Grantee Research Project Results
Final Report: Establishing an AOP for the Role of the Vitamin D Receptor in Developmental Neurotoxicity
EPA Grant Number: R835541Title: Establishing an AOP for the Role of the Vitamin D Receptor in Developmental Neurotoxicity
Investigators: Kullman, Seth W. , Levin, Edward D , Slotkin, Theodore
Institution: Duke University Medical Center , North Carolina State University
EPA Project Officer: Aja, Hayley
Project Period: July 1, 2013 through June 30, 2016 (Extended to January 31, 2018)
Project Amount: $799,496
RFA: Development and Use of Adverse Outcome Pathways that Predict Adverse Developmental Neurotoxicity (2012) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability , Human Health
Objective:
The etiology of a significant number of human diseases, including neurodevelopmental and neurodegenerative diseases, are in part associated with exposures to environmental contaminants and recent trends in toxicological research and indicate a growing interest in chemicals capable of disrupting neuroendocrine mediated signaling. These chemicals comprise a range of natural and synthetic molecules that interact with nuclear hormone receptors altering signaling pathways and regulating adverse effects in multiple organ systems, tissue and cell types. Although the interaction of endocrine disrupting xenobiotics with nuclear receptors such as the Estrogen receptor (ER), Thyroid receptor (TR) and Androgen receptor (AR) has been studied in detail, similar research involving xenobiotic interactions with the Vitamin D receptor (VDR) has remained underexplored. In this study, we have examined the role of vitamin D receptor as a potential target of xenobiotic induced endocrine disruption and provide new insights into the possible mechanisms underlying the complex molecular interactions between VDR and select VDR agonists and antagonists. Goals of this study were to (1) identify and validate VDR as a nuclear receptor target for chemical agents identified through quantitative highthroughput testing (qHTS) formats, including EPA’s ToxCast and Tox21 data sets, and (2) establish linkages between developmental exposures, modulation of VDR signaling and larval/adult neural behavioral consequence in zebrafish.
Summary/Accomplishments (Outputs/Outcomes):
(1) In our initial studies, we established a series of orthogonal assays to confirm high-throughput screening data and novel mechanistic insights into VDR-xenobiotic interactions. High throughput screening (HTS) programs have demonstrated that the Vitamin D receptor (VDR) is activated and/or antagonized by a wide range of structurally diverse chemicals. In this study, we examined the Tox21 qHTS data set generated against VDR for reproducibility and concordance and elucidated functional insights into VDR-xenobiotic interactions. Twenty-one potential VDR agonists and 19 VDR antagonists were identified from a subset of >400 compounds with putative VDR activity and examined for VDR functionality utilizing select orthogonal assays. Transient transactivation assay (TT) using a human VDR plasmid and Cyp241A1 luciferase reporter construct revealed 20/21 active VDR agonists and 18/19 active VDR antagonists. A mammalian-2-hybrid assay (M2H) then was used to evaluate VDR interactions with co-activators and co-regulators. With the exception of a select few compounds, VDR agonists exhibited significant recruitment of co-regulators and co-activators, whereas antagonists exhibited considerable attenuation of recruitment by VDR. A unique set of compounds exhibiting synergistic activity in antagonist mode and no activity in agonist mode was identified. Cheminformatics modeling of VDR-ligand interactions were conducted and revealed selective ligand VDR interactions. Overall, data emphasizes the molecular complexity of ligand-mediated interactions with VDR and suggest that VDR transactivation may be a target site of action for diverse xenobiotics. Subsequent studies were conducted with zebrafish VDR alpha and VDR beta paralogs. In general, the zebrafish VDR alpha paralog recapitulated the xenobiotic responses observed with the human VDR chemical screen. Comparatively, the zebrafish VDR beta paralog exhibited a significant departure in agonists/antagonists activity profiles suggesting that subfunctionalization of these paralogs has occurred.
(2) Based on our initial studies, we observed that select trialkyltins significantly modulated the Vitamin D receptor (VDR) functional activities. Given the known role of trialkyltins as retinoid X receptor (RXR) agonists, we investigated if these compounds could influence VDR transactivation through interaction of the VDR:RXR heterodimer. RXR and VDR engage in a canonical interactions when bound to the ligand 1,25 dihydroxy vitamin D3. This ligand-mediated interaction facilitates both transactivation and transrepression of highly selective gene regulatory networks. The RXR:VDR interaction is thought to be non-permissive, that is, ligands to RXR do not transactivate the receptor heterodimer. This is in contrast to other RXR:NR partnerships such as RXR:PPARg where potent RXR ligands such as organotins can active the receptor heterodimer. In this study, we identify a novel role of organotins wherein both TBT and TPT act as potent VDR antagonists at high nM concentrations and as VDR synergists at low nM concentrations in the presence of vitamin D3. This biphasic activity is observed both in transient transactivation of VDR in Cos7 cells and with endogenous gene expression of CYP24A1 in HL60 cells. Using functional RXR mutants, potent RXR inhibitors and mammalian two-hybrid assays, we demonstrate that the synergistic actions of triorganic tins on VDR transactivation are mediated through RXR. We propose that trialkyltins modify RXR:VDR interactions in a manner that is conditionally permissive and suggest that this previously unrecognized synergistic role of organotins in altering VDR transcription is likely to have important endocrine mediated implications. Over all, we suggest a novel role of trialkyltins to promote conditional permissive transactivation of VDR.
(3) In a third study, we examined the role of yet another well-known non-essential toxic element, cadmium, in disrupting the transactivational activity of VDR. Cadmium exposure poses serious health risks to humans and other vertebrates with established neurodevelopmental toxicity. Unlike other nuclear hormone receptors such as the ER where cadmium is a demonstrated receptor agonist, we demonstrate that cadmium inhibits transactivation of ligand induced VDR in multiple cell types. We demonstrate that cadmium inhibits vitamin D3 induced endogenous gene expression of a potent downstream gene (CYP24A1) regulated by VDR. Given the ability of cadmium to disrupt zinc fingers in the DNA binding domain of nuclear receptors, we investigated the role of cadmium to inhibit VDR binding to DNA as a potential mechanism of receptor inhibition. Results from mutation studies, ChiP and EMSA assays indicate that cadmium likely prevents binding of Cd-bound VDR to canonical VDREs. Interestingly, however, cadmium also facilitated recruitment of coactivator and coregulator when protein:protein interactions were investigated. Taken together, these findings clearly suggest two distinct modes of action of cadmium that likely disrupts VDR transactivation. Overall, we demonstrate that cadmium has the potential to disrupt the vitamin D endocrine system.
(4) Using zebrafish as a model organism, we studied the expression patterns of two vitamin D receptor paralogs (VDRa & VDRb) in adult zebrafish and Japanese medaka brain. The objective in this study was to document the spatiotemporal expression of VDR in the brain and identify putative patterns of expression. Vitamin D receptor expression was observed in the neurons, astrocytes and radial glial cells. In neurons, expression was limited to the neuronal cytoplasm, axon hillock and axons. In radial glial cells and astrocytes, expression was predominantly in the cytoplasm and sporadically in the nucleus within the olfactory bulb, dorsal and ventral telencephalon, dorsal and ventral thalamus, optic tectum, cerebellum, medulla and alongside ventricles throughout the rostral-caudal axis. This expression pattern is consistent with that observed in mouse and human brain and suggest that VDR localization may be highly conserved between evolutionary distant species. No differences were observed in localization between VDRa and VDRb paralogs in both medaka and zebrafish brain suggesting that these two receptors are likely not subfunctionalized at the tissue expression level. Co-localization of VDR was identified in neurons expressing tyrosine hydroxylase TH/DAT, VACHT and GFAP suggesting that VDR may be expressed in dopaminergic neurons within the telecephalon, cholinergic neurons within the purkinje neurons of the cerebellum and glial cells of astrocytic lineage. The spatial clustering of additional TH and VDR positive neurons that do not co-express also is particularly interesting and suggest that select neurons appear to be in close proximity in a manner that would be conducive for paracrine signaling. This mode of signaling whereby VDR potentially regulates development of DA neurons only has been speculated but not reported thus far in literature.
(5) Critical to the use of zebrafish as a complementary model for the study of neurobehavioral toxicity is the development of an efficient, sensitive and validated test battery to characterize behavioral function. We have developed a battery of behavioral tests to assess sensorimotor, cognitive and affective function of zebrafish. Over the past yea,r we have established a series of studies to assess larval zebrafish behavior following exposure to several select VDR agonists/antagonists. In the current set of studies, we examined 10 compounds known to affect VDR function in vitro for neurobehavioral effects in vivo in zebrafish. Zebrafish embryos were exposed to doses of the compounds in their water during the first 5 days post-fertilization. On day 6, the embryos were tested in an alternating light-dark locomotor assay using a computerized video tracking system. We found that all 10 of the compounds produced significant changes in locomotor behavior in exposed zebrafish larvae, although the direction of the effect (i.e., hypo- or hyperactivity) and the sensitivity of the effect to changes in illumination condition varied across the compounds. The nature of the behavioral effects generally corresponded to the effects these compounds have been shown to exert on VDR. In general, we observe that VDR agonists result in marked hypo-activity in this assay. Comparatively, VDR antagonists result in hyperactivity. These studies lay the foundation for further investigation into whether behavioral dysfunction persists into adulthood and if so, which behavioral functions are affected. Zebrafish can be useful for screening compounds identified in high-throughput in vitro assays to provide an initial test for how those compounds would affect construction and behavioral function of a complex nervous system, helping to bridge the gap between in vitro neurotoxicity assays and mammalian models for risk assessment in humans.
(6) Lastly, to aid in facilitating proof of principle, we have developed models of vitamin D deficiency (D-) and vitamin D sufficiency (D+) in zebrafish that can be used as a positive control supporting our initial hypothesis regarding select role(s) of vitamin D in neurodevelopment. In this line of investigation, we have established a F0 population(s) of fish that are either D+ and D-. In behavioral assays, vitamin D deficient larvae exhibit hyperactivity (similar to VDR antagonists) and are active for a longer duration, swim faster and travel a greater distance than both vitamin D sufficient and lab diet larvae who behave similarly. Conversely, at 4 months of age, larvae that were vitamin D deficient as embryos displayed a significantly (p<0.0005) decreased swimming activity (cm/min) and maintain significantly (p<0.0005) lower tank position compared to vitamin D sufficient and lab diet fish in the novel tank diving test. Results from the tap-startle test demonstrate a significantly (p<0.05) slower but greater habituation to startle in the vitamin D deficient fish. Taken together, vitamin D does appear to influence early neurodevelopment and suggest that zebrafish exhibit both short- and long-term behavioral impairments following early developmental vitamin D deficiency.
Conclusions:
Nuclear receptors comprise one of the largest groups of transcription factors among vertebrates and mediate a wide range of physiological functions that are essential for growth, development, differentiation, metabolism, homeostasis and reproduction among other functions. Transcriptional activities of these receptors are predominantly driven through ligand dependent interactions with endogenous ligands. However, a growing body of evidence suggests that in addition to endogenous ligands, a diverse group of exogenous chemicals including environmental contaminants, pharmaceuticals and synthetic molecules can function as receptor agonists or antagonists. In this study, we demonstrate the ability of vitamin D receptor to bind a variety of structurally diverse xenobiotics. We illustrate how structural variations in the exogenous ligands can affect their binding to the ligand-binding domain of VDR and facilitate allosteric modifications of VDR that result in the receptor's ability to interact with the obligate heterodimerzation partner RXR and impact subsequent recruitment of coactivators and/or corepressors, thus affecting the overall transactivational functions of VDR. Given the diverse physiological roles of vitamin D/VDR signaling and the emerging importance of VDR in neurodevelopment and neurodegeneration, identification of exogenous agents that may influence VDR signaling may have profound physiological importance and represent a novel mechanism of “endocrine disruption.” Additionally, we demonstrate that translational in vivo models such as the zebrafish can be utilized to establish mechanistic linkages between in vitro assays describing receptor function and adverse physiological, and/or behavioral outcomes that may facilitate construction of adverse outcome pathways.
Journal Articles on this Report : 9 Displayed | Download in RIS Format
Other project views: | All 24 publications | 9 publications in selected types | All 9 journal articles |
---|
Type | Citation | ||
---|---|---|---|
|
Bailey JM, Oliveri AN, Levin ED. Pharmacological analyses of learning and memory in zebrafish (Danio rerio). Pharmacology, Biochemistry, and Behavior 2015;139(Pt B):103-111. |
R835541 (Final) |
Exit |
|
Benjamin Neelon SE, White AJ, Vidal AC, Schildkraut JM, Murtha AP, Murphy SK, Kullman SW, Hoyo C. Maternal vitamin D, DNA methylation at imprint regulatory regions and offspring weight at birth, 1 year and 3 years. International Journal of Obesity 2018;42(4):587-593. |
R835541 (2017) R835541 (Final) |
Exit Exit |
|
Ideraabdullah FY, Belenchia AM, Rosenfeld CS, Kullman SW, Knuth M, Mahapatra D, Berman M, Levinlo ED, Peterson CA. Maternal vitamin D deficiency and developmental origins of health and disease (DOHaD). Journal of Endocrinology 2019;241(2):R65-R80. DOI:10.1530/JOE-18-0541. |
R835541 (Final) |
not available |
|
LI C, Hits Z, Warrick J, Li J, Geller S, Trantow V, McClean M, Beebe D. Under oil open-channel microfluidics empowered by exclusive liquid repellency. SCIENCE ADVANCES 2020;6(16) |
R835541 (Final) |
not available |
|
Mahapatra D, Franzosa J, Roell K, Kunenemann M, Houck K, Reif D, Fourches D, Kullman S. Confirmation of high-throughput screening data and novel mechanistic insights into VDR-xenobiotic interactions by orthogonal assays. SCIENTIFIC REPORTS 2018;8(8883). |
R835541 (Final) |
Exit Exit |
|
Oliveri A, Levin E. Dopamine D-1 and D-2 receptor antagonism during development alters later behavior in zebrafish. BEHAVIOURAL BRAIN RESEARCH 2019;356:250-256. |
R835541 (Final) |
Exit Exit |
|
Oliveri A, Glazer L, Mahapatra D, Kullman S, Levin E. Developmental exposure of zebrafish to vitamin D receptor acting drugs and environmental toxicants disrupts behavioral function. Neurotoxicology and Teratology 2020;81(106902) |
R835541 (Final) |
not available |
|
Oliveri A, Knuth M, Glazer L, Bailey J, Kullman S, Levin E. Zebrafish show long-term behavioral impairments resulting from developmental vitamin D deficiency. Physiology & Behavior 2020;224(113016) |
R835541 (Final) |
not available |
|
Stutts W, Knuth M, Ekelof M, Mahapatra D, Kullman S, Muddiman D. Methods for Cryosectioning and Mass Spectrometry Imaging of Whole-Body Zebrafish. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2020;31(4):768-772 |
R835541 (Final) |
not available |
Supplemental Keywords:
Vitamin D, developmental neurotoxicity, Tox21, zebrafishProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
- 2017 Progress Report
- 2016 Progress Report
- 2015 Progress Report
- 2014 Progress Report
- Original Abstract
9 journal articles for this project