Nuclear Receptor Assays Research Articles

Epidemiology meets toxicogenomics: Mining toxicologic evidence in support of an untargeted analysis of pesticides exposure and Parkinson’s disease

BackgroundPesticides have been widely used in agriculture for more than half a century. However, with thousands currently in use, most have not been adequately assessed for influence Parkinson’s disease (PD). ObjectivesHere we aimed to assess biologic plausibility of 70 pesticides implicated with PD through an agnostic pesticide-wide association study using a data mining approach linking toxicology and toxicogenomics databases. MethodsWe linked the 70 targeted pesticides to quantitative high-throughput screening assay findings from the Toxicology in the 21st Century (Tox21) program and pesticide-related genetic/disease information with the Comparative Toxicogenomics Database (CTD). We used the CTD to determine networks of genes each pesticide has been linked to and assess enrichment of relevant gene ontology (GO) annotations. With Tox21, we evaluated pesticide induced activity on a series of 43 nuclear receptor and stress response assays and two cytotoxicity assays. ResultsOverall, 59 % of the 70 pesticides had chemical-gene networks including at least one PD gene/gene product. In total, 41 % of the pesticides had chemical-gene networks enriched for ≥ 1 high-priority PD GO terms. For instance, 23 pesticides had chemical-gene networks enriched for response to oxidative stress, 21 for regulation of neuron death, and twelve for autophagy, including copper sulfate, endosulfan and chlorpyrifos. Of the pesticides tested against the Tox21 assays, 79 % showed activity on ≥ 1 assay and 11 were toxic to the two human cell lines. The set of PD-associated pesticides showed more activity than expected on assays testing for xenobiotic homeostasis, mitochondrial membrane permeability, and genotoxic stress. ConclusionsOverall, cross-database queries allowed us to connect a targeted set of pesticides implicated in PD via epidemiology to specific biologic targets relevant to PD etiology. This knowledge can be used to help prioritize targets for future experimental studies and improve our understanding of the role of pesticides in PD etiology.

Effect-Based Trigger Values for Mixtures of Chemicals in Surface Water Detected with In Vitro Bioassays.

Effect-based trigger (EBT) values for in vitro bioassays are important for surface water quality monitoring because they define the threshold between acceptable and poor water quality. They have been derived for highly specific bioassays, such as hormone-receptor activation in reporter gene bioassays, by reading across from existing chemical guideline values. This read-across method is not easily applicable to bioassays indicative of adaptive stress responses, which are triggered by many different chemicals, and activation of nuclear receptors for xenobiotic metabolism, to which many chemicals bind with rather low specificity. We propose an alternative approach to define the EBT from the distribution of specificity ratios of all active chemicals. The specificity ratio is the ratio between the predicted baseline toxicity of a chemical in a given bioassay and its measured specific endpoint. Unlike many previous read-across methods to derive EBTs, the proposed method accounts for mixture effects and includes all chemicals, not only high-potency chemicals. The EBTs were derived from a cytotoxicity EBT that was defined as equivalent to 1% of cytotoxicity in a native surface water sample. The cytotoxicity EBT was scaled by the median of the log-normal distribution of specificity ratios to derive the EBT for effects specific for each bioassay. We illustrate the new approach using the example of the AREc32 assay, indicative of the oxidative stress response, and 2 nuclear receptor assays targeting the peroxisome proliferator-activated receptor gamma and the arylhydrocarbon receptor. The EBTs were less conservative than previously proposed but were able to differentiate untreated and insufficiently treated wastewater from wastewater treatment plant effluent with secondary or tertiary treatment and surface water. Environ Toxicol Chem 2021;40:487-499. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Addressing Data Gaps in Toxicity of Food Additive Chemicals using High-throughput Screening Technology

Background: More than 8,000 chemical additives are permitted in food in the United States, however, most lack adequate health and safety information. High-throughput screening tools, such as Toxicity Forecaster (ToxCast), provide opportunities to quickly assess and prioritize thousands of food additives for further investigation. Given the significant lack of in vivo data for most chemicals, we aimed to characterize the toxicity data available for chemical food additives using ToxCast.Methods: We developed an up-to-date inventory of chemical food additives utilizing information from the U.S. Food and Drug Administration, the Environmental Protection Agency, and private manufacturers. We quantified the number of chemicals with ToxCast data available, and conducted a preliminary analysis of toxicity data for selected phthalates and phthalate alternatives (di(2-ethylhexyl) phthalate (DEHP), diisononyl cyclohexane-1,2-dicarboxylate (DINCH), N,N-Diethylhydroxylamine (DEHA), and bis (2-ethylhexyl) terephthalate (DEHT)).Results: Our updated inventory indicates that there are more than 9,000 chemicals allowed in contact with food, including direct and indirect additives, food packaging chemicals, pesticide residues, and substances generally recognized as safe. Preliminary analyses suggest that approximately 23% of these have ToxCast data available. Comparing bioassay data in ToxCast for selected phthalates and phthalate alternatives, we found DEHP to be the most toxic chemical, with 40 active endpoints related to cell morphology, DNA binding, and nuclear receptors. DEHT had seven bioactive endpoints, followed by DINCH and DEHA each with 3 active endpoints. DEHA was the only plasticizer with no bioactivity for nuclear receptor assays, one important biological pathway for toxicity in humans.Conclusions: ToxCast may be a useful tool for characterizing the toxicity of chemical food additives. Future analyses will focus on more diverse biological endpoints and compare ToxCast results to available in vivo data. These analyses will help advance understanding on the appropriateness of ToxCast for regulatory decision-making.

Evaluation of a multiplexed, multispecies nuclear receptor assay for chemical hazard assessment

Sensitivity to potential endocrine disrupting chemicals in the environment varies across species and is influenced by sequence conservation of their nuclear receptor targets. Here, we evaluated a multiplexed, in vitro assay testing receptors relevant to endocrine and metabolic disruption from five species. The TRANS-FACTORIAL™ system of human nuclear receptors was modified to include additional species: mouse (Mus musculus), frog (Xenopus laevis), zebrafish (Danio rerio), chicken (Gallus gallus), and turtle (Chrysemys picta). Receptors regulating endocrine function and xenobiotic recognition were included, specifically: ERα, ERβ, AR, TRα, TRβ, PPARγ and PXR. The assay, ECOTOX-FACTORIAL™, was evaluated with 191 chemicals enriched with known receptor ligands. Hierarchical clustering of potency values demonstrated strong coherence of receptor families. Interspecies comparisons of responses within a receptor family showed moderate to high concordance for potencies under 50 μM. PPARγ showed high concordance between mammalian species, 89%, but only 63% between mammalian and zebrafish. For chemicals with potencies below 1 μM, concordances were 89–100% for all receptors except PXR. Concordance showed a strong positive relationship to ligand-binding domain sequence similarity and critical amino acid residues obtained by the Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool. In combination with SeqAPASS, ECOTOX-FACTORIAL may provide efficient screening of important receptors to identify species of high priority for effects monitoring.

Assessing species-specific differences for nuclear receptor activation for environmental water extracts

In vitro bioassays are increasingly applied to detect endocrine disrupting chemicals (EDCs) in environmental waters. Most studies use human nuclear receptor assays, but this raises questions about their relevance for evaluating ecosystem health. The current study aimed to assess species-specific differences in the activation or inhibition of a range of human and zebrafish nuclear receptors by different water extracts. Wastewater and surface water extracts were run in transactivation assays indicative of the estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), mineralocorticoid receptor (MR), pregnane X receptor (PXR) and peroxisome proliferator-activated receptor gamma (PPARγ). The transactivation assays were complemented with competitive binding assays for human AR, GR, PR and MR. In most cases, both human and zebrafish nuclear receptor activity were detected in the water extracts. Only some species-specific differences in potency and activity were observed. Water extracts were more active in zebrafish PXR compared to human PXR whereas the opposite was observed for PPARγ. Further, all water extracts inhibited zebrafish PR, while only one extract showed weak anti-progestagenic activity for human PR. Due to these observed differences, zebrafish nuclear receptor assays may be preferable over human nuclear receptor assays to assess the potential risks of EDCs to aquatic organisms. However, recognizing issues with availability of zebrafish nuclear receptor assays and the relatively small differences in responsiveness for many of the human and zebrafish nuclear receptors, including the widely studied ER, the current study supports the continued use of human nuclear receptor assays for water quality monitoring.

Hedgehog signal disruption, gonadal dysgenesis and reproductive disorders: Is there a link to endocrine disrupting chemicals?

Developmental exposure to chemicals that can disrupt sex hormone signaling may cause a broad spectrum of reproductive disorders. This is because reproductive development is tightly regulated by steroid sex hormones. Consequently, non-animal screening methods currently used to test chemicals for potential endocrine disrupting activities typically include steroidogenesis and nuclear receptor assays. In many cases there is a correlation between in vitro and in vivo data examining endocrine disruption, for example between blocked androgen receptor activity and feminized male genitals. However, there are many examples where there is poor, or no, correlation between in vitro data and in vivo effect outcomes in rodent studies, for various reasons. One possible, and less studied, reason for discordance between in vitro and in vivo data is that the mechanisms causing the in vivo effects are not covered by those typically tested for in vitro. This knowledge gap must be addressed if we are to elaborate robust testing strategies that do not rely on animal experimentation. In this review, we highlight the Hedgehog (HH) signaling pathway as a target for environmental chemicals and its potential implications for reproductive disorders originating from early life exposure. A central proposition is that, by disrupting HH signal transduction during critical stages of mammalian development, the endocrine cells of the testes or ovaries fail to develop normally, which ultimately will lead to disrupted sex hormone synthesis and sexual development in both sexes. If this is the case, then such mechanism must also be included in future test strategies aimed at eliminating chemicals that may cause reproductive disorders in humans.

Identification of Estrogen-Related Receptor α Agonists in the Tox21 Compound Library.

The estrogen-related receptor α (ERRα) is an orphan nuclear receptor (NR) that plays a role in energy homeostasis and controls mitochondrial oxidative respiration. Increased expression of ERRα in certain ovarian, breast, and colon cancers has a negative prognosis, indicating an important role for ERRα in cancer progression. An interaction between ERRα and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) has also recently been shown to regulate an enzyme in the β-oxidation of free fatty acids, thereby suggesting that ERRα plays an important role in obesity and type 2 diabetes. Therefore, it would be prudent to identify compounds that can act as activators of ERRα. In this study, we screened ∼10,000 (8311 unique) compounds, known as the Tox21 10K collection, to identify agonists of ERRα. We performed this screen using two stably transfected HEK 293 cell lines, one with the ERRα-reporter alone and the other with both ERRα-reporter and PGC-1α expression vectors. After the primary screening, we identified more than five agonist clusters based on compound structural similarity analysis (e.g., statins). By examining the activities of the confirmed ERRα modulators in other Tox21 NR assays, eliminating those with promiscuous NR activity, and performing follow-up assays (e.g., small interfering RNA knockdown), we identified compounds that might act as endocrine disrupters through effects on ERRα signaling. To our knowledge, this study is the first comprehensive analysis in discovering potential endocrine disrupters that affect the ERRα signaling pathway.

Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma.

Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function.

Differentiating true androgen receptor inhibition from cytotoxicity-mediated reduction of reporter-gene transactivation in-vitro

In vitro effect-based reporter assays are applied as biodetection tools designed to address nuclear receptor mediated-modulation. While such assays detect receptor modulating potential, cell viability needs to be addressed, preferably in the same well. Some assays circumvent this by co-transfecting a second constitutively-expressed marker gene or by multiplexing a cytotoxicity assay. Some assays, such as the CALUX®, lack this feature. The cytotoxic effects of unknown substances can confound in vitro assays, making the interpretation of results difficult and uncertain, particularly when assessing antagonistic activity. It's necessary to determine whether the cause of the reporter signal decrease is an antagonistic effect or a non-specific cytotoxic effect.To remedy this, we assessed the suitability of multiplexing a cell viability assay within the CALUX® transcriptional activation test for anti-androgenicity. Tests of both well-characterized anti-androgens and cytotoxic compounds demonstrated the suitability of this approach for discerning between the molecular mechanisms of action without altering the nuclear receptor assay; though some compounds were both cytotoxic and anti-androgenic. The optimized multiplexed assay was then applied to an uncharacterized set of polycyclic aromatic compounds. These results better characterized the mode of action and the classification of effects. Overall, the multiplexed protocol added value to CALUX test performance.

Differential Effects and Potential Adverse Outcomes of Ionic Silver and Silver Nanoparticles in Vivo and in Vitro

Nanoparticles are of concern because of widespread use, but it is unclear if metal nanoparticles cause effects directly or indirectly. We explored whether polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNPs) cause effects through intact nanoparticles or dissolved silver. Females of the model species fathead minnow (Pimephales promelas) were exposed to either 4.8 μg/L of AgNO3 or 61.4 μg/L of PVP-AgNPs for 96h. Microarray analyses were used to identify impacted receptors and toxicity pathways in liver and brain tissues that were confirmed using in vitro mammalian assays. AgNO3 and PVP-AgNP exposed fish had common and distinct effects consistent with both intact nanoparticles and dissolved silver causing effects. PVP-AgNPs and AgNO3 both affected pathways involved in Na(+), K(+), and H(+) homeostasis and oxidative stress but different neurotoxicity pathways. In vivo effects were supported by PVP-AgNP activation of five in vitro nuclear receptor assays and inhibition of ligand binding to the dopamine receptor. AgNO3 inhibited ligand binding to adrenergic receptors α1 and α2 and cannabinoid receptor CB1, but had no effect in nuclear receptor assays. PVP-AgNPs have the potential to cause effects both through intact nanoparticles and metal ions, each interacting with different initiating events. Since the in vitro and in vivo assays examined here are commonly used in human and ecological hazard screening, this work suggests that environmental health assessments should consider effects of intact nanoparticles in addition to dissolved metals.

Miniaturization of a Panel of High Throughput Yeast-Cell-Based Nuclear Receptor Assays in 384- and 1536-Well Microplates

A panel of luminescent Saccharomyces cerevisiae cell-based nuclear receptor assays, consisting of human estrogen receptors α and β, androgen receptor, and aryl hydrocarbon receptor, was miniaturized from the standard 96-well microplate format to high-throughput 384- and 1536-well microplate formats. In these assays, firefly luciferase lacking the peroxisome targeting sequence was used as a reporter and D-luciferin substrate was pre-mixed with the yeast cells before the incubation step, eliminating cell lysis and substrate addition steps, and allowing multiple readings at any desired time point. All of the assays were highly functional in the 384-well format, and most functioned well in the 1536-well format. The detection limit of the estrogen receptor α assay was even lower in the miniaturized microplate formats than in the original 96-well format. The panel of yeast-cell-based nuclear receptor assays can be used for high-throughput chemical testing and environmental monitoring of potential endocrine-disrupting activity of compounds and samples.

Two Thiophenes Compounds Are Partial Peroxisome Proliferator-Activated Receptor .ALPHA./.GAMMA. Dual Agonists

In our previous study, two synthetic thiophenes such as IMB-05 and IMB-15 were found as peroxisome proliferator-activated receptor gamma (PPARγ) agonists and exhibited beneficial effects on glucose tolerance of diabetic mice in vivo. In the present study, their effect on the transactivity of other nuclear receptors was further investigated. IMB-05 and IMB-15 could not only activated PPARγ but also efficiently activate PPARα in GAL4-hPPARα/γ (ligand binding domain (LBD)) chimeric receptor assay and PPAR response element (PPRE)-luc reporter gene assay with EC(50) values of 1.8-5.2 µM, whereas no activity was observed in other nuclear receptor assays. In addition, the maximal efficacy of IMB-05 and IMB-15 in activating PPARα/γ was approximately 30% of that observed with Wy14643 and rosiglitazone. These data indicate that the two thiophene derivatives are novel class of partial PPARα/γ dual agonists, which may be the mechanism underlying their regulatory effects on glucose homeostasis.

Nuclear and cytosol androgen receptor in androgen dependent dermatoses in female patients.

Determination of cytosol and nuclear androgen receptor (AR) was performed in females with androgen dependent dermatoses. 34 patients with acne, hirsutism and androgenetic alopecia were punch biopsied on day 21 of the menstrual cycle under local anesthesia within correspondent predilection sites. The tissue was snap frozen in liquid nitrogen and stored until receptor assays were performed. Overall evaluation of both, cytosol and/or nuclear AR was positive in 76% of all cases. In 86% of (10 out of 14) females with androgenetic alopecia, in 80% (8 out of 10) of patients with acne and 60% (6 out of 10) of hirsute females cytosol and/or nuclear AR were positive. No significant differences of cytosol androgen receptor levels became evident between the dermatoses. In contrast, nuclear androgen receptor levels showed a trend towards distinct differences with highest levels in hirsutism, followed by androgenetic alopecia and acne. Androgen stimulability thus seems to be superior in the first and minor in the latter. Comparison with previous studies on cytosol androgen receptor in androgen dependent dermatoses shows significantly higher number of positive results by additive nuclear receptor determination. Additional nuclear androgen receptor assay, thus gives a more complete picture of local hormone action.

Comparison of full-length versus ligand binding domain constructs in cell-free and cell-based peroxisome proliferator-activated receptor alpha assays

Peroxisome proliferator-activated receptor alpha (PPARα) is the nuclear receptor responsible for regulating genes that control lipid homeostasis. Because of this role, PPARα has become a target of interest for the development of drugs to treat diseases such as dyslipidemia, obesity, and atherosclerosis. Assays currently employed to determine potency and efficacy of potential drug candidates typically utilize a truncated form of the native receptor, one which lacks the entire N-terminal region of the protein. The amino terminus, containing the regions that encode the ligand-independent activation function AF-1 and DNA binding domains, is highly structured and contributes significantly to the overall tertiary structure of the native protein. We report that differences in PPARα full-length and ligand binding domain constructs result in differences in binding affinity for coactivator peptides but have little effect on potency of agonists in both cell-free and cell-based nuclear receptor assays.

The role of aromatization in the restoration of male rat reproductive behavior.

This study assessed the role of aromatization in the expression of male reproductive behavior by testing the effects of the aromatase inhibitor, fadrozole, on the restoration of male sexual behavior and partner preference in testosterone-treated gonadectomized rats. We measured nuclear estrogen receptor occupation to determine whether fadrozole blocked brain aromatase. In addition, nuclear androgen receptor assays were used to verify that fadrozole does not block androgen receptors. Mini-osmotic pumps fitted to brain infusion cannulas were used to deliver fadrozole (20 micrograms/day) into the right lateral ventricle. The majority of animals receiving fadrozole treatment with two, 10 mm testosterone filled Silastic capsules (T/F group) failed to display any sexual behavior 7 and 13 days following implant surgery. In contrast, animals receiving fadrozole treatment which were implanted with two, 10 mm testosterone capsules and one, 5 mm 1% estradiol capsule (T/F/E group) copulated normally, indicating that fadrozole's inhibition of male sex behavior was specifically due to blocking aromatase activity. Moreover, the animals which received only one, 5 mm 1% estradiol capsule (E group) also failed to exhibit male sexual behavior. Partner preference for either a sexually receptive female or a non-receptive female was measured in a three chambered apparatus for an index of sexual motivation. Repeated measures contrasts on the group x test interaction indicated that the T/F group was not significantly different from the T group. In addition, the E group did not show a preference for the receptive females and was significantly different from the T group. Fadrozole treatment resulted in a 59% decrease in brain nuclear estrogen receptor occupation relative to the T group. Fadrozole had no significant effect on brain nuclear androgen receptor occupation. Our results lend support to the hypothesis that both androgen receptor activation and aromatization are necessary for the restoration of male sexual behavior in rats. However, we found that estradiol is neither necessary nor sufficient for the restoration of partner preference.

Human endometrial nuclear estrogen receptors among Norplant implant users

Summary Twenty-four parous women were included in this study. Sixteen cases were using the Norplant implant contraceptive system for more than 6 months (Group A) while eight cases served as matched controls in the secretory phase (Group B). Norplant implant users belonged to one of two equal subgroups according to whether they experienced regular menstrual bleeding (Subgroup A1) or prolonged episodes of amenorrhoea (Subgroup A2). Endometrial samples were processed for nuclear oestrogen receptor assay and for evaluation of the receptor's binding affinity to oestrogen. The results showed marked reduction in endometrial nuclear oestrogen receptors in Norplant users compared with controls as the majority of users had undetectable receptor levels. This down-regulation effect was significantly more pronounced among women with prolonged episodes of amenorrhoea. The affinity section of the study revealed no significant differences between groups or subgroups probably due to the small number of subjects with detectable receptor concentrations for comparison. These data may help in the understanding of contraceptive mechanisms and menstrual associated problems, and in the development of therapeutic measures.

Hormonal Regulation of Feather Development and Moult on the Level of Feather Follicles

A sensitive and specific nuclear steroid receptor assay was developed by the stabilization (using sodium molybdenate) and solubilization (using sodium thyocyanate) of receptors, which made it possible to simultaneously measure specific progestin (R5020), androgen (R1s81) and oestrogen (E2) binding sites individually, in feather follicles from secondary remiges and rectrices of domestic geese. Saturation analyses and Scatchard plots showed that each binding site represents a unique type of nuclear receptor with high binding affinity and low capacity for the specific steroid. The presence of a specific, common E2 TEST receptor is also supposed. Three parts of feather follicles were studied separately and among these the papilla and pulp seem to be the most active during feather development, but the role of the follicular wall is also important. The steroid binding capacity is sex dependent in both remiges and rectrices. Castration increases the specific androgen, ovarectomy the specific oestrogen binding sites. A strong positive relationship was shown between plasma level of TEST and P4 and the change in their binding sites in follicles during the moulting process. Five stages of feather development were studied. In the resting follicles androgen (males) or oestrogen (females) receptor dominance is typical. At the onset of moult a bi-phasic increase in progestin binding sites takes place together with an increase in the number of oestrogen receptors. High levels of androgen and oestrogen binding sites are found during the rapid growth of new feathers (differentiation of keratin structures) in the period of maximum pulp size. The results indicate a complex multi-steroidal regulation of feather follicle function.

Quantitative determination of nuclear estrogen receptors by an enzyme immunoassay: Applicability and caveats

A method is presented with which approx. 95% of nuclear estrogen receptors appear to be extracted from MCF-7 cells. Since both nuclear isolation and nuclear estrogen receptor extraction take place in a single test tube with only vortex mixing, loss of nuclear material is minimized. The amount of nuclear estrogen receptors in the nuclear extract was determined by direct [ 3H]estradiol labeling of monolayer cultures and with a commercially available estrogen receptor immunoassay (ER-EIA) kit. Since the ER-EIA kit was designed and calibrated for quantitative determination of cytosolic estrogen receptor isolated in low ionic strength buffer, the applicability of the ER-EIA to quantitative determination of estrogen receptor content in high ionic strength nuclear extraction buffer was tested. A linear relationship exists between the amount of nuclear estrogen receptor detected by the immunoassay, the amount of receptor present in serial dilutions of the nuclear extract and the amount of nuclear estrogen receptor detected in cells by [ 3H]estradiol labeling of monolayer cultures, the absolute amount of nuclear estrogen receptors determined by the immunoassay consistently exceeded the amount of receptor detected by [ 3H]estradiol labeling. The possibility that the enzyme immunoassay must be properly calibrated for the specific conditions of the nuclear estrogen receptor assay is disscussed.

Estrogen and progestin receptors in acoustic and spinal neurilemmomas.: Clinicopathologic correlations

Estradiol and progestin receptors were studied in 20 patients with neuraxial Schwann cell tumors, and their presence was correlated to the clinicopathologic features and the amount of preoperative corticosteroid therapy. Based on an arbitrary cutoff value of 200 fmol per gram of tumor as indicative of a positive receptor value in breast cancer, 4 and 13 of the neurilemmoma tissue samples could be considered as positive for estrogen and progesterone receptors, respectively. Whereas there was no convincing correlation between the estrogen and progestin receptor activity and the age, sex, or menopausal status of the patients, overweight patients had significantly higher estrogen and progestin binding values. The correlation between the amount of preoperative prednisone therapy and the amount of [ 3H]estradiol and [ 3H]promegestone binding revealed no dose relationship. Correlating [ 3H]estradiol and [ 3H]promegestone content with the histologic type of the schwannomas (Antoni types A and B, respectively), we were not able to draw conclusions, because of the predominance of Antoni type A over Antoni type B tissues in our material. The necessity of nuclear receptor assays, ligand specificity testing, and in vitro studies is stressed as a prerequisite for answering the questions whether neurilemmomas contain genuine sexual steroid hormone receptors and whether these receptors are regulated via an estrogen-estrogen-receptor system as is the case in classical sexual steroid hormone target tissues.

Applicability of Nuclear Androgen Receptor Quantification to Human Prostatic Adenocarcinoma

It has been proposed that concentrations of nuclear androgen receptor may be predictive of tumor hormone dependence in cases of advanced human prostatic cancer. We have investigated the ability of this receptor population to reflect patient prognosis during endocrine therapy in 12 cases of stage D disease. KCl-extractable, nuclear matrix-bound and total nuclear androgen receptor concentrations showed a significant positive correlation with duration of patient survival (p < 0.05) while cytosolic and total cellular androgen receptor concentrations were not significantly correlated with survival. However, use of selected threshold concentrations of receptors revealed that only cytosolic, nuclear KCl-extractable and total cellular receptors could significantly differentiate long-term and short-term survivors. Even given the small number of patients studied, the potential use of this androgen receptor assay as an index of both tumor hormone-dependence and patient prognosis was evident. Therefore, in order to make these androgen receptor assays more applicable, we attempted to simplify the methods for use on readily available tissues. Similar amounts of nuclear androgen binding were observed in crude and purified nuclear pellets, in nuclei treated with DNase and KCl in differing orders or in nuclei from tissue homogenized using glass or Polytron homogenization procedures. More importantly, nuclear androgen receptor concentrations in specimens of prostatic cancer or benign hyperplasia taken by needle biopsy or transurethral resection involving electrocautery did not differ from those of parallel specimens taken by Thompson cold punch. Simplified nuclear androgen receptor assays of needle biopsy or electrocautery specimens are accurate and should prove clinically applicable.