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. 2020 Aug 26;10(9):1675.
doi: 10.3390/nano10091675.

Gadolinium Oxide Nanoparticles Induce Toxicity in Human Endothelial HUVECs via Lipid Peroxidation, Mitochondrial Dysfunction and Autophagy Modulation

Mohd Javed Akhtar  1 , Maqusood Ahamed  1 , Hisham Alhadlaq  2
Affiliations

Gadolinium Oxide Nanoparticles Induce Toxicity in Human Endothelial HUVECs via Lipid Peroxidation, Mitochondrial Dysfunction and Autophagy Modulation

Mohd Javed Akhtar et al. Nanomaterials (Basel). .

Abstract

In spite of the potential preclinical advantage of Gd2O3 nanoparticles (designated here as GO NPs) over gadolinium-based compounds in MRI, recent concerns of gadolinium deposits in various tissues undergoing MRI demands a mechanistic investigation. Hence, we chose human to measure umbilical vein endothelial cells (HUVECs) that line the vasculature and relevant biomarkers due to GO NPs exposure in parallel with the NPs of ZnO as a positive control of toxicity. GO NPs, as measured by TEM, had an average length of 54.8 ± 29 nm and a diameter of 13.7 ± 6 nm suggesting a fiber-like appearance. With not as pronounced toxicity associated with a 24-h exposure, GO NPs induced a concentration-dependent cytotoxicity (IC50 = 304 ± 17 µg/mL) in HUVECs when exposed for 48 h. GO NPs emerged as significant inducer of lipid peroxidation (LPO), reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and autophagic vesicles in comparison to that caused by ZnO NPs at its IC50 for the same exposure time (48 h). While ZnO NPs clearly appeared to induce apoptosis, GO NPs revealed both apoptotic as well as necrotic potentials in HUVECs. Intriguingly, the exogenous antioxidant NAC (N-acetylcysteine) co-treatment significantly attenuated the oxidative imbalance due to NPs preventing cytotoxicity significantly.

Keywords: ROS; autophagy; lysosome; necrosis; oxidative stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Shape and size of gadolinium oxide (GO) nanoparticles (NPs) characterized by TEM image captured at (A) 50 nm and (B) 20 nm. High resolution TEM image captured at (C) 2 nm depicts crystal plane. (D–F) SEM, EDS and XRD images of GO NPs, respectively.
Figure 2
Figure 2
Potential cytotoxicity in human umbilical vein endothelial cells (HUVECs) due to GO NPs was demonstrated by (A) MTT biochemical assays followed by imaging same cells under settings of (B) phase-contrast and (C) calcein-AM live imaging. (D) Corrected total cellular fluorescence (CTCF) plotting of calcein-AM. For concentration-dependent cytotoxicity (IC50) calculation, a scatter plot in Microsoft Excel was inserted followed by setting the Y-axis to logarithmic. Then a trend line was selected and ‘exponential’ picked. Then ‘display equation’ was used in calculating ICs. IC50s calculations were further verified and confirmed from the online IC50 calculator (https://www.aatbio.com/tools/ic50-calculator) provided by AAT BioQuest, Inc. (CA 94085, USA). Scale bar represents 50 µm (20× objective). * Statistically significant difference than the controls (p < 0.05).
Figure 3
Figure 3
Potential loss of membrane integrity in HUVEC cells due to GO NPs were revealed by (A) quantifying LDH release in culture media, (B) peroxidation in lipid bilayers poly unsaturated fatty acids, followed by (C) imaging of membrane residing BODIPY probe; (D) BODIPY CTCF of only green fluorescence is given, as it is the indicator of lipid peroxidation (LPO) while red fluorescence is uniform in control and treated cells. Scale bar represents 50 µm in each image (20× objective). * Statistically significant difference than the controls (p < 0.05). #—Significantly high BODIPY fluorescence due to GO NPs compared to ZnO NPs (p < 0.05).
Figure 4
Figure 4
Level of oxidative stress was determined by measuring (A) reactive oxygen species (ROS) induction and (B) GSH depletion in HUVEC cells. H2O2 was used as a positive control of oxidant in DCFH-DA probing. MMP was detected by JC-1 in control and treated groups of HUVECs; (C) images captured in tandem for JC-1 monomer (green) and JC-1 aggregate (red); (D) Quantification of MMP is given as ratio of monomer/aggregate. Scale bar represents 25 µm in each image (40× objective). * Statistically significant difference than the controls (p < 0.05). #—Significantly high MMP induction due to GO NPs compared to ZnO NPs (p < 0.05).
Figure 5
Figure 5
Autophagy was determined by a (A) plate reader as well as by (B) direct observation under microscopy. Merging of images was carried out in ImageJ software; (C,D) CTCF for monodansylcadaverine (MDC) and LysoTracker (LTR), respectively. Scale bar represents 25 µm (40× objective). * Statistically significant difference than the controls (p < 0.05). #—Significantly high MDC and LTR activity due to GO NPs compared to ZnO NPs (p < 0.05).
Figure 6
Figure 6
HUVEC cells treated with toxic concentrations of NPs of GO and ZnO for 48 h and apoptosis/necrosis was determined by (A) triple-staining and (B) caspase-3 activity. Cells were stained with Hoechst 33442 (blue color) that stains nucleus of live or dead cell, PI (red color) that stains nucleus of only dead or dying cell and annexinV (green color) that preferentially stain apoptotic cells. Zoomed images (left) represent equal areas carved out from each of PI images to observe morphology of nucleus in greater detail whereas zoomed out images (right) are carved out from each merged image for a better visualization representing the area equal to yellow circles. Scale bar represents 50 µm (20× objective). * Statistically significant difference than the controls (p < 0.05). #—Significantly low caspase-3 activity due to GO NPs compared to ZnO NPs (p < 0.05).
Figure 7
Figure 7
Inhibitory effect of N-acetylcysteine (NAC) cotreatment on (A) ROS-induction, (B) GSH-depletion—and consequently, (C) cytotoxicity that would otherwise be induced by NPs alone. * Statistically significant difference than the controls (p < 0.05). α and β denote significant preventive potential of NAC on ROS generation, GSH decline and cytotoxicity due to GO NPs and ZnO NPs, respectively (p < 0.05).
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