Effect of heat-treatment and the role of phospholipases on Fungizone®-induced cytotoxicity within human kidney proximal tubular (HK-2) cells and Aspergillus fumigatus

The objectives of this study were to determine the effects of heat-treatment on Fungizone® (FZ)-induced cytotoxicity in human kidney (HK-2) cells and fungal isolates of Aspergillus fumigatus, and to determine the possible role of phospholipases (PLA2 and PLC) on heat-treated FZ (HFZ)-associated renal cell toxicity. HK-2 cells were grown at 37 °C in T75 flasks and seeded in 96-well plates at 20,000 cells/well. FZ and HFZ concentrations of 10, 25 and 50 μg/mL of AmpB were prepared. Snake venom PLA2 and PLC (2.15 U/mL) were pre-incubated with HFZ for 1 h prior to addition to the cells. After 18 h of incubation, an MTS assay was performed to assess cell viability through mitochondrial respiration. A spore suspension of A. fumigatus was prepared and 96-well plates were seeded at 500,000 spores/well. HFZ and FZ were prepared as above and incubated with the fungi at 35 °C. After 72 h, the minimum inhibitory concentration (MIC) was determined as the lowest concentration of drug that inhibited visible growth. Student-Newman-Keuls multiple comparisons tests were conducted to determine statistical significance. FZ-induced cytotoxicity was significantly greater than for HFZ in HK-2 cells at amphotericin B (AmpB) concentrations between 10 and 50 μg AmpB/mL (n = 5-9, p < 0.05). HFZ and FZ were found to have similar minimum inhibitory concentration (MIC) ranges for A. fumigatus (0.225-0.25 μg) AmpB/mL; (n = 6). The addition of PLA2 and PLC to 50 μg heat-treated AmpB/mL significantly enhanced the cytotoxicity compared to controls (n = 6, p <  0.05). The presence of the phospholipases did not alter FZ-associated renal cell toxicity. Taken together, these findings suggest heat-treatment significantly decreased FZ-induced cytotoxicity in HK-2 cells without altering toxicity against a reference strain of A. fumigatus. In addition, PLA2 and PLC enhanced the renal toxicity associated with HFZ, but not that of FZ.