Methods to monitor EDCs both in the environment and in humans include measurements of environmental and tissue concentrations, questionnaires, personal monitoring devices, biomarkers, and mathematical models. Obviously, both external (air, water, food, and soil) and internal (blood, urine, breast milk, adipose tissue, and muscle tissue) measurements are required. Generally, identification and measurements of EDC residues utilize instruments similar to those used to detect other environmental contaminants, namely gas and liquid chromatography, high performance liquid chromatography, mass spectrometry, and high-resolution mass spectrometry (HRMS). However, the chemical nature of many EDCs is unknown. Therefore, nonspecific methods that measure biological activity may provide the best indication of EDC contamination. These methods, however, are limited in identifying specific chemicals and are most applicable in screening. Using the biological methods in conjunction with classical analytical processes is desirable. A tiered approach incorporating both is often employed.
Biologically Based Methods:
To screen for EDC activity, methods that test for biological activity are often utilized. Most biologically based methods are in vitro and fall into one of the following categories (often used in conjunction with one another and classical analytical methods such as Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC), or Mass Spectrometry (MS)):
Receptor binding assays: These utilize isolated receptor ligands from test organisms. The ligands are specific for certain cellular receptors and are co-incubated with high-affinity radioligand and various concentrations of test compound. Displacement of radioligand is examined against known active compounds (usually 17b-Estradiol).
Cell proliferation assays: These assays monitor cell proliferation in certain organ cells. Cell proliferation in these cells is characteristic of estrogen action and can be induced by very low concentrations of estrogenic compounds.
Receptor-dependent gene expression assays: These assays measure a compounds ability to stimulate receptor-dependent gene expression in cells. The method often uses transfected mammalian or yeast cells.
Immunoassays: Used successfully for years in the environmental analysis of pesticides, immunoassays are capable of detecting chemicals at remarkably low levels. Classical Analytical methods require extra effort (such as sample concentration) to detect at similar levels. The method is typically inexpensive and can accommodate large sample loads. An example is the Enzyme-Linked Immunosorbent Assay (ELISA). Immunoassays have been used analyze environmental matrices (air, water, food, soil) and biological fluids.
Immunoaffinity Chromatography: This method utilizes a selective antibody coupled to a stationary support within a column. Elution of sample through the column allows the analyte to adhere to the immobilized antibody producing a purified extract that is usually clean enough for detection and quantitation. Immunoaffinity Chromatography can be coupled to HPLC and/or MS to obtain high resolution and selectivity. This has also been used to analyze blood EDC concentrations.
Comparison of Analytical Methodologies
In vitro vs. in vivo:
While in vivo methods are crucial in identifying the connection between exposure and biological effects, they are expensive, cannot accommodate high throughput screening, and cannot characterize individual compounds. In addition, in vivo studies are subject to inter-individual, seasonal, and temporal variability clouding interpretation. In comparison, in vitro bioassays are relatively inexpensive and can be conducted at large throughput, and are performed quickly. Additionally, since the methods are simplified biological systems, the responses are highly sensitive and allow for analysis of individual compounds. The methods also circumvent socio-political and ethical issues associated with in vivo studies.
While useful, in vitro bioassays have limitations. For one, the relevance of the measured response in estimating in vivo activity depends on the mechanism the assay was based on. Consequently, knowledge about the mechanism of action of a compound must be attained prior to analysis of the compound. Also, in vitro bioassays are mechanism specific and do not account for many factors that may effect the mechanism in vivo such as cross-talk between biological pathways, environmental influence, and integration of action through different mechanisms at different tissues. Furthermore, in vivo studies account for metabolic transformations, bioaccumulation, and homeostatic controls that can affect the potency and efficacy of compounds, factors that in vitro studies cannot assess. Thus, a multifaceted approach incorporating all methods must be utilized in monitoring and measuring EDCs in the environment, in animals, and in humans.
In vitro vs. classical analytical techniques:
Classical instrumental analyses are necessary for the identification and quantitation of compounds. However, these methods do not provide information regarding the potency, and efficacy of a compound in modulating endocrine function. In contrast, in vitro bioassays measure mechanically-based biological responses. The methods also allow for a practical integrated measure of the synergistic effects of all compounds in a given sample, whereas instrumental analysis of mixtures are often expensive, difficult, and time consuming. Some bioassays also provide superior sensitivity and allow for detection of compounds that exert effects at levels below analytical detection limits. Additionally, in vitro bioassays are capable of detecting compounds for which there are no analytical methods available.
Monitoring The Agents In The Environment:
Spurred by increasing concern over adverse human health effects due to EDCs, the EPA established the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) in October 1996 to develop an endocrine disruptor screening program. EDSTACs recommendations consisted of a four-step program: (1) initial sorting of chemicals, (2) establishment of screening priorities, (3) Tier 1 screening, and (4) Tier 2 testing.
Step 1, the initial sorting step, separates EDCs into four categories: (1) polymers with an average MW greater than 1000 daltons, (2) chemicals with insufficient data to determine their potential for endocrine activity, (3) chemicals that have sufficient data to bypass screening but not testing, and (4) substances with enough data and can be referred to the appropriate agency for hazard assessment.
Step 2, establishes screening priorities for the categories of chemicals. The EDSTAC recommended a compartment-based priority setting strategy. In the strategy, compartments (groups of chemicals) are defined and the chemicals within each compartment are prioritized. The EDSTAC did not state what the sets should be and has advised the EPA to do so.
Step 3, Tier 1 screening, utilizes a battery of assays in order to evaluate endocrine disruption potential. The EDSTAC recommendation includes the use of the following three in vitro and five in vivo assays.
In vitro Tier 1 assays:
- Estrogen Receptor Binding or Reporter Gene Assay
- Androgen Receptor Binding or Reporter Gene Assay
- Steroidogenisis Assay Using Minced Testes
In vivo Tier 1 assays:
- Rodent 3-day Uterotrophic Assay
- Rodent 20-day Pubertal Female Assay with Thyroid
- Rodent 5-7 day Hershberger Assay
- Frog Metamorphosis Assay
- Fish Gonadal Recrudescense Assay
Samples/Compounds testing negative in Tier 1 screening will be interpreted as having a low potential for disruption of estrogen, androgen, or thyroid systems. Samples/Compounds testing positive in Tier 1 will continue for in depth evaluation in Tier 2.
Step 4, Tier 2 testing, as established in order to identify biological affects due to endocrine disruption. Tier 2 testing also identifies relationships between dose and response. EDSTAC recommended that the most sensitive life stages (in utero or in ovo) be tested, that the tests be multi-generational, and that each major taxonomic group (mammals, birds, fish, amphibians, and invertebrates) be included.
Currently Available Methods for Measuring EDCs in the Environment
Currently, EPA uses and enforces several methods for measurement of most of the 95 known EDCs. The attached tables outline the methods used to characterize specific chemicals with known endocrine disrupting characteristics.
Generally, for organic chemicals, Drinking Water Analyses are performed within the EPA 500 Series Methods, Ground Water and Wastewater Analyses are performed within the EPA 600 Series Methods, and the Soils and Solid Wastes Analyses are performed within the EPA SW846 (8000 Series) Methods. For inorganic chemicals, the EPA 200 Series Methods, the 6000 Series Methods and the 7000 Series Methods are used.
The applicable EPA methods for measuring known endocrine disruptors in various environmental media are presented in the following tables. For more information regarding the specifics of each method, please refer to http://www.epa.gov/.
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