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 17ß-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.

Dysregulation of kisspeptin and neurogenesis at adolescence link inborn immune deficits to the late onset of abnormal sensorimotor gating in congenital psychological disorders.

Neuropsychological syndromes including schizophrenia often do not manifest until late adolescence or early adulthood. Studies attributing a role in brain maintenance to the immune system led us to propose that malfunction of immune-dependent regulation of brain functions at adolescence underlies the late onset of such diseases/syndromes. One such function is sensorimotor gating, the ability to segregate a continuous stream of sensory and cognitive information, and to selectively allocate attention to a significant event by silencing the background (measured by prepulse inhibition; PPI). This activity is impaired in schizophrenia, as well as in several other neuropsychological diseases. Using a model of prenatal immune activation (maternal polyriboinosinic-polyribocytidylic acid (poly I:C) injection), often used as a model for schizophrenia, and in which abnormal PPI has a delayed appearance, we demonstrated a form of immune deficit in the adult offspring. Similar abnormal PPI with a delayed appearance was found in congenitally immune-deficient mice (severe combined immune deficient, SCID), and could be reversed by immune reconstitution. This functional deficit correlated with impairment of both hippocampal neurogenesis and expression of the gene encoding kisspeptin (Kiss1) that manifested at adulthood. Moreover, exogenous administration of a kisspeptin-derived peptide partially reversed the gating deficits in the SCID mice. Our results suggest that a form of congenital immune deficiency may be a key factor that determines manifestation of developmental neuropsychological disorders with onset only at early adulthood.

The influence of various regions of the FOXP2 sequence on its structure and DNA-binding function.

FOX proteins are a superfamily of transcription factors which share a DNA-binding domain referred to as the forkhead domain. Our focus is on the FOXP subfamily members, which are involved in language and cognition amongst other things. The FOXP proteins contain a conserved zinc finger and a leucine zipper motif in addition to the forkhead domain. The remainder of the sequence is predicted to be unstructured and includes an acidic C-terminal tail. In the present study, we aim to investigate how both the structured and unstructured regions of the sequence cooperate so as to enable FOXP proteins to perform their function. We do this by studying the effect of these regions on both oligomerisation and DNA binding. Structurally, the FOXP proteins appear to be comparatively globular with a high proportion of helical structure. The proteins multimerise via the leucine zipper, and the stability of the multimers is controlled by the unstructured interlinking sequence including the acid rich tail. FOXP2 is more compact than FOXP1, has a greater propensity to form higher order oligomers, and binds DNA with stronger affinity. We conclude that while the forkhead domain is necessary for DNA binding, the affinity of the binding event is attributable to the leucine zipper, and the unstructured regions play a significant role in the specificity of binding. The acid rich tail forms specific contacts with the forkhead domain which may influence oligomerisation and DNA binding, and therefore the acid rich tail may play an important regulatory role in FOXP transcription.

Schizophrenia: from the brain to peripheral markers. A consensus paper of the WFSBP task force on biological markers.

Objective. The phenotypic complexity, together with the multifarious nature of the so-called "schizophrenic psychoses", limits our ability to form a simple and logical biologically based hypothesis for the disease group. Biological markers are defined as biochemical, physiological or anatomical traits that are specific to particular conditions. An important aim of biomarker discovery is the detection of disease correlates that can be used as diagnostic tools. Method. A selective review of the WFSBP Task Force on Biological Markers in schizophrenia is provided from the central nervous system to phenotypes, functional brain systems, chromosomal loci with potential genetic markers to the peripheral systems. Results. A number of biological measures have been proposed to be correlated with schizophrenia. At present, not a single biological trait in schizophrenia is available which achieves sufficient specificity, selectivity and is based on causal pathology and predictive validity to be recommended as diagnostic marker. Conclusions. With the emergence of new technologies and rigorous phenotypic subclassification the identification of genetic bases and assessment of dynamic disease related alterations will hopefully come to a new stage in the complex field of psychiatric research.

Age-dependent spatial memory loss can be partially restored by immune activation.

Aging is often associated with a decline in hippocampus-dependent spatial memory. Here, we show that functional cell-mediated immunity is required for the maintenance of hippocampus-dependent spatial memory. Sudden imposition of immune compromise in young mice caused spatial memory impairment, whereas immune reconstitution reversed memory deficit in immune-deficient mice. Analysis of hippocampal gene expression suggested that immune-dependent spatial memory performance was associated with the expression of insulin-like growth factor (Igf1) and of genes encoding proteins related to presynaptic activity (Syt10, Cplx2). We further showed that memory loss in aged mice could be attributed to age-related attenuation of the immune response and could be reversed by immune system activation. Homeostatic-driven proliferation of lymphocytes, which expands the existing T cell repertoire, restored spatial memory deficits in aged mice. Thus, our results identify a novel function of the immune system in the maintenance of spatial memory and suggest an original approach for arresting or reversing age-associated memory loss.

High throughput adjustable 96-well plate assay for androgen receptor binding: a practical approach for EDC screening using the chimpanzee AR.

The issue as to whether natural and man-made chemicals interfere with endocrine function has raised concerns. This interference could be biologically significant even at very low doses if the chemicals interact deleteriously with hormone receptors at low concentrations. Therefore, the United States Environmental Protection Agency (USEPA) Office of Coordination and Policy (OSCP) requested that a nonhuman mammalian androgen receptor binding assay be developed for possible use in their Endocrine Disruptor Screening Program (EDSP). Ideally, this assay would be high throughput, not use animals as a source of receptor protein, easily deployed throughout the scientific community, utilize reagents available to both the public and private sector, and have the potential for future automation. We developed a highly modified 96-well plate assay which meets these criteria. It employs a baculovirus expressed recombinant primate androgen receptor which is publically available and exploits the unique ability of some mammalian androgen receptors to remain biologically active after guanidine hydrochloride (GdnHCl) solubilization. This GdnHCl treated receptor remains soluble and requires no additional purification prior to use. We provide a very detailed description of the assay protocol itself, and similarly detailed method for producing and solubilizing the receptor.

Substitution of synthetic chimpanzee androgen receptor for human androgen receptor in competitive binding and transcriptional activation assays for EDC screening.

The potential effect of receptor-mediated endocrine modulators across species is of increasing concern. In attempts to address these concerns, we are developing androgen and estrogen receptor binding assays using recombinant hormone receptors from a number of species across different vertebrate classes. The United States Environmental Protection Agency (USEPA) Office of Science Coordination and Policy (OSCP) requested that we develop a nonhuman mammalian receptor-binding assay for possible use in their Endocrine Disruptor Screening Program (EDSP). Since the chimpanzee androgen receptor is very similar to that of humans and thus possesses properties which could be exploited in future endocrine studies, we synthesized and expressed this gene in eukaryotic expression plasmids, baculovirus expression vectors and replication deficient adenovirus. In all ligand-binding and transcriptional activation assays tested, the chimpanzee receptor performed essentially identically to the human receptor. This suggests that the chimpanzee gene could substitute for the human gene in endocrine screening assays.

Generation of recombinant baculovirus via liposome-mediated transfection.

Baculovirus expression vectors have become a popular method of producing recombinant proteins. Production of recombinant virus requires the transfection of both the native viral DNA and a transfer plasmid into insect cells where recombination takes place. While several methods of transfecting insect cells exist, we have found liposome-mediated transfection to be highly efficient. Here we detail the protocols and medium needed for efficient, simple transfection of Spodoptera frugiperda cells.

Development of two androgen receptor assays using adenoviral transduction of MMTV-luc reporter and/or hAR for endocrine screening.

The discovery of xenobiotics that interfere with androgen activity has highlighted the need to assess chemicals for their ability to modulate dihydrotestosterone (DHT)-receptor binding. Previous test systems have used cells transfected with plasmid containing a reporter gene. Here we report the use of transduction for gene delivery and assessment of the modulation of DHT-induced gene activation. Transduction, the ability of replication-defective viruses to deliver biologically competent genes, is a well understood biological process, which has been utilized to repair defective genes in humans as well as to express exogenous genes in rodent models. Human breast carcinoma cells (MDA-MB-453) containing endogenous copies of the androgen (hAR) and glucocorticoid (GR) receptors were transduced with replication-defective human adenovirus type 5 containing the luciferase (Luc) reporter gene driven by the AR- and GR-responsive glucocorticoid-inducible hormone response element found with the mammary tumor virus LTR (Ad/MLUC7). In a second set of experiments, CV-1 cells were transduced as above with MMTV-luc and also hAR. Cells were subcultured in 96-well plates, transduced with virus, exposed to chemicals, incubated for 48 h, lysed, and assayed for luciferase. Luc gene expression was induced in a dose-dependent manner by DHT, estradiol, and dexamethasone (MDA only) and inhibited by AR antagonist hydroxyflutamide (OHF), hydroxy-DDE, HPTE (2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane), a methoxychlor metabolite, and M1 and M2 (vinclozolin metabolites). The transduced cells responded to AR agonists and antagonists as predicted from our other studies, with a very robust and reproducible response. Over all replicates, 0.1 nM DHT induced luc expression by about 45-fold in CV-1 cells (intra-assay CV = 20%) and 1micromolar OHF inhibited DHT by about 80%. In the transduced MDA cells, 0.1 nM DHT induced luc by about 24-fold (intra-assay CV = 33%), which was inhibited by OHF by about 85%. DHT-induced luciferase activity peaked in both cell lines between 1 and 100 nM, displaying about 64- and 115-fold maximal induction in the CV-1 and MDA 453 cells, respectively. For agonists, a two-fold induction of luc over media control was statistically significant. For AR antagonists, a 25-30% inhibition of DHT-induced luc expression was typically statistically significant. Comparing the two assays, the transduced CV-1 cells were slightly more sensitive to AR-mediated responses, but the transduced MDA 453 cells were more responsive to GR agonists. In summary, these assays correctly identified the endocrine activity of all chemicals examined and displayed sensitivity with a relatively low variability and a high-fold induction over background. Adenovirus transduction for EDC screening has the potential to be employed in a high-throughput mode, and could easily be applied to other cell lines and utilized to deliver other receptors and reporter genes.

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.

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.

Effect of baculovirus on selected vertebrate cells.

Risk assessment of viral pesticides is required by federal statute. Subdivision M of the Pesticide Testing Guidelines includes mammalian cell culture studies for viral agents. Using these tests we have demonstrated baculovirus toxicity to human fibroblast and lung cells, as well as monkey kidney cells. However, virus was not toxic to toad cells. Furthermore, toxicity was associated with the virus particle and was not removed by dialysis of the preparation, gradient purification or psoralen inactivation of the virus. However, heat-inactivated virus was not toxic.

Axial skeletal and Hox expression domain alterations induced by retinoic acid, valproic acid, and bromoxynil during murine development.

Retinoic acid (RA) alters the developmental fate of the axial skeletal anlagen. "Anteriorizations" or "posteriorizations," the assumption of characteristics of embryonic areas normally anterior or posterior to the affected tissues, are correlated with altered embryonal expression domains of Hox genes after in utero RA treatment. These "homeotic" changes have been hypothesized to result from alterations of a "Hox cod" which imparts positional identity in the axial skeleton. To investigate whether such developmental alterations were specific to RA, or were a more general response to xenobiotic exposure, CD-1 pregnant mice were exposed to RA, valproic acid (VA), or bromoxynil (Br) during organogenesis. Additionally, the expression domains of two Hox genes, Hoxa7 and Hoxa10, were examined in gestation day (GD) 12.5 embryos obtained from control, RA, VA, or Br, treated gravid dams exposed on GD 6, 7, or 8. The anterior expression boundary of Hoxa7 is at the level of the C7/T1 vertebrae and that of Hoxa10 is at L6/S1. Compound-induced changes in the incidence of skeletal variants were observed. These included supernumerary cervical ribs (CSNR) lateral to C7, 8 vertebrosternal ribs, supernumerary lumbar ribs (LSNR) lateral to L1, extra presacral vertebrae, and the induction of vertebral and/or rib malformations. RA and VA administration on GD 6 caused posteriorization in the cervico-thoracic region (CSNR) while GD 8 exposure to any of the three compounds resulted in anteriorizations in the thoraco-lumbar area (LSNR and an increase in the number of presacral vertebrae). These effects occurred across regions of the axial skeleton. Analysis of gene expression demonstrated changes in the anterior boundaries of Hoxa7 expression domains in embryos treated on GD 6 and 8 with RA. VA and Br did not induce any statistically significant alterations in Hoxa7 and none of the compounds caused alterations in Hoxa10 expression domains. The studies indicate that RA GD 6 treatment-induced Hoxa7 shifts were rostral (posteriorization) while the RA-induced GD 8 anterior expression boundary shift was caudal (anteriorization), correlating with the axial skeletal changes noted. These data suggest that xenobiotic compounds such as VA and Br may induce similar axial skeletal changes by affecting different components of the developmental processes involved in the patterning of the axial skeleton.