Food Additive Titanium Dioxide (E171) Increases Intracellular Labile Fe2+ Levels and Induces Oxidative Stress and Mitochondrial Dysfunction in H9c2 Cardiomyoblasts

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  Figure 1. E171 increases intracellular ferrous iron levels in H9c2 cardiomyoblasts. Representative bright-field images and fluorescence micrographs showing intracellular iron levels detected using the fluorescent probe BioTrackerTM FerroOrange in H9c2 cells. Orange fluorescence corresponds to intracellular labile Fe2+ levels. Cells were exposed to E171 or treated with ferrous sulfate (FeSO4) as a positive control for Fe2+ accumulation. Quantification of fluorescence intensity (arbitrary fluorescence units, AUF) confirms a significant increase in intracellular labile Fe2+ levels following E171 exposure, with FeSO4 producing a comparable elevation consistent with its role as a positive control. Data are presented as mean ± SD (n = 3 independent experiments). Statistical significance was determined using one-way ANOVA followed by appropriate Tukey analysis (* p < 0.05, ** p < 0.005). CT = non-exposed cells.

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  Figure 2. E171 increases ROS production in H9c2 cardiomyoblasts. Representative bright-field and fluorescence images showing intracellular ROS levels in H9c2 cells following exposure to E171. ROS generation was assessed using fluorescent probes for general oxidative species (DCF) and for detecting superoxide anion (DHE). Cells were stained with H2DCFDA, where green fluorescence reflects intracellular ROS generation, and with dihydroethidium (DHE), where red fluorescence indicates intracellular superoxide (O2•−) production. Fluorescence microscopy revealed a marked increase in ROS levels in cells treated with E171 compared with control cells. Quantification of fluorescence intensity (arbitrary fluorescence units, AFU) confirmed a significant increase in both DCF and DHE signals following E171 exposure. Data are presented as mean ± SD from 3 independent experiments. Statistical significance was determined using Student’s t-test (** p < 0.001; **** p < 0.0001). CT = non-exposed cells.

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  Figure 3. E171 disrupts glutathione redox homeostasis in H9c2 cardiomyoblasts. Intracellular glutathione levels were quantified in H9c2 cells following exposure to E171. (A) Total glutathione levels (GSH + GSSG), (B) reduced glutathione (GSH), (C) oxidized glutathione (GSSG), and (D) the GSH/GSSG ratio were determined and normalized to protein content. While GSH + GSSG and GSH levels were not significantly altered compared with control conditions, exposure to E171 significantly increased GSSG. Consequently, the GSH/GSSG ratio was markedly reduced, indicating disruption of the cellular redox balance. Data are presented as mean ± SD from 3 independent experiments. Statistical significance was determined using Student’s t-test (* p < 0.05, ** p < 0.005).

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  Figure 4. E171 induces mitochondrial ultrastructural alterations in H9c2 cardiomyoblasts. Transmission electron microscopy (TEM) micrographs showing representative mitochondrial morphology in control (CT) and E171-treated cells. (A) Control cells exhibiting preserved mitochondrial ultrastructure. (B) Higher magnification of control mitochondria displaying intact outer membranes and well-defined cristae. (C) E171-treated cells showed accumulation of electron-dense aggregates and altered mitochondrial morphology. (D) Higher magnification of E171-treated cells demonstrates mitochondrial abnormalities, including loss of cristae, membrane rupture, and increased electron density. Boxes indicate the areas enlarged in the high-magnification TEM images shown in panels B and D. (E) Quantification of mitochondrial area measured from TEM micrographs. The mitochondrial area was measured using ImageJ software. (F) Percentage distribution of mitochondria classified as conserved, without cristae (No-cristae), membrane rupture, or electron-dense. Percentages were calculated relative to the total number of mitochondria analyzed per group. A total of 20 mitochondria from 13 independent cells (CT) and 17…

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  Figure 5. E171 impairs mitochondrial respiration in H9c2 cardiomyoblasts. Mitochondrial respiratory function was evaluated in H9c2 cells by oxygen consumption rate (OCR) parameters under control conditions (CT), in the presence of Quercetin (Q), after exposure to E171, and in cells treated with both quercetin and E171 (Q + E171). Respiratory parameters analyzed included total respiration, oxidative phosphorylation (OxPhos), electron transport system capacity (chain transporter (CT)), no mitochondrial (No mit), and Leak. Exposure to E171 significantly reduced several mitochondrial respiratory parameters compared with control conditions, indicating impaired mitochondrial bioenergetics. Q treatment alone did not significantly affect mitochondrial respiration or prevent mitochondrial dysfunction induced by nanoparticle exposure. Data are presented as mean ± SD (n = 3 independent experiments). Statistical significance was determined using one-way ANOVA followed by appropriate Tukey analysis (* p < 0.05, ** p < 0.005, *** p < 0.0005). ns = no significant.

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  Figure 6. Integrative schematic representation of the effects of E171 on H9c2 cardiomyoblasts. Exposure to E171 increased intracellular ferrous iron (Fe2+) levels and reactive oxygen species (ROS), disrupted the glutathione redox balance (increased GSSG and decreased GSH/GSSG ratio), and induced mitochondrial structural alterations characterized by swelling and cristae disorganization. Functionally, mitochondrial respiration was significantly reduced, as evidenced by decreased total respiration, oxidative phosphorylation (OxPhos), electron transport chain capacity (CT), and non-mitochondrial respiration (No mit), while proton leak remained unchanged. Treatment with (Q, 10 μM) did not significantly ameliorate mitochondrial ultrastructural alterations or mitochondrial respiratory parameters under the experimental conditions tested.