Session summaries (Posted: August 20, 2002)

1. Policy/Research Review
2. Models and Testing Protocols
3. Theme Session
4. Environmental Research

Session summaries

• Policy/Research Review: The possible human and wildlife health effects of endocrine disruptors spurred government and industry to begin research and testing programs in the late 1990s. The lack of information about endocrine disrupting chemicals (EDC) led to programs that would decipher the biology, identify health risks, and design testing and screening programs. Session speakers presented updates on several of these programs, especially advances in developing screening tests.
  • Long-term Research Strategies for Endocrine Disruptors in the US Environmental Protection Agency (EPA), Elaine Francis, US EPA: The US EPA's multi-year plan, developed in 2000, coordinates the agency's EDC programs for the next seven years. The long-term goals include conducting research to better understand EDCs impacts, exposures, and other scientific questions; assess risk to human and wildlife health; and develop screening and testing methods and approaches to prevent/reduce exposures.

  • International Test Guidelines Development at the OECD, M-C Huet, Organization for Economic Co-operation and Development (OECD): Internationally, the OECD founded the Task Force on Endocrine Disrupter Testing and Assessment in 1998 to work with member countries to enhance communication among members, coordinate testing and assessment of EDs worldwide, and produce internationally accepted test guidelines for assessing EDCs in mammals and non-mammals. So far, a testing framework has been established; several toxicology validation tests are being assessed; and meetings about fish, bird, and amphibian testing have been held.

  • Endocrine Disruptors and Aquatic Organisms: Update on the European Chemical Industry Research Strategy, Tom Hutchinson, AstraZeneca Global Safety, Health, and Environment: Private sector - working in conjunction with government agencies - are also focusing on devising precise testing methods and protocols for EDCs. The European chemical industry formed the Endocrine Modulating Steering Group to define appropriate fish, amphibian, and bird assays for short-term and long-term effects. Tests for estrogens, anti-estrogens, and anti-androgens are at various stages of development and include a fish screening assay, a fish development test, and a fish reproductive study.

• The Quest for Field Deployable Biomarkers for Endocrine Active Compounds in Wildlife, Pat Guiney, S.C. Johnson and Son Inc., Racine, Wisconsin, USA: To better understand the endocrine, reproductive, and developmental health of humans and wildlife and the possible role of EDCs in these systems, the American Chemistry Council formed the Endocrine Technical Implementation Panel (TIP). The panel oversees long-term collaborative research among industry, government, and university groups worldwide. The research links laboratory studies documenting changes in organisms with population effects in the wild. The ultimate goal is to apply new information to better understand risk, develop testing methods, and communicate the results. Several studies are underway including: 1) understanding fence lizard behavior and biology to determine if reptiles would make good testing models for EDC effects; 2) developing methods to assess behavior, biochemical, and reproductive changes in wild house finch and using these real-life effect parameters with captive laboratory birds; 3) conducting a multi-year study on ecosystem changes and population effects by exposing an entire lake to a synthetic estrogen and measuring long-term changes in the aquatic animal populations; 4) developing models to bridge laboratory and field studies using a mix of animals (alligators, mussels, fish), endpoints (genetic, geochemical, individual, populations), and mixtures of contaminants; and 5) using DNA and biochemical factors from frogs to understand chemical effects on metamorphosis and to model thyroid hormone disruption from EDCs.

• QSAR-based Approaches for Predicting Estrogen Receptor Binding Affinities of HPV-Inerts, John Walker, Toxic Substance Control Act (TSCA) Interagency Testing Committee: The computational approach known as Quantitative Structure Activity Relationships (QSARs) measure a chemical's ability to bind to estrogen receptors. Two-QSAR based approaches were used to predict binding affinity of 419 high production volume inert chemicals. More than half (287) did not bind to the ER, about one-fourth (109) had very weak binding, while the remainder had weak (18) to moderate (5) binding. The results suggest QSAR may not be a good predictor of activity because receptor binding may be only one mechanism of EDC activity.

• Endocrine Disruptor Mechanisms: Beyond Receptor Binding, JP Giesy, Michigan State University, East Lansing, Michigan, USA: Most research to date has focused on EDCs binding to hormone receptors and altering cellular functions. Receptor binding, though, is only one mechanism of action for EDCs. The compounds can be direct mimics - such as agonists, antagonists, and partial antagonists that bind to receptors - or indirect mimics - substances that alter signalling pathways or hormone synthesis by inducing enzymes directly or indirectly thus affecting hormone concentrations. Because of this, many of the screens and tests being developed may not catch all possible EDCs, giving false negatives that deem compounds safe when they may not be. For instance, some triazine herbicides, such as atrazine, do not bind to the ER but are estrogenic, possibly because the chemicals increase production of the enzyme aromatase, which in turn increases estradiol and lowers testosterone levels. Other compounds can alter conversion of the nucleotide cyclic-AMP to AMP thus changing aromatase and estradiol concentrations. Still others, such as dioxin-like contaminants, can interfere with pathways that eventually lead to altered receptor actions.

• Models and Testing Protocols: Developing viable and accurate animal models to predict EDC activity and health consequences is underway. Speakers presented results of quail, fathead minnow, and clawed frog research.
  • Reproductive consequences of EDCs in Birds: Can Laboratory Studies be Extrapolated to Free-ranging Species?, Mary Ann Ottinger, University of Maryland, College Park, Maryland, USA: Precocial bird species, such as Japanese quail, are already well-developed when they hatch and may be more sensitive to EDCs than non-precocial species. A series of experiments determined that the quail can serve as a good model species for assessing EDC effects in multiple generations and developing observable and analytical ways to determine effects on wild birds. Several reliable indicator endpoints were found including reproductive success (fertility, hatching, and offspring survival), endocrine measures (plasma hormones and gonad weight/histology), neuroendcorine measures (catecholamines, aromatase and GnRH-1), and behavior measures (reproductive and movement deficiencies).

  • Identification of Endocrine Disruptors Using a Short-term Reproductive Assay with the Fathead Minnow, Gerald Ankley, US EPA, Duluth, Minnesota, USA: A short-term, reproductive assay using adult fathead minnows has accurately and consistently predicted EDC actions through one or more endpoints. Therefore, the EPA plans to use the minnow test to fulfill screening and testing requirements. The tests effectiveness was assessed with several endpoints (blood vitellogenin, hormone concentrations, fertility, fecundity, and offspring survival) using different rates of exposure and a variety of EDCs with known and different modes of action (estrogen agonists estradiol and methoxychlor, androgen agonists methyltestosterone and trenbolone, androgen antagonists vinclozolin and flutamide, and the hormone metabolism inhibitor fadrozole).

  • Development of an Amphibian Metamorphosis Model for Detecting Thyroid Axis Disruption, Joseph Tietge, US Environmental Protection Agency, Duluth, Minnesota, USA: Amphibian metamorphosis - the changing of larvae into adults - is controlled by thyroid hormones. Although several thyroid biochemical indicators and morphology changes were examined, no one method accurately assessed EDC developmental effects. This research, though, increases understanding of how amphibians develop. Future efforts will focus on developing an integrated approach to test chemicals for thyroid axis disruption using multiple gene expression, biochemical indicators, and developmental endpoints.

• Theme Session: Pete Myers, co-author of Our Stolen Future: An explosion of research, money, and interest in endocrine disrupting chemicals occurred in the late 1990s. This surge was partly fueled by past conferences, such as the Racine, Wisconsin, Wingspread Conferences and the Estrogen in the Environment conferences, and publication of the book Our Stolen Future in 1996. The United States, Japan, and Europe are currently spending $100 million to identify EDCs, determine health effects and risks, and reduce exposure. Much more is known today about EDCs because of the increased research. Some major trends surrounding the compounds include:
  1. EDCs are not just estrogens. The compounds represent a host of hormonal personas including estrogens, anti-estrogens, androgens, anti-androgens, progesterones, and thyroid hormones. Chemically-mediated signals - important from ecosystems to cells - are vulnerable to this wide range of hormonal activity. For example, a recent research paper found several EDCs interrupted molecular signals between legume plant roots and special nitrogen fixing bacteria living in the roots leading to severely decreased levels of essential nitrogen fixing activities, unhealthy plants, and in some cases, plant death.

  2. EDCs have multiple mechanisms of action, including binding and activating hormone receptors, altering rates of synthesis or metabolism, and interfering with gene activation.

  3. EDCs come in all shapes and sizes and are not just the classic persistent bioaccumulative toxins such as DDT and DDE. All compounds listed by the United Nations Stockholm Convention are EDCs and new PBTs, such as polybrominated diphenyl ethers (flame retardants) and perfluorinated sulfonates, are garnering attention to determine their hormonal activity. Additionally, many modern use, short-lived chemicals such as bisphenol A, nonylphenol, and others are EDCs.

  4. Exposure is ubiquitous because the chemicals are everywhere. A recent US government study found most US citizens carry phthalates in their bodies. The highest amounts were discovered in women of reproductive age - probably because some cosmetics contain the substances. The study raised concerns because women of child-bearing age and their developing children are most vulnerable to EDCs.

  5. Low-dose effects are real. Several recent studies show that fetal exposure to low levels of certain chemicals can alter development. For example, a recent study found that exposing unborn rodents to extremely low levels of bisphenol A (2 parts per billion) alters prostate development. In another study, 10 parts per billion of atrazine reduced frog larynx size (a hormonally-controlled male sex characteristic) and 1 part per billion caused frogs to have both male and female gonads. Background levels - or those found in the everyday environment - of suspected and known EDCs may also affect health. Unborn Dutch children exposed to relatively high background levels of PCBs had more illness by 3.5-years-old than other children not exposed to the same prebirth levels.

  6. Chemical mixtures are the rule. Exposure occurs to many different substances, not one chemical at a time. Current research employing traditional dose response curves may not offer the best approach to decipher the complexities of multichemical exposure.

  
  Based on these trends conceptional shifts are occurring, placing more emphasis on an ever broadening suite of signals and endpoints, more focus on mixtures, and an increasing interest in the precautionary principle because standard epidemiology studies may not be able to determine health effects soon enough. Some important future research opportunities may include: the affects of contaminated food and water; atrazine's health and developmental effects; non-Hodgkin's lymphoma and its relation to contaminants that may hamper the immune system; leptin hormone disruption as a factor in human obesity; interference with chemically mediated symbiosis in coral and plant root systems; and pollution of the scientific process by vested interests, investigator in-fighting, and public perception of the truth.

• Environmental Research. Several speakers presented environmental research results dealing with a number of organisms and ecological systems including snails, fish, frogs, sewage, and lakes.
  • Evidence of Endocrine Disruption in Neogastropods Due to TBT Ten Years After the 1989 Regulation, Doug Bright, Royal Roads University, Victoria, British Columbia, Canada: The organotin tributyltin (TBT), a marine paint additive, is a known hormone disruptor responsible for imposex (penile development in females) in marine snails and other types of gastropods. Imposex measurements and whole body concentrations of three species of Nucella snails sampled near Vancouver Island in 1999 were compared with data from 1987 and 1993. Comparisons were made to determine the effectiveness of a 1989 regulation banning TBT on vessels less than 25 meters in length. Overall, imposex incidence decreased but was still largely observed in animals living in international shipping harbors, near high ship traffic areas, and in other places well away from harbors, which have imposex rates of 64 - 100 percent. The researchers conclude that the regulations are helping curb imposex in some areas but large ocean vessels are probably still providing a fresh source of organotins, especially TBT, MBT, and DBT that are causing imposex in the short-lived (less than two years) animals.

  • Endocrine Disruptors in New York City Municipal Sewage Effluent, Anne McElroy, State University of New York: New York City sewage effluent contains endocrine disrupting chemicals to varying degrees depending on the level of the plant's treatment. Larvae and juvenile fish were exposed from 4 to 21 days to effluent from one plant with secondary treatment and two with full treatment. Researchers found that the larval fish are more sensitive to the effluent substances than juveniles. Larvae exposed to all three effluents had elevated vitellogenin and estrogen receptor activity while juvenile fish had higher receptor activity, but only in response to the less treated sewage. Culprits may be natural and synthetic estrogens and nonylphenol ethoxylates. Low hormone levels (5 - 40 ng/l) in the effluent suggest other types of substances are contributing to the effects. The researchers are analyzing the effluent to determine its content.

  • Identification of Environmental Endocrine Disruptors in Boston Harbor Seawater - Initial Results, Raymond Siegener, University of Massachusetts - Boston, USA: Using caffeine as a tracer, researchers analyzed estrogenic compounds in effluent from the Deer Island Sewage Treatment Plant, Boston's 300-million-gallons a day, state-of-the-art sewage treatment facility. Caffeine concentrations were followed from raw sewage to release into the marine environment after treatment to determine flow and dissipation of effluent into the natural environment. Following that, E-screen and A-screen assays determined that nonylphenols, known hormone mimics, are widely distributed in the saltwater environments of Boston Harbor and Massachusetts Bay.

  • Effects of Ethynylestradiol on Fish: Preliminary Results from a Whole-lake Experiment, Karen Kidd, Department of Fisheries and Oceans Canada, Winnipeg, Manitoba, Canada: To determine fish population effects of exposure to estrogens, researchers added environmentally-relevant amounts of ethynylestradiol (the synthetic estrogen in birth control pills) to an established lake at the Experimental Lakes Area in Northwestern Ontario. Data on vitellogenin, gonadal development, thyroid and sex hormones, and population parameters on a number of fish species are being analyzed after the first year's addition of estrogen to the lake. The data will be compared to pre-treatment values to evaluate health and population effects. Preliminary results show that the male protein vitellogenin is 80 times higher in experimental fish than in reference fish and that the protein is also elevated in male fish.

  • Morphologically Intermediate Papilla Syndrome (MIPS) in UK Populations of Sand Goby: Endocrine Disruption?, MF Kirby, CEFAS, United Kingdom: Two species of sand gobies were sampled from marine estuaries in the United Kingdom to asses endocrine disruption in wild fish populations. Wild male fish showed no increased levels of vitellogenin, mRNA, or the presence of ovotestis. They did, however, develop the female trait of terminal villi on the uro-genital papilla, a male organ used to transfer sperm. The condition, nicknamed "morphologically intermediate papilla syndrome" (MIPS), was more common (more than half of fish sampled) in males living near known estrogen contamination. Laboratory studies on juvenile fish also link MIPS to estrogenic actions. Because the fish are found worldwide and most species have papillae, this morphological marker may be an important, global monitoring tool.

  • Atrazine Disrupts Sex differentiation in the African Clawed Frog (Xenopus laevis) at Ecologically Relevant Doses, Tyrone Hayes, University of California, Berkeley, USA. Low-levels of atrazine, a common, heavily used herbicide, adversely affected male frog sexual characteristics at several concentrations - even below the EPA's drinking water standard of 3 parts per billion. Developing frog larvae were exposed to .1, .4, .8, 1, 10, 25, and 200 parts per billion of atrazine. Low-levels (1 part per billion and above) of the herbicide permanently reduced the size of the male larynx (important in male sexual behavior). Laryngeal growth depends on androgen so, in this case, atrazine's anti-androgen effects are probably the result of altered sex steroid levels (the pesticide affects the gonads and interrupts testosterone production or release). Gonadal abnormalities, such as multiple sex organs, occurred at all exposure concentrations while mixed testis and ovaries occurred at 1 part per billion and above. The researcher also found ovaries in the testis of 100 percent of wild male frogs sampled in Wisconsin, Illinois, Iowa, and Nebraska. Atrazine's mixed grab bag of feminized and masculinized traits also includes loss of breeding pads and feminized cloacas. Atrazine is widely used in the Midwest to control weeds on cropland. It is applied in spring when the atrazine-laden runoff water from snow and spring storms can collect in ponds and streams where frog eggs are developing.