The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Proceedings for TestSmart -- Endocrine Disruptors

Breakout Group V - In Vitro Assays For Non-Receptor Mediated Endpoints

Chair

Robert Brueggemeier
Ohio State University Health Sciences

Panel Members

Michael Blanton
Battelle

Daniek Doerge
NCTR

Susan C. Laws
United States Environmental Protection Agency

Douglas Stocco
Texas Tech University

Breakout Session V addressed the topic of "In Vitro Assays For Non-Receptor Mediated Endpoints." The breakout session focused on these four questions:

The breakout session began with short presentations by panel members. Dr. Susan Laws, EPA, outlined the Endocrine Disruptor Screening Program (EDSP) to develop comprehensive toxicological and ecotoxicological screens and tests for identifying and characterizing the endocrine effects of various environmental contaminants, industrial chemicals, and pesticides. The program's aim is to develop a two-tiered approach, e.g., a combination of in vitro and in vivo mammalian and ecotoxicological screens (Tier 1) and a set of in vivo tests (Tier 2) for identifying and characterizing endocrine effects of pesticides, industrial chemicals, and environmental contaminants. The proposed Tier 1 Screening Battery consists of in vitro screens (ER binding/reporter gene assay, AR binding/reporter gene assay, and steroidogenesis assay using minced testis) and in vivo screens (rodent 3-day uterotrophic assay, rodent 20-day pubertal female assay, rodent 5-7 day Hershberger assay, frog metamorphosis assay, and fish reproductive screening assay). Alternative Tier 1 Screening assays are also proposed if the pubertal female assay is not utilized. The alternate assays include a 20-day pubertal male assay, an aromatase assay, and a rodent in utero-lactation assay. Validation of the individual screens and tests is required, and the purpose of validation is to establish the assay's reliability (an objective measure of a method's intra- and interlaboratory reproducibility) and the assay's relevance (how strong is the link between the readout of the assay and the mechanism of the toxic effect). The process is science-driven, and addresses the scientific principles of objectivity and experimental design.

Two panelists discussed in vitro assays that may be used to measure the effects of potential endocrine disruptors on steroid hormone biosynthesis. Dr. Douglas Stocco, Texas Tech University, discussed early steps in steroidogenesis as a potential site for effects of endocrine disruptors. Steroid hormones are not stored in endocrine tissues such as the adrenal cortex, testis, and ovary; rather, these hormones are synthesized from cholesterol and secreted. Steroidogenesis is initiated by the action of a pituitary trophic hormone and involves the conversion of cholesterol to pregnenolone via side chain cleavage catalyzed by the mitochondrial cytochrome P450 enzyme complex. A critical protein for this process is the steroidogenic acute regulatory protein (StAR), which facilitates the movement of cholesterol to the inner mitochondrial membrane which is necessary for side chain cleavage catalysis. StAR protein is tightly regulated, and Dr. Stocco described studies in a rat Leydig cell line, the MA-10 line, demonstrating the rapid induction of the StAR protein following LH treatment. Recently, he examined the effects of certain endocrine disruptors on StAR protein induction in the MA-10 cells. Lindane treatment of the cells suppressed mRNA and protein levels of StAR and resulted in decreased conversion of cholesterol to pregnenolone. These results suggest that certain endocrine disruptors may affect StAR protein levels and thus alter steroid hormone biosynthesis. The cell-based assay is sensitive and may be suitable as an in vitro screening assay; however, availability of the MA-10 cells may limit widespread use.

Dr. Robert Brueggemeier, Ohio State University, discussed aromatase as a second in vitro enzyme system that may be used to measure the effects of potential endocrine disruptors on steroid hormone biosynthesis. Aromatase is the cytochrome P450 enzyme complex present in the endoplasmic reticulum that converts androgens into estrogens. Aromatase is found in the ovary in the adult female, in the placenta during pregnancy, and in the male testis. In both sexes, aromatase is also present in certain regions of the brain and in adipose tissues. The regulation of aromatase is complex in various tissues, and several tissue-specific promoter regions have been identified upstream from the aromatase gene. An in vitro aromatase assay could easily be utilized as an alternative screening method in the Tier 1 Screening Battery to assess the potential effects of various environmental toxicants on aromatase activity. Both in vitro subcellular (microsomal) assays and cell-based assays are available for measuring aromatase activity. The in vitro subcellular assay using human placental microsomes, is commonly used to evaluate the ability of pharmaceuticals and environmental chemicals to inhibit aromatase activity. In addition, human JEG-3 choriocarcinoma cell culture lines, originally isolated from cytotrophoblasts of malignant placental tissues, have been used as in vitro systems for measuring the effects of compounds on aromatase activity. The cell lines have an additional advantage of determining the ability of endocrine disruptors to induce or suppress aromatase enzyme levels. Numerous flavonoids and related phytoestrogen derivatives demonstrate aromatase inhibition with IC50 values in the micromolar range, both in placental microsomal assays and in JEG-3 cells. Dr. Brueggemeier discussed the effects of endocrine disruptors on aromatase expression. In one report, toxaphene at 10 µM has been reported to decrease in aromatase expression SK-BR-3 breast cancer cell. In another report, atrazine, simazine, and propazine at 30 mM resulted in dose-dependent induction of aromatase activity in adrenocortical carcinoma cells, but no induction was observed in MCF-7 breast cancer cells. Finally, a possible high throughput assay measuring microsomal aromatase inhibition has been reported, and Dr. Brueggemeier reported data from the analysis of flavonoids using this assay. Thus, both an in vitro microsomal enzyme assay and available cell-based aromatase assays are sensitive, are routinely used, and are amendable for an in vitro enzyme system that may be used to measure the effects of potential endocrine disruptors.

Thyroid hormone biosynthesis was the subject of a presentation by Dr. Daniel Doerge, National Center for Toxicological Research, as an in vitro assay that may be used to measure the effects of potential endocrine disruptors. The critical enzyme for the biosynthesis of thyroxine (T4) and triiodothyronine (T3) is thyroid peroxidase, which catalyzes the iodination of tyrosine residues coupled to thyroglobulin and the condensation of the iodotyrosine residues to produce T4 and T3 coupled to thyroglobulin. Dr. Doerge discussed enzyme studies with the isoflavone, genistein, and described the ability of genistein at low concentrations to act as a suicide substrate of thyroid peroxidase and inactivate enzyme activity. Although this enzyme is very sensitive in vitro to genistein, paradoxically the administration of genistein in vivo did not alter levels of thyroid hormones nor induce hypothyroidism in treated animals. Therefore, this study illustrates the importance of the relevance of an in vitro test to the mechanism of the toxic effect it measures and to its proposed uses.

Mr. Michael Blanton, Battelle Pacific Northwest Laboratories, gave the final presentation and updated the session on the validation of a QSAR approach to identify ER-binding endocrine disruptors with in vitro estrogen receptor binding assays. This validation process is still underway and results are not available at this time. Mr. Blanton described the various components of assay validation process, and problems encountered in the development of standardized assays. Important considerations include study design, protocol development, assay result reproducibility among various individuals performing the assays, test compound solubility, and assay reliability.

General discussion among panelists and session participants addressed other potential in vitro assays. Possible biochemical and/or subcellular assays include transport protein binding assays, trophic hormone (ACTH, LH, FSH, TSH) binding assays, growth factor receptor binding assays, promoter and gene regulation assays, cell signaling assay systems (cAMP, protein kinase A, protein tyrosine kinase), and metabolism (Phase I and Phase II pathways). Several of these types of assays are routinely performed in drug development efforts in both academia and the pharmaceutical industry; thus, these assays may be amenable for in vitro tests for endocrine disruptors. Possible cellular and/or apical assays that evaluate effects on cell-cell communication, on cell proliferation, and on organogenesis may also be amenable to screening and testing of endocrine disruptors. Critical questions pertaining to the use of such in vitro cell assays in the EDSTP include assay development, assay standardization, and the specificity of the readout (e.g., how closely is the endpoint linked to effects of interest). Other potentially limiting factors included issues of chemical uptake (solubility), potential effects of metabolites, lack of feedback regulatory mechanisms that exist in whole animal systems.

Important issues that need to be addressed for every proposed assay is the relevance of an in vitro test to the mechanism of the toxic effect it measures and to its proposed uses. Therefore, it may be feasible to reduce animal testing with appropriate in vitro assays for endocrine disruptors; however, animal testing cannot be completely replaced due to the necessity of demonstrating the relevance of in vitro assays to in vivo toxic effects of endocrine disruption.