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 m echanisms induced by gliotoxin in LLC-PK1 cells, a porcine renal proximal t ubular cell line. Gliotoxin at 100 ng/ml did not show a cytotoxic effect, b ut unmasked a dose-dependent cell death induced by TNF-alpha. TNF-alpha-ind uced cell death in the presence of gliotoxin was associated with hypodiploi d nuclei and activation of caspase-3-like proteases. Blockade of caspases b y boc-aspartyl (OMe)-fluoromethylketone and z-DEVD.fmk inhibited TNF-alpha- induced cell death. As the concentrations of gliotoxin were increased, glio toxin killed the cells directly in a dose-dependent manner. Further analyse s of DNA fragmentation, hypodiploid nuclei, mitochondrial membrane potentia l, and plasma membrane integrity revealed that cell death proceeded via apo ptosis, Gliotoxin-induced apoptosis was associated with dose-dependent and time-dependent activation of caspase-3-like proteases. Boc-aspartyl (OMe)-f luoromethylketone attenuated the killing effect. Gliotoxin also increased t he intracellular levels of reactive oxygen species as measured by how cytom etry. N-acetylcysteine, a well-known antioxidant, completely abolished the gliotoxin-induced caspase-3-like activity, cytotoxicity, and reactive oxyge n species. In conclusion, (1) gliotoxin at 100 ng/ml unmasks the ability of TNF-alpha-induced apoptosis, and the effect of TNF-alpha is mediated by ca spase-3-like proteases; and (2) at higher concentrations gliotoxin itself i nduces cell death, which is via apoptosis and dependent on caspase-3-like a ctivity and reactive oxygen species.