Superfund Research Program
Monitoring Xenoestrogen Exposure in Largemouth Bass
Whether released into the atmosphere, onto land, or into rivers, environmental contaminants often come to rest in the aquatic ecosystem. Because of their exposure in aquatic ecosystems, fish can provide an early warning of effects that may later become apparent in other wildlife and ultimately in humans. Many Superfund sites are contaminated with compounds that can act as weak estrogens, disturbing normal endocrine systems by mimicking hormones or altering the synthesis, metabolism, or activity of hormones. Scientists are accumulating evidence that even low-level exposure to specific pollutants can disrupt the endocrine system of fish. Such disruption can result in decreases in immune and stress responses, energy metabolism, and osmoregulatory ability and result in reproductive damages such as reduced fertility, reduced hatchability, reduced viability of offspring, impaired hormone activity, and altered sexual behavior. Many of these physiological changes are mediated through the action of estrogen receptors. The discovery of multiple receptors in fish raises questions regarding the mechanisms of action of estrogen and the contaminants.
University of Florida (UF) project investigators have discovered and cloned three separate estrogen receptors (ER's) in largemouth bass. These receptors have >90% similarity in their DNA binding domains (the areas on the ER's that bind to DNA) but only 60-70% similarity in their ligand binding domains (the areas on the ER's that bind to steroids or other compounds). This variation in ligand binding domain structures could explain how different estrogen mimics might bind differently to the various estrogen receptors. To discern the role the three estrogen receptors play in the normal reproductive process, the UF scientists first determined the tissue distribution of the three receptors. They used real time polymerase chain reaction (PCR) to measure levels of the respective mRNAs in liver, gonad, brain and pituitary tissues. PCR is an enzymatic reaction that can precisely and rapidly amplify a small segment of DNA millions of times. The results indicate that all three ER's are present in all tissues, but at different levels. Moreover, their expression in liver and gonadal tissues changes as a function of the reproductive cycle in females, suggesting they have specific and distinct roles. How exposure to environmental estrogens may modulate their expression remains to be tested.
The UF group is also investigating the feasibility of applying array technology as a monitoring tool for detecting the presence and distribution of estrogenic compounds in aquatic habitats. Simply, an array is a grid of DNA spots - each with a unique DNA sequence. A macroarray is a "high-density" array where hundreds to thousands of spots are bound to a membrane. A microarray is a "very high density" array, where thousands to tens of thousands of spots are bound to a glass slide. When exposed to a preparation of radiolabeled or fluorescently labeled nucleic acid, each spot will hybridize (join with a DNA strand to form a double-stranded molecule) only to its complementary DNA (cDNA). Any spot that is bound to cDNA will produce a signal, allowing researchers to identify expression patterns of genes following specific treatments.
In the liver of adult female fish, normal cycles of endogenous estrogen (17b-estradiol or E2) activate genes, instigating the transcription of specific mRNAs that encode proteins required for reproduction. These include the three estrogen receptors, the egg yolk precursor protein vitellogenin (Vtg), and vitelline envelope proteins (ZP's), among others. Male and juvenile fish also possess these genes, but under normal conditions only females produce sufficient estrogen to induce transcription of the genes and subsequent protein synthesis. Finding these mRNAs in male fish is an indication of exposure to an estrogenic substance.
The UF scientists are developing a genetic tool that could be used to monitor the environmental distribution of endocrine disrupting compounds that mimic estrogen. The UF researchers are challenging the macroarray system with controls as well as samples from male fish exposed to E2, the strong estrogen mimics ethinyl estradiol (EE2) and diethylstilbesterol (DES), and the weak estrogen mimics nonylphenol (NP) and methoxychlor.
Their analytical technique involves a series of steps:
- Preparation of DNA for the macroarray. Genes involved in reproduction including vitellogenin, estrogen receptors, and vitelline envelope proteins are isolated using differential display (DD). DD works by selectively converting successive subsets of mRNAs into cDNAs, and then "displaying" these cDNA fragments by gel electrophoresis. mRNA will only be present for expressed genes. The DNA is then amplified via PCR.
- Printing DNA onto the macroarray. The samples are robotically spotted in duplicate onto neutral nylon membranes.
- Total hepatic mRNA is extracted from livers of control and exposed animals and is radiolabeled. cDNA is then generated from the mRNA.
- The arrays are exposed to the radiolabeled cDNA to allow hybridization to occur.
- Hybridized spots on the array will produce a detectable signal.
The UF researchers have shown that this macroarray technique is very reproducible - there is very little variation between duplicate arrays and the variation between animals treated with the same exposures is very low.
The UF macroarray technique reveals a characteristic expression pattern of up and down-regulated genes or "fingerprint" for fish exposed to E2. This pattern is consistent with previously reported patterns obtained using DD analysis. In addition, the array technique reveals a similar expression pattern in samples from fish exposed to both the strong and weak estrogenic compounds, indicating that this technique is capable of evaluating exposure to these contaminants. The UF researchers have also shown that their macroarray technique can be used as a tool to quantitate exposure - Vtg and ZP expression levels increased in a dose dependent manner with low to high concentrations of EE2.
Based on the high throughput of array technology, fish macroarrays can serve as one method to detect the extent of exposure of animals to endocrine disrupting compounds in the environment. The UF scientists plan to conduct studies to field validate the existing estrogen responsive array and to expand the array to include other endocrine endpoints. Coupled with the presence of three distinct estrogen receptors that have disparate ligand binding domains and regulate specific as well as overlapping sets of genes, it may be possible to determine unique gene induction fingerprints for each environmental estrogen.
In addition to serving as a monitoring tool to evaluate exposure, fish macroarrays can provide information on biochemical pathways that are modulated by environmental contaminants and may be able to pinpoint mechanisms of action. This knowledge will be valuable in studies to examine the impact of these compounds on not only the reproductive system, but also on other endocrine-mediated pathways in the brain and cardiovascular systems
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To learn more about this research, please refer to the following sources:
- Bowman CJ, Denslow ND. 1999. Development and validation of a species- and gene-specific molecular biomarker: Vitellogenin mRNA in largemouth bass (Micropterus salmoides). Ecotoxicology 8:399-416.
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