Browsing by Author "Kullman, Seth W"
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Item Open Access Characterization of Fxr Alpha in Medaka and Its Involvement in Hepatobiliary Injury(2009) Howarth, Deanna LynneThe liver is a primary target for toxicants and/or their metabolites. Selected fish species now serve as model organisms for laboratory investigations of toxic responses in the liver. One such model is the Japanese medaka (Oryzias latipes), a small freshwater teleost with a robust history of usage in liver and biliary toxicity studies. The structural components of the medaka hepatobiliary system have been well-described by recent studies in two- and three-dimensional contexts, but efforts to characterize the molecular mechanisms underlying critical medaka liver functions during normalcy remain sparse. This dearth of information makes it difficult to definitively characterize toxic responses in this model organism. A crucial transcription factor underlying proper hepatobiliary function in both mammalian and non-mammalian species is the farnesoid X receptor alpha (FXRα), a member of the nuclear receptor superfamily that plays a key role in bile acid homeostasis. This dissertation describes the function of medaka fxrα during both normalcy and toxicity.
To achieve this overall objective, in vitro techniques were first employed to study the function of medaka fxrα. Two isoforms of fxrα that differ in the AF1 domain, Fxrα1 and Fxrα2, were isolated from liver cDNA and are the result of alternative splicing of one gene locus. Fxrα2 responded significantly to C24 bile acids and the synthetic FXRα agonist GW4064. On the other hand, Fxrα1, despite having an identical ligand-binding domain to that of Fxrα2, showed no response to any agonists tested by transient transactivation assays. Furthermore, Fxrα2 interacted with nuclear receptor coactivators PGC-1α and SRC-1 in mammalian two-hybrid assays while Fxrα1 did not. These findings point to a significant importance of the AF1 domain to overall receptor structure and function.
Following in vitro functional characterization, in vivo experiments using medaka larvae were performed to determine fxrα's function during normalcy. Quantitative, real-time PCR data demonstrated that Fxrα1 is highly expressed in adult liver, while Fxrα2 is expressed predominantly in gut. Fxrα1's expression was higher than Fxrα2 in embryos and larvae at all developmental timepoints tested. In vivo exposures of medaka hatchlings to GW4064 at various doses significantly altered expression of defined FXRα targets, including: bile salt export protein (BSEP), small heterodimer partner (SHP), and cytochrome P450 7A1 (CYP7A1). Surprisingly, numerous sublethal hepatic alterations to hepatocytes and bile preductular epithelial cells (BPDECs) were observed following exposure to GW4064; alterations included: lipid accumulation, glycogen depletion, mitochondrial swelling and rupture of mitochondrial membranes, disruption of endoplasmic reticulum, and apoptosis. Significant lipid accumulation, as revealed by oil red O whole mount staining of larvae, was also noted at lower doses of GW4064. These findings were the first observations of sublethal hepatotoxicity of GW4064; to date, no studies in the mammalian literature reported alterations following its administration.
Because of studies in the mammalian literature that demonstrated alleviation of cholestatic injury induced via the classic hepatotoxicant α-naphthylisothiocyanate (ANIT) by GW4064, it was originally hypothesized that a similar finding would be observed in medaka coexposed to these compounds. However, because of GW4064's ability to induce sublethal hepatic alterations in medaka, it was anticipated that its coadministration with ANIT would result in enhanced toxicity rather than alleviation as described in rodent models. However, despite the sublethal alterations induced by 1 uM GW4064, alleviation of toxicity following exposure to 15 uM ANIT was observed. Surprisingly, reduction of GW4064's toxicity was also observed in larvae exposed to both compounds. These investigations of fxrα function are an important and essential component in furthering our understanding of hepatobiliary toxicity in small aquarium fish models of human liver disease. These collective findings have created molecular underpinnings necessary for understanding medaka hepatobiliary function during normalcy and toxicity.
Item Open Access Developmental exposure of zebrafish to vitamin D receptor acting drugs and environmental toxicants disrupts behavioral function.(Neurotoxicology and teratology, 2020-09) Oliveri, Anthony N; Glazer, Lilah; Mahapatra, Debabrata; Kullman, Seth W; Levin, Edward DVitamin D receptor (VDR) signaling is important for optimal neurobehavioral development. Disruption of VDR signaling by environmental toxicants during early development might contribute to the etiology of behavioral dysfunction. In the current set of studies, we examined ten compounds known to affect VDR function in vitro for neurobehavioral effects in vivo in zebrafish. Zebrafish embryos were exposed to concentrations of the compounds in their water during the first 5 days post-fertilization. On day 5, the embryos were tested in an alternating light-dark locomotor assay using a computerized video tracking system. We found that most 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. These studies lay a foundation for further investigation to determine 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.Item Open Access Developmental exposure to pesticides that disrupt retinoic acid signaling causes persistent retinoid and behavioral dysfunction in zebrafish.(Toxicological sciences : an official journal of the Society of Toxicology, 2024-03) Hawkey, Andrew B; Shekey, Nathan; Dean, Cassandra; Asrat, Helina; Koburov, Reese; Holloway, Zade R; Kullman, Seth W; Levin, Edward DEarly developmental exposure to environmental toxicants may play a role in the risk for developing autism. A variety of pesticides have direct effects on retinoic acid (RA) signaling and as RA signaling has important roles in neurodevelopment, such compounds may cause developmental neurotoxicity through an overlapping adverse outcome pathway. It is hypothesized that a pesticide's embryonic effects on retinoid function may correspond with neurobehavioral disruption later in development. In the current studies, we determined the effects of RA-acting pesticides on neurobehavioral development in zebrafish. Buprofezin and imazalil caused generalized hypoactivity in the larval motility test, whereas chlorothalonil and endosulfan I led to selective hypoactivity and hyperactivity, respectively. With buprofezin, chlorothalonil, and imazalil, hypoactivity and/or novel anxiety-like behaviors persisted in adulthood and buprofezin additionally decreased social attraction responses in adulthood. Endosulfan I did not produce significant adult behavioral effects. Using qPCR analyses of adult brain tissue, we observed treatment-induced alterations in RA synthesis or catabolic genes, indicating persistent changes in RA homeostasis. These changes were compound-specific, with respect to expression directionality, and potential patterns of homeostatic disruption. Results suggest the likely persistence of disruptions in RA signaling well into adulthood and may represent compensatory mechanisms following early life stage exposures. This study demonstrates that early developmental exposure to environmental toxicants that interfere with RA signaling causes short as well as long-term behavioral disruption in a well-established zebrafish behavioral model and expand upon the meaning of the RA adverse outcome pathway, indicating that observed effects likely correspond with the nature of underlying homeostatic effects.Item Open Access Maternal vitamin D deficiency and developmental origins of health and disease (DOHaD).(The Journal of endocrinology, 2019-03) Ideraabdullah, Folami Y; Belenchia, Anthony M; Rosenfeld, Cheryl Susan; Kullman, Seth W; Knuth, Megan; Mahapatra, Debrata; Bereman, Michael; Levin, Edward D; Peterson, Catherine AnnVitamin D is an essential nutrient that is metabolized in the body to generate an active metabolite (1,25(OH)2D) with hormone-like activity and highly diverse roles in cellular function. Vitamin D deficiency (VDD) is a prevalent but easily preventable nutritional disturbance. Emerging evidence demonstrates the importance of sufficient vitamin D concentrations during fetal life with deficiencies leading to long-term effects into adulthood. Here, we provide a detailed review and perspective of evidence for the role of maternal VDD in offspring long term health, particularly as it relates to Developmental Origins of Health and Disease (DOHaD). We focus on roles in neurobehavioral and cardiometabolic disorders in humans and highlight recent findings from zebrafish and rodent models that probe potential mechanisms linking early life VDD to later life health outcomes. Moreover, we explore evidence implicating epigenetic mechanisms as a mediator of this link. Gaps in our current understanding of how maternal VDD might result in deleterious offspring outcomes later in life are also addressed.Item Open Access Zebrafish show long-term behavioral impairments resulting from developmental vitamin D deficiency.(Physiology & behavior, 2020-10) Oliveri, Anthony N; Knuth, Megan; Glazer, Lilah; Bailey, Jordan; Kullman, Seth W; Levin, Edward DVitamin D has been shown in a wide variety of species to play critical roles in neurodevelopment. Vitamin D deficiency disrupts development of the brain and can cause lasting behavioral dysfunction. Zebrafish have become an important model for the study of development in general and neurodevelopment in particular. Zebrafish were used in the current study to characterize the effects of developmental vitamin D deficiency on behavioral function. Adult zebrafish that had been chronically fed a vitamin D deficient or replete diets were bred and the offspring were continued on those diets. The offspring were behaviorally tested as adults. In the novel tank diving test the vitamin D deficient diet significantly lowered the vertical position of fish indicative of more anxiety-like behavior. In the novel tank diving test swimming activity was also significantly decreased by vitamin D deficiency. Startle response was increased by developmental vitamin D deficiency during the early part of the test. No significant effects of vitamin D deficiency were seen with social affiliation and predatory stimulus avoidance tests. These results indicate a phenotype of vitamin D deficiency characterized by more anxiety-like behavior. This result was relatively specific inasmuch as few or no behavioral effects were seen in other behavioral tests.