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

Abstract

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 (PLA 2 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 PLA 2 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 PLA 2 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, PLA 2 and PLC enhanced the renal toxicity associated with HFZ, but not that of FZ.