A Practical Approach, Second Edition=Ronald D. Ho.pdf

496 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITIONtransfections and reduce the variability associated with this technique. However, stably transformedcell lines utilizing endogenous or transformed androgen receptor combined with a stably transformedhormone-responsive luciferase (or other) tagged reporter were not readily available untilrecently. These are genetically modified cell lines that are derived by transfection followed byantibiotic selection and clonal expansion. Ideally, this methodology produces a stable populationof cells that will respond uniformly to stimuli. 36–41Transcriptional activation assays have several strengths. They utilize a well-defined mechanismof action to distinguish agonists from antagonists and can be performed using mammalian cells.Thus, the results are likely applicable to humans. Transient transformation and viral-transductionassays are highly responsive (i.e., give high levels of induction in comparison with vehicle controls)and provide more control over the specificity of response. For example, androgen receptors orglucocorticoid receptors can be transfected into cells separately, and the responses can be assessedindependently. Both adenoviral-transduced and stable cell lines are amenable to a 96-well plateformat and give a consistent response with low variability, thus making them attractive for adaptationto high throughput systems (HTS). Generally, in vitro transcriptional activation assays are costeffective and, with proper training and equipment, are rapid and easy to perform. Cell lines derivedfrom human tissue can be used to develop these assays, so it is human receptors that are used inthe assessments. In addition, only a small amount of test chemical is needed to run multiple assays.Transcriptional activation assays also have some limitations. Compared with traditional receptorbinding assays that use cytosolic preparations, there is a requirement for cell culture equipment,techniques, and training. The presence of more than one receptor in the cells (especially if usingcells with endogenous receptors) that can activate the hormone response element of the reportermay be viewed as a limitation. This requires additional assays using selective competitors todifferentiate individual receptor activity. Transient transfection assays require separate transformationsfor each set of assays conducted, can have high interassay variability, and are generally notsuited for high throughput systems. Stable cell lines have many advantages, as discussed above,but require a considerable investment of time and resources to produce each cell line. Anotherconcern is the possibility of false negatives and false positives. These systems assess the testchemical, but if a particular test chemical requires metabolic activation to produce its hormonallyactive metabolite, it could produce a false negative in the assay. It is also necessary to assess theissue of cytotoxicity when working with cell-based assays. In tests for antagonist activity, decreasesin reporter activity could be due to either the test chemical or general cellular toxicity from thecompound. The incorporation of cell toxicity tests into routine chemical testing is an importantextension of the method. Otherwise, a test chemical could be falsely identified as an antagonist.C. SteroidogenesisThe EDSTAC final report recommended two assays for the evaluation of effects of environmentalchemicals on steroidogenesis. The first, a sliced testis protocol, was designed to measure testosteroneproduction in vitro from testicular parenchymal fragments. The second, an aromatase assay designedto assess the potential effect of toxicant exposure on the conversion of testosterone to estradiol,was recommended as an alternate approach. Thus, the focus on steroidogenesis was on two majorbiologically active hormones, testosterone and estradiol, both generated in the later portion of thegonadal steroidogenic pathway (Figure 11.4).The rationale for recommending the sliced testis assay was based on a number of previousstudies that demonstrated the utility of the method for identifying toxicant-induced endocrinealterations. 42,43 In general, this method measures testosterone release in vitro under both nonstimulatedand human chorionic gonadotropin (hCG)-stimulated conditions over a period of hoursfollowing testis decapsulation. The assay may be conducted using testes from animals followingin vivo exposure, or it may evaluate in vitro testosterone production by using testes from animalswith no prior exposure to the test chemical. Since a number of variations in the procedure have© 2006 by Taylor & Francis Group, LLC