Mechanism of action of the antifibrogenic compound gliotoxin in rat liver cells

Abstract

Gliotoxin has been shown to promote a reversal of liver fibrosis in an animal model of the disease although its mechanism of action in the liver is poorly defined. The effects of gliotoxin on activated hepatic stellate cells (HSCs) and hepatocytes have therefore been examined. Addition of gliotoxin (1.5 M) to culture-activated HSCs resulted in its rapid accumulation, resulting in increased levels of glutathione and apoptosis without any evidence of oxidative stress. In contrast, although hepatocytes also rapidly sequestered gliotoxin, cell death only occurred at high (50-M) concentrations of gliotoxin and by necrosis. At high concentrations, gliotoxin was metabolized by hepatocytes to a reduced (dithiol) metabolite and glutathione was rapidly oxidized. Fluorescent dye loading experiments showed that gliotoxin caused oxidative stress in hepatocytes. Antioxidants-but not thiol redox active compounds-inhibited both oxidative stress and necrosis in hepatocytes. In contrast, HSC apoptosis was not affected by antioxidants but was potently abrogated by thiol redox active compounds. The adenine nucleotide transporter (ANT) is implicated in mitochondrial-dependent apoptosis. HSCs expressed predominantly nonliver ANT isoform 1, and gliotoxin treatment resulted in a thiol redox-dependent alteration in ANT mobility in HSC extracts, but not hepatocyte extracts. In conclusion, these data suggest that gliotoxin stimulates the apoptosis of HSCs through a specific thiol redox-dependent interaction with the ANT. Further understanding of this mechanism of cell death will aid in finding therapeutics that specifically stimulate HSC apoptosis in the liver, a promising approach to antifibrotic therapy. Supplementary material for this article can be found on the HEPATOLOGY website

Gliotoxin causes apoptosis and necrosis of rat Kupffer cells in vitro and in vivo in the absence of oxidative stress: Exacerbation by caspase and serine protease inhibition

“…These observations contrast a recent study reporting comparable gliotoxin-induced apoptosis of Kupffer cells and HSCs in normal and cirrhotic livers (54). The reason for this discrepancy may be that liver slices were incubated for 8h with gliotoxin in vitro (54), while we and others (1,24) investigated the effect of gliotoxin in vivo. We also found significant gliotoxin-induced apoptosis of hepatocytes in the fibrotic liver but not the normal liver.…”

“…Although 1.5-7.5 μM gliotoxin was found to cause apoptosis of culture-activated and immortalized HSCs respectively in the absence or at low level of oxidative stress (2,24), higher concentrations induce oxidative stress and necrotic death of the immortalized HSCs (2). Necrosis of hepatocytes induced by high concentrations of gliotoxin (>10 μM) was prevented by antioxidants catalase and superoxide dismutase (24). However, gliotoxin did not stimulate SO and H 2 O 2 generation in Kupffer cells, and several antioxidants did not prevent gliotoxininduced death of these cells.…”

“…We also found significant gliotoxin-induced apoptosis of hepatocytes in the fibrotic liver but not the normal liver. Since hepatocytes undergo necrosis, but not apoptosis, upon treatment with high (>10 μM) concentration of gliotoxin in vitro (1,24), these results suggest that hepatocytes are either modified during fibrosis to become sensitive to pro-apoptotic effect of gliotoxin or that gliotoxin may release proapoptotic stimuli from nonparenchymal cells that then cause apoptosis of hepatocytes.…”

The epipolythiodioxopiperazine (ETP) class of fungal toxins: distribution, mode of action, functions and biosynthesis

“…It also causes a redox-dependent change in electrophoretic mobility of a component of the mitochondrial permeability transition pore, the adenine nucleotide transporter (ANT). This transition pore is an essential mediator of apoptosis modulated by mitochondria (Orr et al, 2004). The ANT is sensitive to oxidizing agents and has two cysteine residues which may be targets of gliotoxin (McStay et al, 2002).…”

“…Gliotoxin also can selectively kill activated hepatic stellate cells in rats, which is a model system for liver fibrosis (Dekel et al, 2003;Wright et al, 2001). This morbidity is caused by selective induction of apoptotic cell death in stellate cells through redox-dependent effects on the adenine nucleotide transporter (Orr et al, 2004). However, the usefulness of ETPs such as gliotoxin as antimicrobial agents, and as clinical immunosuppressive agents, will be limited by their toxicity.…”