1999 Progress Report: Use of a Transgenerational Model to Evaluate Threshold Effects of Endocrine Disrupting Chemicals in Fish

EPA Grant Number: R827098
Title: Use of a Transgenerational Model to Evaluate Threshold Effects of Endocrine Disrupting Chemicals in Fish
Investigators: Foran, Christy M. , Benson, William H. , Nimrod, Alison C.
Current Investigators: Foran, Christy M. , Slattery, Marc
Institution: University of Mississippi Main Campus
Current Institution: University of Mississippi
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1998 through September 30, 2001
Project Period Covered by this Report: October 1, 1998 through September 30, 1999
Project Amount: $406,024
RFA: Exploratory Research - Environmental Biology (1998) RFA Text |  Recipients Lists
Research Category: Biology/Life Sciences , Economics and Decision Sciences , Health , Ecosystems

Objective:

The first of five model endocrine disruptors is currently being tested on reproduction in Japanese medaka. The five model chemicals were chosen based on their distinct mechanisms of action. To meet the objective set forth in the grant, we have exposed medaka to purported endocrine disruptors during two critical life stages: sexual differentiation and reproduction.

Octylphenol (OP), cadmium (Cd), atrazine (ATZ), and vinclozolin (VZ) will serve as model endocrine disruptors because of their distinct mechanisms of action. In addition, ethinylestradiol (EE2), a potent estrogen used in oral contraceptives, will be tested. EE2 is distinguished from OP, another estrogen (E2) receptor agonist, because of its 1,000- to 10,000-fold greater affinity for the receptor, and because of its potential entry into the environment post-usage via sewage treatment plants (Desbrow et al., 1996; Aherne and Briggs, 1989; for review, see Arcand-Hoy et al., 1998).

We have begun our assessment of Cd on reproductive function in medaka through exposure of larvae and adult pairs. The adults raised as a result of these exposures (F2 from adult exposure, or mature animals from hatchling exposure) are currently being assessed for changes in reproductive endpoints. These animals are exposed for 14 days at concentrations of 10 µg/L as a maximum tolerated value, 5 µg/L as an environmentally realistic concentration, and 1 µg/L (1/5 the environmental concentration) as the low concentration selected to determine the sensitivity of the response. We determined the concentration necessary to result in 50 percent mortality of medaka hatchlings within 96 hours to be 16 µg/L (LC50).

Progress Summary:

The most important progress in this stage of the research project is the development of reliable assays for most of the biochemical endpoints outlined in the grant proposal.

Reproduction. Pairs of males and females (10 pairs for each exposure group x 3 exposure groups/treatment x (4 concentrations + 1 control) treatments = 150 pairs for each chemical) are housed in separate chambers. The chambers are randomly placed in a single water bath to maintain constant temperature (26 C). Each chamber is examined and eggs are collected. Following egg collection, exposure solutions are renewed for adult exposure.

Egg Development, Hatchability, and Larvae Survival. Collected eggs are separated gently. A subset of the collected eggs is measured with a digital image analyzer to calculate egg size. The remaining eggs are incubated in six-well culture plates with hatching solution and examined daily to record development, hatching, and death of embryos. Following hatch, survival is monitored after 15 days (swim-up occurs at 5 days).

Plasma Hormone Assays. We have developed a method for collection of serum from adult medaka and subsequent analysis of estradiol (E2) and testosterone (T) concentrations using enzyme-linked immunoassay (EIA; EIA method follows Munro and Lasley, 1988). Blood is collected from the aorta of an anesthetized medaka by cutting the isthmus and inserting a heparinized capillary tube. Up to 5 µL of plasma is collected from a single animal, allowed to clot, and spun down to collect the serum; two to three fish are pooled for steroid determination. An antibody against the hormone is bound to the wells of a 96-well microtiter plate. The excess antibody is washed out, and the well is filled with a sample or standard containing the steroid and a constant concentration of steroid conjugated to horseradish peroxidase (HRP). The unbound steroids are washed out and a substrate for HRP is added. The HRP reaction produces a colored product that is measured using a plate reader. Plasma concentrations of estradiol can be determined by this method; however, despite the sensitivity of the assay (0.1 ng/mL), plasma concentrations of testosterone are undetectable from medaka.

Pituitary Function. It has been shown that medaka, both males and females, can be injected with bovine gonadotropin-releasing hormone (GnRH) similarly to that described in the literature (Singh and Singh, 1991). Following 48 hours of GnRH exposure, plasma steroid levels are measured with EIA. Preliminary studies indicate that GnRH injection significantly increases steroid production in untreated animals (Thompson and Benson, unpublished results).

Ex vivo Steroidogenesis. Steroidogenesis is assessed by incubating excised testes and ovaries in tissue culture medium in the presence of 25-hydroxycholesterol. Appearance of E2 and T in the media is measured using EIA. Preliminary studies indicate that incubation of gonads with 25-hydroxychloesterol results in the production of both of these steroids (Thompson and Benson, unpublished results).

Histology. Whole medaka are stunned on ice and fixed in 4 percent paraformaldehyde. Paraffin-embedded fish are sectioned, stained with hematoxylin and eosin, and examined for gonad morphology.

Vitellogenin Production. The relative serum and hepatic vitellogenin concentration from medaka can be determined using a Western blot and an antibody recognizing medaka vitellogenin obtained from N. Denslow (University of Florida). Plasma collected by the method described above is allowed to clot and spun down to collect serum. Although the amount of serum collected is small, the protein concentration is high enough to produce a sample for Western blot analysis.

Vitellogenin Location. Paraffin sections of medaka will be probed with a vitellogenin antibody following a similar protocol to Ortego et al. (1994). Several protocols for immunohistochemistry in medaka are being tried, and experiments with the most effective antibody concentrations and blocking conditions are being conducted.

Hepatic Hormone Receptor Levels. Estrogen receptor and androgen receptor protein levels in hepatic cytosol can be examined using Western blot. Both the cytosolic and microsomal preparations have been examined to determine which fraction gives the best reproducibility; cytosolic liver fractions produce a more reliable signal to the estrogen receptor antibody. Preliminary results with an androgen receptor antibody (E. Wilson, University of North Carolina Medical Center) were inconsistent; we are trying other tissues as a source of reliable androgen receptor measurements, as well as searching for a source for another androgen receptor antibody. Preliminary results are the optimization and validation of the biochemical assays that have been developed. Additionally, in the course of method development, several experiments have been conducted using these methods. We have looked at the influence of Cd on reproductive adults (Thompson and Benson, unpublished results). Adult medaka exposed in pairs to 1 µg/L, 5 µg/L, or 10 µg/L of Cd for 14 days showed no impairment of reproduction in terms of total egg production, proportion of eggs fertilized, egg size, hatching success, or sex ratio of the resulting offspring relative to controls. A time course experiment describing the vitellogenic response of male Japanese medaka to E2 was completed to determine the onset of vitellogenin induction in the liver and plasma, as well as the decrease in response with extended exposure (Thompson and Benson, 1999). A collaboration with researchers at Trent University investigating the transgenerational effects of DDT exposure in medaka resulted in a description of a "priming" influence of in ovo exposure of males to DDT on the response to subsequent, adult exposure (Metcalfe et al., in press). Male medaka exposed in ovo to o, p'-DDT were treated for 4 days with E2 as adults. The concentration of vitellogenin in the liver of these re-exposed males was significantly higher than that of E2-treated males with no previous DDT exposure; however, no reproductive impairment was detected with transgenerational DDT exposure.

Future Activities:

The goal for Year 2 of the "Threshold Effects" grant is to finish the collection of all endpoint data from Cd-exposed animals and their offspring. This will necessitate the completion of the development of all the proposed endpoints. Additionally, exposure of medaka to EE2 and VZ will be accomplished in Year 2. No major changes in the use of equipment, techniques, or materials are anticipated.

A full-time research technician has been hired to perform exposures and biochemical assays, and to maintain animals during grow-out phases.

In the first year, most of the required method development as well as the adult exposure to Cd was completed; therefore, the anticipated timeline has been adhered to so far. The testing of subsequent chemicals should take less time than has been required with our initial experiments.


Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other project views: All 20 publications 8 publications in selected types All 7 journal articles
Type Citation Project Document Sources
Journal Article Foran CM, Bennett ER, Benson WH. Developmental evaluation of a potential non-steroidal estrogen: triclosan. Marine Environmental Research 2000;50(1-5):153-156. R827098 (1999)
R827098 (2000)
R827098 (2001)
  • Abstract from PubMed
  • Full-text: Science Direct
    Exit
  • Abstract: Science Direct
    Exit
  • Other: Science Direct PDF
    Exit
  • Journal Article Foran CM, Bennett ER, Benson WH. Exposure to environmentally relevant concentrations of different nonylphenol formulations in Japanese medaka. Marine Environmental Research 2000;50(1-5):135-139. R827098 (1999)
    R827098 (2000)
    R827098 (2001)
  • Abstract from PubMed
  • Full-text: Science Direct
    Exit
  • Abstract: Science Direct
    Exit
  • Other: Science Direct PDF
    Exit
  • Journal Article Metcalfe TL, Metcalfe CD, Kiparissis Y, Niimi AJ, Foran CM, Benson WH. Gonadal development and endocrine responses in Japanese medaka (Oryzias latipes) exposed to o,p′-DDT in water or through maternal transfer. Environmental Toxicology and Chemistry 2000;19(7):1893-1900. R827098 (1999)
    R827098 (2000)
    R827098 (2001)
  • Abstract: Wiley InterScience Abstract
    Exit
  • Supplemental Keywords:

    water, toxicology, heavy metals, ecological effects., RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, Toxics, Geographic Area, Environmental Chemistry, Health Risk Assessment, pesticides, State, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Risk Assessments, Biochemistry, Physical Processes, Biology, Endocrine Disruptors - Human Health, predictors of reproductive toxicity, testosterone, fish, altered sexual development, endocrine disrupting chemicals, Japanese medaka, plasma hormone levels, exposure, Mississippi (MS), cellular level endocrine status, atrazine, estrogen receptors, gonad morphology, transgenerational model, concentration-effect resposnses, epidemiologic studies, animal reporductive impairment, exposure assessment

    Progress and Final Reports:

    Original Abstract
  • 2000 Progress Report
  • Final