2001 Progress Report: Frog Deformities: Role of Endocrine Disruptors During DevelopmentEPA Grant Number: R827398
Title: Frog Deformities: Role of Endocrine Disruptors During Development
Investigators: Gardiner, David M. , Blumberg, Bruce
Institution: University of California - Irvine
EPA Project Officer: Carleton, James N
Project Period: October 1, 1999 through September 30, 2002
Project Period Covered by this Report: October 1, 2000 through September 30, 2001
Project Amount: $1,194,536
RFA: Endocrine Disruptors (1999) RFA Text | Recipients Lists
Research Category: Economics and Decision Sciences , Endocrine Disruptors , Health , Safer Chemicals
The objective of this research project is to assess the significance of endocrine disruptors that activate retinoid signaling pathways for their role in causing limb developmental deformities in frogs, and to understand their mechanism of action to assess their implications for human health.
There are multiple developmental windows during Xenopus and Rana early limb bud development during which disruption of retinoid signaling induces multiple phenotypes. During this past year, we have completed our experiments on the effects of a known activator of RAR-mediated signaling (tetrahydro tetramethyl napthalenyl propenyl benzoic acid (TTNPB)) on limb bud stages of developing Xenopus tadpoles. Equivalent studies in Rana sylvatica also have been completed. A manuscript is in preparation for publication. We have determined that all the phenotypes observed in wild populations of frogs can be induced in the laboratory by TTNPB exposure at appropriate stages of development.
We have confirmed and extended our results from pilot experiments indicating that there are multiple developmental windows of sensitivity during limb bud development, and that these windows are remarkably short. Treatment at all stages is developmentally toxic at high doses and long exposures, as evidenced by the death of larvae a few weeks after exposure. At lower dosages, survival is high; and, between Stg. 50-53, the rate of malformation is high. Treatment at Stg. 48/49 has no observed phenotypic effects on surviving tadpoles. Treatment at Stg. 50/51 induces duplicated limb buds. Treatment at Stg. 51/52 induces bony triangles. Treatment at Stg. 52/53 induces the loss of distal limb structures. All effects are restricted to the hindlimbs as observed in the wild. Equivalent results have been obtained for studies of Rana.
This past year, we have begun to identify the downstream target genes of TTNPB involved in the induction of each of the various limb malformations. We are using the assay paradigm that we have developed to expose tadpoles to appropriate doses of TTNPB at appropriate stages to induce the different limb deformities. We then are using reverse transcriptase-polymerase chain reaction (RT-PCR) to screen for changes in expression of several genes identified as likely targets of RAR-mediated signaling. We currently are examining the stage-specific expression of molecular markers of limb development, such as BMP-4, Shh, Gdf-5, Xhoxa11, Xhoxa13, Meis-2, Wnt-14, Xhoxd-11 and Xhoxd-13.
Analytical high performance liquid chromatography (HPLC) is guiding fractionation and identification of RAR activating compounds. Solid-phase resin (XAD-2) extracts of lake water from the Minnesota Crow Wing B (CWB) site now have been fractionated over four sequential HPLC columns. Fractions were tested for transactivation of RAR activity using GAL-RARs and UAS-luciferase reporter constructs transiently transfected into Cos-7 cells. The activity profile was used to guide the fractionation. The activity is consistently present in water samples.
After initial fractionation on preparative and semi-preparative C18 reverse phase columns, the activity now has been resolved into three distinct peaks of activity with baseline separation using a C18 analytical reverse phase column with mixed solvent elution. Approximately 10 peaks readily are observed in the ultraviolet (UV) chromatogram with UV maxima around less than 200, 230, and 280 nm.
Electrospray mass spectroscopy (ES-MS) indicates a number of prevalent ions (Table 1) present in the fractions. Exact masses have been obtained for all ions and putative molecular formulae deduced. At least two series of ethoxyl polymer derivatives (series A and B) separated by 44 a.m.u. can be detected. A second series of ions 399.12/473.14 and 397.16/471.17 may be related compounds differentiated by the saturation of a double bond.
The resolution of the activity into three distinct peaks, the similarity in UV absorbance spectra, and the presence of polyethoxyl (n = 3-11) species suggest that the RAR activities may be a man- made polymer derivative or geometric isomers.
Further mass spectroscopic analyses using HPLC ES-MS/MS and EI GC-MS techniques currently are being used to confirm and solve the structures of these ions.
Table 1. Prevalent ions present in the CWB XAD-2 active fractions
Mass Peaks (m/z+) detected:
|399.12/473.14||Related to 397.12/473.14 by saturation of a double bond ?|
|331.15/375.17/419.18||Ethoxyl polymer series A|
|551.23/629.26/683.28/727.31/771.30||Ethoxy polymer series B|
Using the parameters established from our studies of TTNPB effects on stages of Xenopus limb development, we have established a developmental toxicology assay to screen the activity of a large number of known pesticides. Chemicals were selected by a variety of criteria, including known effects on retinoid-sensitive signaling pathways and known presence in water samples from the CWB site. These chemicals include atrazine, cyanazine, trifluralin, azimphos-ethyl, permethrin, triphenyltin acetate, phenothiazine, carbaryl, nonylphenol, and MCPA. Of these, two have tested positive in the preliminary screens and we are working to confirm the reproducibility of these findings. An additional preliminary finding is that a native species, the American toad Bufo americanus, is more sensitive to the effects of one of the suspect teratogens than is Xenopus laevis. We are continuing to determine whether or not this is the case, because such a finding would necessitate further comparative studies to determine the extent to which environmental concentrations of such a teratogen would pose an environmental and/or human health risk. Experiments with 4-heptylbenzoic acid, nonylphenol and preliminary studies of joint toxicity of phenothiazine, permethrin, azimphos ethyl and p-terphenyl are in progress with Rana pipiens tadpoles.
Our continued field studies this year have indicated that populations of amphibians continue to decline, and that new populations of deformed frogs continue to be discovered. Intensive field surveys at the CWB site (tape recorder to monitor calling by breeding frogs and wading census) throughout the spring and summer, revealed that Rana pipiens still are nearly absent (only three adults, no choruses recorded). The previously low breeding populations of adults R. clamitans, R. septentrionalis, and Bufo americanus also now are absent. Continued weekly CWB surveys through prospective mink, green, and leopard frog metamorphosis period (about 5/23-8/22) to collect data on newly metamorphosed animals indicated that the frequency of malformations is lower than in previous years (8.8 percent for mink frogs and 6.3 percent for green frogs). No leopard frog metamorphs were seen. The array of malformation types also was somewhat different, with the predominant type being abnormal jaws, followed by animals with unresorbed tails with adult pigment (whereas in previous years the predominant types of malformations were skin webbings and hindlimb malformations). We also surveyed eight other sites where malformed frogs were observed in previous years, and noticed that although the frequency was significant, it was lower than in the past (< 5 percent as compared to 5-10 percent).
Future Activities:We will continue to screen pesticides that are known to be used in areas where populations of deformed frogs are found. We are particularly focused on two that have given positive results in our Xenopus limb bud teratogenesis assay. Experiments with 4-heptylbenzoic acid, nonylphenol, and preliminary studies of joint toxicity of phenothiazine, permethrin, azimphos ethyl, and p-terphenyl are in progress with Rana pipiens tadpoles. Because these pesticide formulations include various inert ingredients, we also have begun to test for the teratogenic activity of several of those chemicals as well.
We have completed the pilot experiments to optimize the techniques for collecting RNA and using RT-PCR on exposed and control limb buds to test for effects on expression of target genes. During Year 3, we will use these techniques to identify genes that are targets of the disruption of TTNPB-sensitive signaling pathways. We currently are examining the stage-specific expression of molecular markers of limb development such as BMP-4, Shh, Gdf-5, Xhoxa11, Xhoxa13, Meis-2, Wnt-14, Xhoxd-11, and Xhoxd-13. We then will examine the effect of exposure to teratogenic doses of TTNPB.
We continue to use the solid-phase extraction protocol developed to sample sufficiently large volumes of lake water to purify the RAR-activating chemical. Further mass spectroscopic analyses using HPLC ES-MS/MS and EI GC-MS techniques currently are being used to confirm and solve the structures of these ions.
Once we have solved the structure of the RAR-activating chemical, we will use our frog limb bud teratogenesis assays to determine the parameters under which the identified compound(s) do or do not induce malformations.
Having identified the RAR-activating chemical, we will use mass spectroscopy to assay water samples from a number of sites in areas of the country where deformed frogs are found. We have already collected and archived samples from several such sites, and if the chemical has a reasonably long environmental half-life, we will be able to determine its prevalence over the years of the current research program.