Leon Carlos, Taylor Ross, Bartlett Karen H, Wasan Kishor M
Division of Pharmaceutics and Biopharmaceutics, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, BC, Canada V6T 1Z3.
Int J Pharm. 2005 Jul 14;298(1):211-8. doi: 10.1016/j.ijpharm.2005.04.018.
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 degrees C in T75 flasks and seeded in 96-well plates at 20,000 cells/well. FZ and HFZ concentrations of 10, 25 and 50 microg/mL of AmpB were prepared. Snake venom PLA2 and PLC (2.15 U/mL) were pre-incubated with HFZ for 1h 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 degrees 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 microg 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 microg) AmpB/mL; (n = 6). The addition of PLA2 and PLC to 50 microg 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.
本研究的目的是确定热处理对两性霉素B（FZ）诱导的人肾（HK-2）细胞和烟曲霉真菌分离株细胞毒性的影响，并确定磷脂酶（PLA2和PLC）在热处理的FZ（HFZ）相关肾细胞毒性中可能发挥的作用。HK-2细胞在37℃下于T75培养瓶中培养，并以每孔20,000个细胞接种到96孔板中。制备了浓度为10、25和50μg/mL两性霉素B的FZ和HFZ。在添加到细胞之前，将蛇毒PLA2和PLC（2.15 U/mL）与HFZ预孵育1小时。孵育18小时后，进行MTS试验以通过线粒体呼吸评估细胞活力。制备烟曲霉的孢子悬液，并以每孔500,000个孢子接种到96孔板中。如上述制备HFZ和FZ，并在35℃下与真菌孵育。72小时后，将最低抑菌浓度（MIC）确定为抑制可见生长的最低药物浓度。进行Student-Newman-Keuls多重比较试验以确定统计学显著性。在两性霉素B（AmpB）浓度为10至50μg AmpB/mL之间时，FZ诱导的HK-2细胞毒性显著大于HFZ（n = 5 - 9，p < 0.05）。发现HFZ和FZ对烟曲霉具有相似的最低抑菌浓度（MIC）范围（0.225 - 0.25μg）AmpB/mL；（n = 6）。与对照组相比，向50μg热处理的AmpB/mL中添加PLA2和PLC显著增强了细胞毒性（n = 6，p < 0.05）。磷脂酶的存在未改变FZ相关的肾细胞毒性。综上所述，这些发现表明热处理显著降低了FZ在HK-2细胞中诱导的细胞毒性，而不改变对烟曲霉参考菌株的毒性。此外，PLA2和PLC增强了与HFZ相关的肾毒性，但未增强FZ的肾毒性。
