Browsing by Subject "medaka"

The 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.

Endocrine disrupting chemicals (EDCs) are ubiquitous and often act as xenoestrogens with the ability to disrupt estrogen signaling through differential binding to the various estrogen receptors. Exposure to these xenoestrogens has led to detrimental effects on male reproduction. In fish, observed effects include sex reversal, presence of testicular oocytes, altered courting behavior, vitellogenin synthesis in males, altered fertility and gonadal histopathology. Understanding how xenoestrogens exert their effects is complicated by the existence of multiple estrogen receptors (ESR1, ESR2a, ESR2b, and GPER), coupled with their ability for crosstalk and differential binding capability of selective estrogen receptor modulators (SERMS). Additionally, estrogen can signal through both classic genomic signaling and nongenomic signaling. Furthermore, the importance of estrogen signaling in normal male reproduction is just beginning to be understood. The primary goal of this dissertation was to assess the implications of aberrant estrogen signaling on male reproductive capacity, testicular morphology and gene expression changes in the small aquarium model fish, medaka, by investigating effects of a general estrogen receptor agonist, ethinylestradiol (EE2), and those of a G-protein estrogen receptor (GPER) specific agonist, G-1. This was assessed through breeding experiments, histological assessment of testicular morphology and microarray assessment of testicular gene expression changes following exposure to EE2 and G-1. Finally, a comparison of altered testicular morphology between EE2 and G-1 induced changes was further assessed using a variety of histological techniques. The findings demonstrate that a 14-day exposure to EE2 impaired male reproductive capacity and altered testicular morphology and gene expression in a time- and dose-dependent manner. The testicular morphologic alterations observed include increased germ cell apoptosis, decreased germinal epithelium and thickening of the interstitium. These morphologic changes were highly associated with gene expression changes. A pathway analysis of the differentially expressed genes emphasized genes and pathways associated with apoptosis, cell proliferation, collagen production/extracellular matrix organization, and protein ubiquitination among others. Comparatively, a 14-day exposure to G-1 did not affect male reproductive capacity but did alter testicular morphology and gene expression. The histological analysis found an increased cellularity of the interstitium leading to thickened interstitium but no change in germinal epithelium. The microarray data indicate differential expression in genes most commonly involved in cell cycle, cell proliferation, apoptosis, transcription, translation, and ubiquitination. Finally, an assessment of the testicular histological phenotypes following EE2 and G-1 exposure indicate different morphologic changes led to thickened interstitium observed in the two exposures. In EE2 exposed fish, thickening of interstitium was associated with increased collagen deposition on the periphery of the organ while the interior thickening was primarily due to the collapse of intralobular space associated with decreased germinal epithelium. In the G-1 exposed fish, the thickened interstitium was due to increased cellularity. A modest increase in cell proliferation was observed contributing to the increase in interstitial cells, however, it is also possible that there is a decrease in normal apoptosis and cell turnover as well. These findings highlight the importance of anchoring gene expression changes with morphology and ultimately proper tissue/organ function as well as the potential differences in effects that may occur with EDCs and SERMs.