Assay Endocrine cells

Okamoto, Y., Murakami, H., and Nishida, M. (1997) Detection of interleukin 6 producing cells among various organs in normal mice with an improved enzyme linked immunospot (ELISPOT) assay. Endocrine Journal, 44, 349 355. 

Studies also suggested that IL-6 causes endothelial cell dysfunction and decrease of prostacyclin production. Soluble IL-6 receptors seem to play a modulating and enhancing role in IL-6 activity (FI 8). The incidence of detection appears to be less influenced by the method of assay, as either ELISA or bioassay techniques yield consistent results, with a high correlation between these techniques. IL-6 values may be more constant and endocrine-like than those values of TNF and IL-1. 

An example of a TIE approach is that described by Desbrow et al. [7]. In this work, the endocrine disrupting activity detected in effluents of seven UK WWTPs by means of a yeast-based screening assay [52] was mainly attributed to the presence of estradiol, estrone, and ethynylestradiol. However, to assess the estrogenic activity different bioassays may be used, e.g., the yeast-based recombinant estrogen receptor-reporter assay (YES), the MCF-7 cell proliferation (E-screen), and the estrogen receptor-mediated chemically activated... 

Cheng KW (1975) A radioreceptor assay for follicle-stimulating hormone. J Clin Endocrin Metab 41 581-589 Foulds LM, Robertson DM (1983) Electrofocusing fractionation and characterization of pituitary follicle-stimulating hormone from male and female rats. Mol Cell Endocrinol 31 117-130... 

Many other cell lines have been used to address specific types of toxicity. Some of these have reached a level of validation that is sufficient for in vitro screening purposes. For example, the 3T3 NRU assay was regarded as an acceptable screen for hazard identification of potential phototoxicity [27,28], This assay utilized the in vitro 3T3 cell line as a generic cell line and Neutral Red uptake as a cytotoxicity measurement. The compound in question is subjected to UV irradiation to assess the effect of UV rays on compound induced toxicity to 3T3 cells. Another example is the use of the MCF-7 cell proliferation assay as an in vitro screen for endocrine disrupters [29],... 

NICEATM. NICEATM pre-screen evaluation of the in vitro endocrine disruptor assay (Robotic MCF-7 cell proliferation assay of estrogenic activity), chttp // iccvamniehsnihgov/methods/endodocs/CCiPrescreenEvalpdf>. Accessed November 1, 2006. 

False-positive results of ER assays (ER-positive tumor but no response to endocrine therapy) are more common than are false-negative results. The most frequent explanation is heterogeneity of tumor with biopsy of a site that is not representative of the other tumor deposits. In addition to this problem, evidence exists that some tumor cells have receptor defects distal to the initial binding step (e.g., variant cells are able to bind steroid in the cytoplasm but not transport the receptor to the nucleus). 

Another important source of information on the status of alternative test development, with particular emphasis on the requirements for cosmetics testing, is a review paper published in 2011 by Adler and coauthors [9], Table 1 summarizes those relevant for reproductive toxicity. Several assays refer to the detection of endocrine effects on steroidogenesis based on a variety of cell types, and, as already mentioned, they will be dealt with in another chapter of this book. The other tests can be subdivided in placental toxicity/transport, preimplantation toxicity, female and male toxicity, and developmental toxicity. The tests that are suitable for detecting developmental toxicity include the EST, the whole-embryo assay, the micromass test (all three already described above), the zebrafish embryo teratogenicity assay, and the frog embryo teratogenesis assay (FETAX). 

For investigating endocrine disruption of estrogenic compounds, in vitro models are well established and some are well validated. For other tissues, such as adrenal and thyroid, there is less concurrence about test systems and models, but a number of useful cell models are available. In vitro assays provide results that are sometimes limited in scope or impact, but such results can synergize... 

Mechanistic assays Various systems, e.g. cell lines, cell-free extracts, mitochondria, nuclear extracts GAP junction inhibition, oxidative stress measure, protein-binding activity, endocrine-disrupting activity, oxygen consumption, metabolite levels, etc. Mechanistic studies Non-genotoxic carcinogenicity, weight of evidence... 

In this section we will summarize the status of the currently available methodologies for determining serum levels of products that reflect the endocrine function of the thymus. It had been hoped that specific abnormalities in blood levels of thymic hormones would provide important information for diagnosing specific immunodeficiency disorders. However, this has not turned out to be the case, and even though the currently available assays can frequently detect abnormalities on the part of the endocrine thymus, there have been no specific serum alterations that are diagnostic of any particular immunodeficiency disorder, and no strong correlations exist between low or high serum thymic hormone levels and the immune status of the patient as defined in T cell functional assays. 

After intervening endocrine treatment, ERP and PRP levels significantly decrease. This might reflect a selective pressure on ERP+ and PRP+ carcinoma cells. Therefore, recurrent lesions should be assayed for ERP and PRP levels if possible. However, prospective studies should clarify the prognostic importance of receptor levels in metastatic tissue as well as the role of intervening chemotherapy, especially for sequential PRP determination. 

The use of in vivo bioassays (e.g., hyperglycemia) as a measure of insulin in an intact animal or organ is largely historic because the hormones can be measured directly, but in vitro bioassays or bioassays using endocrine responsive tissue cell cultures can be useful in screening compounds. These assays can measure changes of adenylate cyclase, intracellular calcium, and phosphoinositol metabolites. 

Within the framework of SPEED 98, Japan Environment Agency conducted PLC and/or FLC with 28 of 67 chemicals listed [3]. The results showed that only 3 chemicals, i.e., 4-nonylphenol, 4-t-octylphenol, and bisphenol A were suggested as EDCs. We also conducted sex reversal assay or PLC test (56-day exposure) to evaluate the effects of some pesticides (malathion, benomyl, cypermethrin, permethrin, esfenvalerate, fenvalerate) listed in SPEED 98. The results showed that these pesticides gave no endocrine-disrupting effects even at one-seventh to one-twentieth of the acute toxicity values (Table III). The effect of benomyl on hatchability is likely to be caused by inhibition of cell division. Based on these findings, it is unlikely that these chemicals listed affected as EDCs. 

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