Metabolomics of V2O5 nanoparticles and V2O5 nanofibers in human airway epithelial BEAS-2B cells

Abstract

Vanadium is a toxic metal listed by the IARC as possibly carcinogenic to humans. Manufactured nanosize vanadium pentoxide (V₂O₅) materials are used in a wide range of industrial sectors and recently have been developed as nanomedicine for cancer therapeutics, yet limited information is available to evaluate relevant nanotoxicity. In this study we used high-resolution metabolomics to assess effects of two V₂O₅ nanomaterials, nanoparticles and nanofibers, at exposure levels (0.01, 0.1, and 1 ppm) that did not cause cell death (i.e., non-cytotoxic) in a human airway epithelial cell line, BEAS-2B. As prepared, V₂O₅ nanofiber exhibited a fibrous morphology, with a width approximately 63 ± 12 nm and length in average 420 ± 70 nm; whereas, V₂O₅ nanoparticles showed a typical particle morphology with a size 36 ± 2 nm. Both V₂O₅ nanoparticles and nanofibers had dose-response effects on aminosugar, amino acid, fatty acid, carnitine, niacin and nucleotide metabolism. Differential effects of the particles and fibers included dibasic acid, glycosphingolipid and glycerophospholipid pathway associations with V₂O₅ nanoparticles, and cholesterol and sialic acid metabolism associations with V₂O₅ nanofibers. Examination by transmission electron microscopy provided evidence for mitochondrial stress and increased lysosome fusion by both nanomaterials, and these data were supported by effects on mitochondrial membrane potential and lysosomal activity. The results showed that non-cytotoxic exposures to V₂O₅ nanomaterials impact major metabolic pathways previously associated with human lung diseases and suggest that toxico-metabolomics may be useful to evaluate health risks from V₂O₅ nanomaterials.

Keywords: Lung metabolism; Metabolic disruption; Nanofiber; Nanoparticles; Nanotoxicity; Risk assessment; Vanadium.

Fig. 1.

Physicochemical characterization of V₂O₅ nanofibers and nanoparticles. (A) TEM micrographs. (B) SEM micrographs. (C) UV–Vis absorption spectrum. (D) XRD pattern with (001) phase identified. (E) TEM micrographs for lattice visualization with fast Fourier transform. nf V₂O₅: V₂O₅ nanofiber; np V₂O₅: V₂O₅ nanoparticle.

Fig. 2.

Cytotoxicity and intracellular vanadium levels in BEAS-2B cells treated with V₂O₅ nanomaterials. Panels A, B and C provide results for V₂O₅ nanoparticles and Panels D, E and F provide results for V₂O₅ nanofibers. Cytotoxicity was quantified using WST1 assay after 24 h incubation and expressed as cell survival, presented as a percentage of respective control values (A, B, D, E). Cellular V⁵¹ was measured using ICP-MS following 24 h treatment. Abbreviations: nfV₂O₅: V₂O₅ nanofiber; npV₂O₅: V₂O₅ nanoparticle.

Fig. 3.

Metabolic effects of V₂O₅ nanoparticles in BEAS-2B cells. (A) Manhattan plot of 678 metabolic features selected by linear regression with V₂O₅ nanoparticle dose (p < 0.05) from HILIC(+) chromatography. (B). One-way hierarchical cluster analysis (HCA) of 678 selected metabolic features altered by V₂O₅ nanoparticles. (C) PLS-DA of 678 selected metabolites altered by V₂O₅ nanoparticles exposure at p < 0.05. np V₂O₅: V₂O₅ nanoparticle.

Fig. 4.

Metabolic pathways altered by V₂O₅ nanoparticles exposure. (A) Pathway analysis revealed 17 pathways associated with V₂O₅ exposure. Red bars indicate upregulated pathways, blue bars indicate downregulated pathways, grey bars indicate non-specific direction. (B) Network of the major activity modules is shown with metabolites associated with those significantly altered pathways. (C) Top 25 metabolites from PLS-DA altered by V₂O₅ nanoparticles exposure using significant HILIC features with p < 0.05. Annotated metabolites are indicated by pound sign (#). Selected annotated metabolites are individually plotted in (D-H): (D). 3-Dehydroxycarnitine (m/z 146.1175, rt 34s); (E). Palmitoylglycine (m/z 314.26875, rt 23s); (F) 2-Amino-4-hydroxy-3-methylpentanoic acid (m/z 148.0969, rt 272s); (G) LysoPE(18:2, 0:0) (m/z 478.2826, rt 198s); and (H) N-Gluconyl ethanolamine phosphate (m/z 320.0756, rt 249s). Statistical significance for each condition vs. control is indicated by asterisks, with *p < 0.05, and **p < 0.01. np V₂O₅: V₂O₅ nanoparticle.

Fig. 5.

Alterations in metabolites by V₂O₅ nanofiber exposure. (A). Manhattan plot of associated metabolic features (p < 0.05) from HILIC (+) chromatography. (B). HCA of metabolites from HILIC (+) chromatography altered by V₂O₅ nanofiber at p < 0.05. (C) PLS-DA of selected metabolites from HILIC (+) chromatography altered by V₂O₅ nanofiber exposure at p < 0.05. Warm color and positive z-score indicates higher abundance in (A). nf V₂O₅: V₂O₅ nanofiber.

Fig. 6.

Metabolic pathways altered by V₂O₅ nanofiber exposure. (A) Pathway analysis revealed 15 pathways were enriched with V₂O₅ exposure. Red bars indicate upregulated pathways, blue bars indicate downregulated pathways, grey bars indicate non-specific direction. (B) Network of the major activity modules is shown with metabolites associated with those significantly altered pathways. (C) Top 25 metabolites from PLS-DA altered by V₂O₅ nanofiber exposure using significant Hilic(+) features with p < 0.05. Annotated metabolites are indicated by pound sign (#). Selected annotated metabolites are individually plotted in (D-H): (D). S-Adenosylhomocysteine (m/z 385.1286, rt 76s); (E). Butyrylcholine (m/z 175.157, rt 76s); (F) CTP (m/z 481.9768, rt 87s); (G) 3-AMP (m/z 348.07019, rt 281s); and (H) uracil (m/z 157.0255, rt 281s). Significance for each condition vs. control is indicated by asterisks, with *p < 0.05, and **p < 0.01. nf V₂O₅: V₂O₅ nanofiber.

Fig. 7.

Representative TEM images of control BEAS-2B (A-B), 1 ppm V₂O₅ nanoparticles (C-D), and 1 ppm V₂O₅ nanofiber (E-F) after 24 h. Autophagic vacuoles including autophagosomes and autolysosomes were also noted in the V₂O₅ nanoparticle exposed cells (blue arrows). Fusion of lysosomes was commonly observed in both V₂O₅ nanoparticles or V₂O₅ nanofiber exposed cells (green arrows). Internalization of V₂O₅ nanoparticles or nanofibers was found in lysosome (red arrowheads). Note that it is technically challenging to capture a full picture of fibers in a cell from a monolayer, especially at such low concentration. Increased cell debris in lysosomes indicates stimulated breakdown of proteins and other cellular components (purple arrowheads). Swollen and dilated mitochondria with disrupted cristae structure were noted in both V₂O₅ nanoparticles or V₂O₅ nanofibers exposed cells (yellow arrows). Increased endoplasmic reticulum activity in both V₂O₅ nanoparticles or V₂O₅ nanofibers is indicated by black stars. Normal mitochondria are indicated by black arrows. Scale bars are indicated in μm.

Fig. 8.

Intracellular lysosomal activity in BEAS-2B cells following V₂O₅ nanofiber and nanoparticles exposure at 1 ppm. Representative images are shown in (A). Quantitative analyses are shown in (B). Bafilomycin A1 (1x) was used as a positive control that inhibits lysosomal function. nf V₂O₅: V₂O₅ nanofiber; np V₂O₅: V₂O₅ nanoparticle; SQS: self-quenched substrate.

Fig. 9.

Mitochondrial membrane potential measured by JC-1 dye in BEAS-2B cells following V₂O₅ nanofiber and nanoparticles exposure at 1 ppm. Representative images are shown in (A). Quantitative analyses are shown in (B). H₂O₂ (1 mM) was used as a positive control. nf V₂O₅: V₂O₅ nanofiber; np V₂O₅: V₂O₅ nanoparticle.