2):443-449. |
R831300 (Final) R829358 (Final) |
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Wang HY, Olmstead AW, Li H, LeBlanc GA. The screening of chemicals for juvenoid-related endocrine activity using the water flea Daphnia magna. Aquatic Toxicology 2005;74(3):193-204. |
R831300 (2005) R831300 (Final) R832739 (2008) |
Exit Exit |
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Wang YH, Wang G, LeBlanc GA. Cloning and characterization of the retinoid X receptor from a primitive crustacean Daphnia magna. General and Comparative Endocrinology 2007;150(2):309-318. |
R831300 (2006) R831300 (Final) R832739 (2006) R832739 (2007) R832739 (Final) |
Exit Exit Exit |
Supplemental Keywords:
hazard assessment, endocrinology, computational toxicology,, RFA, Scientific Discipline, Health, PHYSICAL ASPECTS, ENVIRONMENTAL MANAGEMENT, POLLUTANTS/TOXICS, Environmental Chemistry, Health Risk Assessment, Endocrine Disruptors - Environmental Exposure & Risk, Chemicals, Risk Assessments, endocrine disruptors, Biochemistry, Physical Processes, Endocrine Disruptors - Human Health, Biology, Risk Assessment, bioindicator, biomarkers, assays, exposure, animal model, EDCs, exposure studies, endocrine disrupting chemicals, sexual development, mechanistic screening, endocrine disrupting chemcials, animal models, human growth and development, toxicity, estrogen response, invertebrates, invertebrate model, estrogen receptors, hormone production, androgen, assessment technology, ecological risk assessment model, analysis of chemical exposure, exposure assessment, estuarine crustaceansRelevant Websites:
http://www.tox.ncsu.edu/faculty/leblanc/ Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.