Primary Impacts of the Fungal Toxin Sporidesmin on HepG2 Cells: Altered Cell Adhesion without Oxidative Stress or Cell Death

Abstract

The fungal metabolite sporidesmin is responsible for severe necrotizing inflammation of biliary tract and liver of livestock grazing on pasture containing spores of Pithomyces chartarum that synthesizes the toxin. The toxin is secreted into bile causing the erosion of the biliary epithelium accompanied by inflammation and damage to surrounding tissues. Toxicity has been suggested to be due to cycles of reduction and oxidation of sporidesmin leading to oxidative damage from the formation of reactive oxygen species. The current work is the first test of the oxidative stress hypothesis using cultured cells. Oxidative stress could not be detected in HepG2 cells incubated with sporidesmin using a dichlorodihydrofluorescein diacetate assay or by use of two-dimensional electrophoresis to search for oxidized peroxiredoxins. There was also no evidence for necrosis or apoptosis, although there was a loss of cell adhesion that was accompanied by the disruption of intracellular actin microfilaments that have known roles in cell adhesion. The results are consistent with a model in which altered contact between cells in situ leads to altered permeability and subsequent inflammation and necrosis, potentially from the leakage of toxic bile into surrounding tissues. There is now a need for the further characterization of the damage processes in vivo, including the investigation of altered permeability and mechanisms of cell death in the biliary tract and other affected organs.

Keywords: HepG2 cells; cell adhesion; hepatobiliary injury; oxidative stress; sporidesmin; two-dimensional electrophoresis.

Figure 1

Structure of sporidesmin (sporidesmin A). The reactivity of the sulfur bridge is associated with the molecular environment of the dioxopiperazine nucleus (circled).

Figure 2

Differential interference contrast (DIC) images of HepG2 cells incubated with 1 µg/mL sporidesmin for 3 h. Cells at time 0 (a), 1 (b), 2 (c), and 3 (d) h. Time lapse movies are in the Supplementary Data. There was the rounding up and shrinkage of individual cells prior to the detachment of individual cells and sheets of cells.

Figure 3

(A) Confocal microscopy of actin (a,b) or cytokeratin (c,d) filaments in HepG2 cells. Control cells (a,c) incubated without sporidesmin contained cytoplasmic filaments of actin (arrow) and a network of cytokeratin filaments. Cells incubated with 1 µg/mL sporidesmin for 2 h showed (b) a loss of cytoplasmic actin filaments without the collapse of the cytokeratin network (d). (B) Cells stained with Annexin-V-FLUOS upper panels to detect apoptosis or necrosis. Lower panels are the corresponding DIC images. Positive staining for apoptosis or necrosis was not detected in the control cells (e) or in adherent (f) or detached (g) cells after exposure to 1 µg/mL sporidesmin for 3 h. There was positive control staining of apoptotic cells (h) in cultures that had been incubated with yessotoxin.

Figure 4

Dichlorodihydrofluorescein diacetate (DCFDA) assay for the detection of reactive oxygen species (ROS) in cells incubated with 1 µg/mL sporidesmin or 25 or 100 µM hydrogen peroxide. Enhanced fluorescence from the oxidation of dichlorofluorescein was detected in cultures containing hydrogen peroxide but did not increase above basal levels in cultures containing sporidesmin. Results are the means and SDs of triplicates. Results for controls without ethanol (not shown) overlaid the ethanol controls.

Figure 5

The two-dimensional electrophoresis of proteins from HepG2 cells that had been incubated with 1 µg/mL sporidesmin or 500 µM hydrogen peroxide for 3 h. The differential in-gel electrophoresis (DIGE) labelling design facilitates the detection of changes in protein abundance compared to control incubates with or without 0.1% ethanol. Extracts of cells incubated with hydrogen peroxide showed multiple changes including a decrease in the abundance of native peroxiredoxins with a gain of acidic isoforms that are expected to be the products of cysteine oxidation. Lower panels show the approximately 20 kDa mass regions containing peroxiredoxins. Circled peroxiredoxin 6 isoforms correspond to the oxidized (left) and native (right) proteins that, respectively, increased or decreased after culture with hydrogen peroxide. There were no detected changes in the abundance of any protein spots from cells that had been incubated with sporidesmin.

Figure 6

Proposed model for changes in cell contact and adhesion. Sporidesmin in the biliary tract or circulation alters contact between cells leading to enhanced permeability and subsequent tissue damage.