Fullerenol cytotoxicity in kidney cells is associated with cytoskeleton disruption, autophagic vacuole accumulation, and mitochondrial dysfunction

Abstract

Water soluble fullerenes, such as the hydroxylated fullerene, fullerenol (C₆₀OHx), are currently under development for diagnostic and therapeutic biomedical applications in the field of nanotechnology. These molecules have been shown to undergo urinary clearance, yet there is limited data available on their renal biocompatibility. Here we examine the biological responses of renal proximal tubule cells (LLC-PK1) exposed to fullerenol. Fullerenol was found to be cytotoxic in the millimolar range, with viability assessed by the sulforhodamine B and trypan blue assays. Fullerenol-induced cell death was associated with cytoskeleton disruption and autophagic vacuole accumulation. Interaction with the autophagy pathway was evaluated in vitro by Lysotracker Red dye uptake, LC3-II marker expression and TEM. Fullerenol treatment also resulted in coincident loss of cellular mitochondrial membrane potential and ATP depletion, as measured by the Mitotracker Red dye and the luciferin-luciferase assays, respectively. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by co-treatment with the autophagy inhibitor, 3-methyladenine. In vitro fullerenol treatment did not result in appreciable oxidative stress, as measured by lipid peroxide and glutathione content. Based on these data, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction and loss of mitochondrial capacity. As nanoparticle-induced cytoskeleton disruption, autophagic vacuole accumulation and mitochondrial dysfunction are commonly reported in the literature, the proposed mechanism may be relevant for a variety of nanomaterials.

Figure 1. Hydrodynamic Size Analysis by DLS

Shown are the intensity and volume distributions of fullerenol-PBS (red) and fullerenol-10 mM NaCl (green) preparations. Each distribution line is the average of 12 independent measurements. Intensity-weighted average was used to determine hydrodynamic size, while volume distribution data was used to determine relative amounts.

Figure 2. SRB Viability

LLC-PK1 cells were treated for 24 and 48 hrs with 0.0002– 60 mM fullerenol. Cell viability was determined at each time point by the SRB assay. Data are presented as percent media control cell viability. Values correspond to the mean ± SE, N=3.

Figure 3. Fullerenol Actin Confocal Analysis

LLC-PK1 cells grown on cover slips were treated for 24 hrs with media (negative control), low dose fullerenol (0.6 mM), high dose fullerenol (3 mM), or were pre-treated with nocodazole (50.0 µM) for 2 hrs. All cells were stained with 1 unit (fluorescence) of Oregon Green 488 phalloidin dye/Hoechst nuclear stain/coverslip (20 µL of 6.6 uM methanolic Oregon Green stock solution/5 µg Hoechst/mL of 1% BSA-PBS /coverslip) for 20 min at ambient temperature, and were attached to microscope slides prior to confocal analysis. Images were acquired at 40× (oil) magnification.

Figure 4. TEM Photomicrographs

LLC-PK1 cells were treated for 6 hrs with media (negative), Hank’s balanced salt starvation media (positive), or 0.03 mM fullerenol (treated). Black inlet boxes show autophagic vacuoles, consisting of double layered membranes containing cellular debris. Image scale bar lengths are as follows: Black scale bar is 10 µm, negative inset bar is 1 µm, positive inset bar is 10 nm and fullerenol inset bar is 2 µm. Asterisk indicates mitochondrial damage. Images were acquired at 5,000–20,000× magnification.

Figure 5. Lysotracker Response

LLC-PK1 cells were treated for 6, 24 or 48 hrs with 0.01–6 mM fullerenol. Data are presented as the percent control Lysotracker Red fluorescence normalized to Celltracker Green fluorescence (percent control Lysotracker red fluorescence/percent control Celltracker green fluorescence). Values correspond to mean ± SE of 3 individual samples. Asterisks indicate statistical difference from control (ANOVA, Dunnett’s post hoc, p < 0.05).

Figure 6. LC3 Immunoblot

LLC-PK1 cells were treated for 6 hrs (A) and 24 hrs (B) with media (negative), starvation buffer (positive), or 6 mM fullerenol (treated) in duplicate. Cell lysate proteins were separated by SDS-PAGE, transferred to PVDF membrane, and probed for LC3 reactive proteins. LC3-I and II are labeled on the immunoblots.

Figure 7. Fullerenol ATP Assay

LLC-PK1 cells were treated for 24 and 48 hrs with 0.2–60 mM fullerenol. ATP content was determined at each time point by the luciferin-luciferase assay. Data are presented as percent media control. Values correspond to the mean ± SE, N=3.

Figure 8. Fullerenol plus 3-MA Mitochondrial Confocal Analysis

LLC-PK1 cells grown on cover slips were treated for 24 hrs with media (negative control), low dose fullerenol (0.6 mM), or high dose fullerenol (3 mM), with or without pretreatment with 2 mM 3-methyladenine (3-MA) for 2 hrs, prior to 1 mM 3-MA cotreatment. All cells were stained with 200 nM Mitotracker Red, fixed with 4 % formaldehyde, and were attached to microscope slides prior to confocal analysis. Images were acquired at 148× (oil) magnification under identical detector settings.

Figure 9. Fullerenol plus 3-MA Lysotracker Assay

LLC-PK1 cells were pre-treated with 2 mM 3-methyladenine (3-MA) for 2 hrs, prior to 0.01–6 mM fullerenol and 1 mM 3-MA cotreatment for an additional 6, 24 and 48 hrs. Data are presented as the percent control Lysotracker Red fluorescence normalized to Celltracker Green fluorescence (percent control Lysotracker red fluorescence/percent control Celltracker green fluorescence). Values correspond to mean ± SE of 3 individual samples.

Figure 10. Fullerenol plus 3-MA ATP Assay

LLC-PK1 cells were treated for 24 and 48 hrs with 0.2–60 mM fullerenol, with or without pre-treatment with 2 mM 3-methyladenine (3-MA) for 2 hrs prior to 1 mM 3-MA cotreatment. ATP content was determined at each time point by the luciferin-luciferase assay. Data are presented as percent media control ATP level. Values correspond to the mean ± SE, N = 3. Asterisks indicate statistical difference from fullerenol alone treatment (Student’s t-test, p < 0.05).