Exposure to the synthetic FXR agonist GW4064 causes alterations in gene expression and sublethal hepatotoxicity in eleutheroembryo medaka (Oryzias latipes)

Abstract

The small freshwater teleost, medaka (Oryzias latipes), has a history of usage in studies of chronic toxicity of liver and biliary system. Recent progress with this model has focused on defining the medaka hepatobiliary system. Here we investigate critical liver function and toxicity by examining the in vivo role and function of the farnesoid X receptor alpha (FXRalpha, NR1H4), a member of the nuclear receptor superfamily that plays an essential role in the regulation of bile acid homeostasis. Quantitative mRNA analysis of medaka FXRalpha demonstrates differential expression of two FXRalpha isoforms designated Fxralpha1 and Fxralpha2, in both free swimming medaka embryos with remaining yolk (eleutheroembryos, EEs) and adults. Activation of medaka Fxralpha in vivo with GW4064 (a strong FXRalpha agonist) resulted in modification of gene expression for defined FXRalpha gene targets including the bile salt export protein, small heterodimer partner, and cytochrome P450 7A1. Histological examination of medaka liver subsequent to GW4064 exposure demonstrated significant lipid accumulation, cellular and organelle alterations in both hepatocytes and biliary epithelial cells of the liver. This report of hepatobiliary injury following GW4064 exposure extends previous investigations of the intrahepatic biliary system in medaka, reveals sensitivity to toxicant exposure, and illustrates the need for added resolution in detection and interpretation of toxic responses in this vertebrate.

Fig. 1

(A) Quantitative, real-time PCR data showing expression of two medaka Fxrα isoforms in tissues of 6 month old OR males (black bars) and females (gray bars). Relative mRNA levels were measured in brain (B), gill (Gi), gut (Gu), heart (H), kidney (K), liver (L), skeletal muscle (M), spleen (S), and gonad (testis: T; ovary: O). Data was normalized to 18S rRNA levels and is represented as the mean relative mRNA level ± SEM (n = 5). (B) Relative mRNA level of Fxrα1 (black bars) and Fxrα2 (gray bars) within medaka embryos and EEs as measured by qPCR. Data was normalized to 18S rRNA levels and is represented as the mean relative mRNA level ± SEM (n = 3 pooled samples).

Fig. 2

Quantitative, real-time PCR data showing alteration in Bsep (A), Shp (B), and Cyp7a1 (C) expression in medaka EEs 10–12 dpf following exposure to GW4064 at 0.01, 0.1, 1, 5, or 10 μM. Data was normalized to β-actin and is represented as the mean mRNA level ± SEM (n = 6). A significant change in Bsep, Shp and Cyp7a1 was noted compared to DMSO controls (*p<0.05; **p<0.01; ***p<0.0001). Expression of Mrp2 (D) and Ntcp (E) were not significantly altered as a result of GW4064 exposure.

Fig. 3

Quantitative, real-time PCR data showing the alteration in Bsep (A) and Shp (B) expression (in medaka EEs 10–12 dpf) as a result of exposure to 5 μM GW4064 over time. Data was normalized to β-actin levels and is represented as the mean mRNA level ± SEM (N = 3). A significant difference in Bsep and Shp expression is noted after 24, 48, and 72 h of exposure to GW4064 (gray bars) but not at 6 h compared to time-matched controls (black bars). Significant differences between expression among dosed individuals at different time points is noted by brackets (*p<0.05; **p<0.01; ***p<0.0001).

Fig. 4

Oil red O whole mount staining of EEs exposed to control (DMSO) and GW4064 for 48 h. Bar = 100 μm. Inset image in the left panel shows positive staining oil droplet (od) in control, Retained in larval development, this structure serves as an internal control and was located in the rostral peritoneal cavity between liver (L) and septum transversum. Y = yolk sac. Larger panels show liver fields from individuals exposed to DMSO, 1, 5, and 10 μM GW4064, demonstrating a dose-dependent increase in lipid accumulation (red stained vacuoles) as a result of GW4064 exposure. Morphometric analyses confirmed the apparent increase in lipid vacuoles (see Results section).

Fig. 5

Representative images from paraffin-embedded liver sections (5 μm) of control and treated (10 μM GW4064) medaka EEs stained with H&E. Scale bar = 10 μm. (A) Control liver at 48 h showing tubules of hepatocytes with darkly staining nuclei and unstained regions of the cytoplasm. Gu: gut; Bw: body wall. (B) Liver from fish treated with 10 μM GW4064 for 48 h. Disruption of the hepatic tubules and pronounced vacuolation (arrowheads) of parenchymal cells are seen. Bv, blood vessel.

Fig. 6

Representative higher-resolution images (3 μm) from control (DMSO, vehicle) and treated (10 μM GW4064) medaka EEs embedded in GMA and stained with H&E. Scale bar = 10 μm. (A) Control (DMSO) liver at 48 h shows normal morphology for hepatocytes and bile preductular epithelial cells (BPDECs) (black arrowheads) which intervene between longitudinal arrays of hepatocytes in a single hepatic tubule. Canaliculi are not resolved with this technique. Representative elements of a transitional biliary passageway (i.e.: hepatic tubule lumen, or bile preductule) are shown in the rectangle at right of field. In upper right hand corner is an endothelial cell and its associated sinusoid. White arrowhead at left hand margin of rectangle denotes nucleated erythrocyte in sinusoid. Between the two sinusoids (Bv) are hepatocytes in a double row configuration. A blue-gray transitional biliary passageway (bile preductule) is denoted by Bp. Note biliary epithelial cell at bottom right corner (black arrowhead) illustrating that these passageways are lined by both hepatocytes and transitional biliary epithelial cells (bile preductular epithelial cells). Hepatocyte cytoplasm contains meandering basophilic structures corresponding to rough endoplasmic reticulum of hepatocytes—see Fig. 8. Basophilic areas contrast with pink stained areas of hepatocyte cytoplasm, denoting glycogen depots. (B) Liver from EE exposed to 10 μM GW4064 for 48 h. Lower right hand corner of micrograph shows interface of liver surface with intestinal wall (dashed line). The remainder of the field is hepatic parenchyma with occasional microcirculatory elements (sinusoids). Hepatocytes (round nuclei with prominent nucleoli) and surrounded by lightly stained cytoplasm containing variously sized clear vacuoles (white arrowheads). Biliary epithelial cells (BPDECs) with elongated nuclei and dark and beaded cytoplasm (black arrowheads).

Fig. 7

Representative images from semithin (500 nm) liver sections of control or 10 μM GW4064 treated EEs, stained with toluidine blue. Scale bar = 10 μm. (A) Control section. Left upper corner is rostral surface of liver and adjacent sinus venosus. Exocrine pancreas is at right lower corner of field. Remainder is hepatic parenchyma and microvasculature. Hepatocytes are free of vacuolation, stain gray-blue, and contain single nucleus with prominent nucleolus. Three biliary epithelial cells (BPDECs) are shown (arrow points to center cell). By comparison to hepatocytes, BPDECs are lightly stained. (B) Liver section from individual exposed to 10 μM GW4064 for 48 h. S = sinusoid. Black arrowheads denote BPDECs surrounding lumen or biliary passageway and containing numerous vacuolar alterations. Hepatocytes contain single prominent nucleus with altered (elongated) nucleolus. Note numerous aquamarine lipid vacuoles in hepatocytes. Two of these are denoted (white arrowheads). Black lines on these vacuoles are due to section artifact (chatter). M = enlarged mitochondria. (C) Liver section from medaka larva exposed to 10 μM GW4064 for 72 h. Large lipid inclusions (Li) are visible throughout hepatic parenchyma. Small clear vacuoles (white arrowheads) are present, some containing membranous debris. Nucleolar fragmentation (Nu) and BPDEC alteration (black arrowheads) are also observed. Bv: blood vessel.

Fig. 8

Transmission electron micrographs of EE liver. (A) Control. This micrograph (11.2k magnification) shows fine structure of hepatocytes and two BPDECs. Together, these form a bile passageway (transitional passageway, or bile preductule). Adjacent hepatocytes are joined by junctional complexes (white arrowheads). Note numerous plasma membranes processes from hepatocytes into the biliary lumen. In addition, junctions are seen between BPDECs and hepatocytes (white arrowheads). Control hepatocyte morphology includes parallel arrays of rough endoplasmic reticulum, extensive glycogen depots (G), numerous mitochondria (black arrowheads) and occasional residual bodies (Rb). Note the large hepatocyte nuclei with rounded electron dense nucleoli. Bar = 1 μm. (B) Electron micrograph (5600× magnification) of EE liver from individual exposed to 10 μM GW4064 for 48 h. Hepatocytes show altered contour and presence of clear and lipid-containing vacuoles, (white arrowheads and Li, respectively). Endoplasmic reticulum shows marked cisternal swelling. Nuclear change is apparent in altered morphology of hepatocellular nucleoli. Hepatocellular mitochondria (black arrowheads) are enlarged and abnormally shaped. Triangular shaped nucleus at the center of the field is a BPDEC. BPDECs were targets for GW4064 as shown by the numerous lipid vesicles and phagocytosed apoptotic vesicles (Av). Bar = 3 μm.