Mary C. Cardon

Competitive binding comparison of endocrine-disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and human.

Typically, in vitro hazard assessments for the identification of endocrine-disrupting compounds (EDCs), including those outlined in the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Tier 1 Screening protocols, utilize mammalian receptors. Evidence, however, exists that fish sex steroid hormone receptors differ from mammalian receptors both structurally and in their binding affinities for some steroids and environmental chemicals. Most of the binding studies to date have been conducted using cytosolic preparations from various tissues. In the present study, we compare competitive binding of a set of compounds to full-length recombinant rainbow trout androgen receptor alpha (rtAR), fathead minnow androgen receptor (fhAR), and human androgen receptor (hAR), each expressed in COS cells. Saturation binding and subsequent Scatchard analysis using [3H]R1881, a high-affinity synthetic androgen, revealed an equilibrium dissociation constant (Kd) of 0.11 nM for the rtAR, 1.8 nM for the fhAR, and 0.84 nM for the hAR. Compounds, including endogenous and synthetic steroids, known mammalian antiandrogens, and environmental compounds, were tested for competitive binding to each of the three receptors. Overall, agreement existed across receptors as to binding versus nonbinding for all compounds tested in this study. Minor differences, however, were found in the relative order of binding of the compounds to the individual receptors. Studies such as these will facilitate the identification of EDCs that may differentially affect specific species and aid in the development and support of future risk assessment protocols.

Environmental Gestagens Activate Fathead Minnow (Pimephales promelas) Nuclear Progesterone and Androgen Receptors in Vitro

Gestagen is a collective term for endogenous and synthetic progesterone receptor (PR) ligands. In teleost fishes, 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) are the predominant progestogens, whereas in other vertebrates the major progestogen is progesterone (P4). Progestins are components of human contraceptives and hormone replacement pharmaceuticals and, with P4, can enter the environment and alter fish and amphibian reproductive health. In this study, our primary objectives were to clone the fathead minnow (FHM) nuclear PR (nPR), to develop an in vitro assay for FHM nPR transactivation, and to screen eight gestagens for their ability to transactivate FHM nPR. We also investigated the ability of these gestagens to transactivate FHM androgen receptor (AR). Fish progestogens activated FHM nPR, with DHP being more potent than 20β-S. The progestin drospirenone and P4 transactivated the FHM nPR, whereas five progestins and P4 transactivated FHM AR, all at environmentally relevant concentrations. Progestins are designed to activate human PR, but older generation progestins have unwanted androgenic side effects in humans. In FHMs, several progestins proved to be strong agonists of AR. Here, we present the first mechanistic evidence that environmental gestagens can activate FHM nPR and AR, suggesting that gestagens may affect phenotype through nPR- and AR-mediated pathways.

Metabolite profiling and a transcriptional activation assay provide direct evidence of androgen receptor antagonism by bisphenol A in fish.

Widespread environmental contamination by bisphenol A (BPA) has created the need to fully define its potential toxic mechanisms of action (MOA) to properly assess human health and ecological risks from exposure. Although long recognized as an estrogen receptor (ER) agonist, some data suggest that BPA may also behave as an androgen receptor (AR) antagonist. However, direct evidence of this activity is deficient. To address this knowledge gap, we employed a metabolomic approach using in vivo exposures of fathead minnows (FHM; Pimephales promelas ) to BPA either alone or in a binary mixture with 17β-trenbolone (TB), a strong AR agonist. Changes in liver metabolite profiles in female FHM in response to these exposures were determined using high resolution (1)H NMR spectroscopy and multivariate and univariate statistics. Using this approach, we observed clear evidence of the ability of BPA to mitigate the impact of TB, consistent with an antiandrogenic MOA. In addition, a transcriptional activation assay with the FHM AR was used to confirm the AR antagonistic activity of BPA in vitro. The results of these in vivo and in vitro analyses provide strong and direct evidence for ascribing an antiandrogenic MOA to BPA in vertebrates.

Activation of Southern White Rhinoceros (Ceratotherium simum simum) Estrogen Receptors by Phytoestrogens: Potential Role in the Reproductive Failure of Captive-Born Females?

The captive southern white rhinoceros (SWR; Ceratotherium simum simum) population serves as an important genetic reservoir critical to the conservation of this vulnerable species. Unfortunately, captive populations are declining due to the poor reproductive success of captive-born females. Captive female SWR exhibit reproductive problems suggested to result from continual ovarian follicular activity and prolonged exposure to endogenous estrogen. However, we investigated the potential role of exogenous dietary phytoestrogens in the reproductive failure of SWR by cloning and characterizing in vitro phytoestrogen binding and activation of recombinant SWR estrogen receptors (ESR). We compared those characteristics with recombinant greater one-horned rhinoceros (GOHR; Rhinoceros unicornis) ESR, a species that receives similar captive diets yet reproduces relatively well. Our results indicate that phytoestrogens bind rhino ESR in a manner similar to other vertebrate species, but there are no differences found in phytoestrogen binding affinity of SWR ESR compared with GOHR ESR. However, species-specific differences in ESR activation by phytoestrogens were detected. The phytoestrogen coumestrol stimulated greater maximal activation of SWR ESR1 than GOHR ESR1. SWR ESR2 were also more sensitive to phytoestrogens and were activated to a greater extent by both coumestrol and daidzein. The concentrations in which significant differences in ESR activation occurred (10(-7) to 10(-5) m) are consistent with circulating concentrations measured in other vertebrate species. Taken together, these findings suggest that phytoestrogens potentially pose a risk to the reproductive health of captive SWR. However, additional studies are needed to further clarify the physiological role of dietary phytoestrogens in the reduced fertility of this species.

Comparison of chemical binding to recombinant fathead minnow and human estrogen receptors alpha in whole cell and cell-free binding assays†

Mammalian receptors and assay systems are generally used for in vitro screening of endocrine-disrupting chemicals with the assumption that minor differences in amino acid sequences among species do not translate into significant differences in receptor function. Objectives of the present study were to evaluate the performance of two different in vitro assay systems (a whole cell and a cell-free competitive binding assay) in assessing whether binding of chemicals differs significantly between full-length recombinant estrogen receptors from fathead minnows (fhERalpha) and those from humans (hERalpha). It was confirmed that 17beta-estradiol displays a reduction in binding to fhERalpha at an elevated temperature (37 degrees C), as has been reported with other piscine estrogen receptors. Several of the chemicals (17beta-estradiol, ethinylestradiol, alpha-zearalanol, fulvestrant, dibutyl phthalate, benzyl butyl phthalate, and cadmium chloride) displayed higher affinity for fhERalpha than for hERalpha in the whole cell assay, while only dibutyl phthalate had a higher affinity for fhERalpha than for hERalpha in the cell-free assay. Both assays were effective in identifying strong binders, weak binders, and nonbinders to the two receptors. However, the cell-free assay provided a less complicated and more efficient binding platform and is, therefore, recommended over the whole cell binding assay. In conclusion, no strong evidence showed species-specific binding among the chemicals tested.

Development of a competitive binding assay system with recombinant estrogen receptors from multiple species

In the current study, we developed a new system using full-length recombinant baculovirus-expressed estrogen receptors which allows for direct comparison of binding across species. Estrogen receptors representing five vertebrate classes were compared: human estrogen receptor alpha (hERalpha), quail estrogen receptor alpha (qERalpha), alligator estrogen receptor alpha (aERalpha), salamander estrogen receptor alpha (sERalpha), and fathead minnow estrogen receptor alpha (fhERalpha). Saturation binding analyses indicated 17beta-estradiol (E2) dissociation constants (Kd) were 0.22+/-0.02nM for hERalpha, 0.28+/-0.04nM for sERalpha, 0.44+/-0.04nM for aERalpha, 0.58+/-0.10nM for qERalpha, and 0.58+/-0.05nM for fhERalpha. Binding specificity to each of the receptors was evaluated using E2, dihydrotestosterone (DHT), corticosterone (C), and ethinylestradiol (EE). E2 and EE were strong binders in all species with IC50s ranging from 0.65nM with hERalpha to 1.01nM with sERalpha for E2 and from 0.68nM with sERalpha to 1.20nM with qERalpha for EE. DHT was a weak binder with IC50s ranging from 3.3microM with hERalpha to 39microM with fhERalpha, and C did not bind any of the receptors at concentrations up to 100microM. This system provides a convenient in vitro approach for directly comparing chemical binding to estrogen receptors across multiple species without the need to sacrifice animals.

Insect virus: assays for viral replication and persistence in mammalian cells

Viral pesticidal agents must be evaluated for potential health hazards prior to utilization. Assessment of the likelihood of replication in humans has included in vitro exposure of human cells to the potential pesticidal agent. Previous in vitro evaluation strategies have lacked positive controls. Thus, negative results, interpreted as no effect of the virus on human cells, could reflect basic deficiencies in the testing protocols. We designed a testing scheme for viral pesticides and used it to test the nuclear polyhedrosis virus of Autographa californica. Tests were aimed at evaluating potential replication or gene expression in primate cells. Parallel tests were run utilizing identical protocols with primate viruses known to produce the biological effect being evaluated. Thus protocols described were tested with positive viral controls.

Occurrence and In Vitro Bioactivity of Estrogen, Androgen, and Glucocorticoid Compounds in a Nationwide Screen of United States Stream Waters

In vitro bioassays are sensitive, effect-based tools used to quantitatively screen for chemicals with nuclear receptor activity in environmental samples. We measured in vitro estrogen (ER), androgen (AR), and glucocorticoid receptor (GR) activity, along with a broad suite of chemical analytes, in streamwater from 35 well-characterized sites (3 reference and 32 impacted) across 24 states and Puerto Rico. ER agonism was the most frequently detected with nearly all sites (34/35) displaying activity (range, 0.054-116 ng E2Eq L-1). There was a strong linear relationship (r2 = 0.917) between in vitro ER activity and concentrations of steroidal estrogens after correcting for the in vitro potency of each compound. AR agonism was detected in 5/35 samples (range, 1.6-4.8 ng DHTEq L-1) but concentrations of androgenic compounds were largely unable to account for the in vitro activity. Similarly, GR agonism was detected in 9/35 samples (range, 6.0-43 ng DexEq L-1); however, none of the recognized GR-active compounds on the target-chemical analyte list were detected. The utility of in vitro assays in water quality monitoring was evident from both the quantitative agreement between ER activity and estrogen concentrations, as well as the detection of AR and GR activity for which there were limited or no corresponding target-chemical detections to explain the bioactivity. Incorporation of in vitro bioassays as complements to chemical analyses in standard water quality monitoring efforts would allow for more complete assessment of the chemical mixtures present in many surface waters.

Rapid efficient production of baculovirus expression vectors

Recombinant baculoviruses have been used to produce foreign proteins and have the potential to be safe, efficacious insecticides. Isolation of recombinant virus is usually by plaque phenotype. Typical recombination rates are less than 1%, thus requiring time consuming inspection of hundreds of individual plaques. We describe a method of generating recombinants which requires less time than current protocols and frequently produces recombinants at rates exceeding 30%. This protocol employs liposome-mediated transfection, reduced post-transfection incubation times, linearized parental virus which produces occlusion positive plaques in clones of the parental genotype, and colorimetric detection of recombinants. This protocol allows the initial, and frequently the final, isolation of recombinants in 7 days.

Detection of androgenic activity in emissions from diesel fuel and biomass combustion

The present study evaluated both diesel fuel exhaust and biomass (wood) burn extracts for androgen receptor-mediated activity using MDA-kb2 cells, which contain an androgen-responsive promoter-luciferase reporter gene construct. This assay and analytical fractionization of the samples were used as tools to separate active from inactive fractions, with the goal of identifying the specific compounds responsible for the activity. A significant androgenic response was detected from the diesel emission. High-performance liquid chromatographic fractionation of the sample indicated that significant androgenic activity was retained in three fractions. 4-Hydroxybiphenyl was identified from the most active fraction using gas chromatography/mass spectroscopy. This purified compound was then tested at doses from 1 nM to 100 microM. 4-Hydroxybiphenol exhibited antagonist activity at low concentrations and agonist activity at high concentrations. A competitive-binding assay confirmed binding to the androgen receptor, with a median inhibitory concentration for radioligand binding of approximately 370 nM. Significant androgenic activity also was detected in the wood burn samples, but we were unable to identify the specific chemicals responsible for this endocrine activity. The present study demonstrates that in vitro bioassays can serve as sensitive bioanalytical tools to aid in characterization of complex environmental mixtures.