Gliotoxin-Induced Cytotoxicity Proceeds via Apoptosis and Is Mediated by Caspases and Reactive Oxygen Species in LLC-PK1 Cells

Renal failure associated with aspergillosis is caused by pathogenic fungi. Gliotoxin is a toxic epipolythiodioxopiperazine metabolite produced by the pathogens. The present study investigated the cytotoxicity and underlying mechanisms induced by gliotoxin in LLC-PK1 cells, a porcine renal proximal tubular cell line. Gliotoxin at 100 ng/ml did not show a cytotoxic effect, but unmasked a dose-dependent cell death induced by TNF-a. TNFa‐induced cell death in the presence of gliotoxin was associated with hypodiploid nuclei and activation of caspase-3‐like proteases. Blockade of caspases by boc-aspartyl (OMe)-fluoromethylketone and z-DEVD.fmk inhibited TNF-a‐induced cell death. As the concentrations of gliotoxin were increased, gliotoxin killed the cells directly in a dose-dependent manner. Further analyses of DNA fragmentation, hypodiploid nuclei, mitochondrial membrane potential, and plasma membrane integrity revealed that cell death proceeded via apoptosis. Gliotoxin-induced apoptosis was associated with dose-dependent and time-dependent activation of caspase-3‐like proteases. Boc-aspartyl (OMe)-fluoromethylketone attenuated the killing effect. Gliotoxin also increased the intracellular levels of reactive oxygen species as measured by flow cytometry. N-acetylcysteine, a well-known antioxidant, completely abolished the gliotoxin-induced caspase-3‐like activity, cytotoxicity, and reactive oxygen species. In conclusion, (1) gliotoxin at 100 ng/ml unmasks the ability of TNF-a‐induced apoptosis, and the effect of TNF-a is mediated by caspase-3‐like proteases; and (2) at higher concentrations gliotoxin itself induces cell death, which is via apoptosis and dependent on caspase-3‐like activity and reactive oxygen species.