Age modulates Fe3O4 nanoparticles liver toxicity: dose-dependent decrease in mitochondrial respiratory chain complexes activities and coupling in middle-aged as compared to young rats

Abstract

We examined the effects of iron oxide nanoparticles (IONPs) on mitochondrial respiratory chain complexes activities and mitochondrial coupling in young (3 months) and middle-aged (18 months) rat liver, organ largely involved in body iron detoxification. Isolated liver mitochondria were extracted using differential centrifugations. Maximal oxidative capacities (V(max), complexes I, III, and IV activities), V(succ) (complexes II, III, and IV activities), and V tmpd, (complex IV activity), together with mitochondrial coupling (V(max)/V0) were determined in controls conditions and after exposure to 250, 300, and 350 μ g/ml Fe3O4 in young and middle-aged rats. In young liver mitochondria, exposure to IONPs did not alter mitochondrial function. In contrast, IONPs dose-dependently impaired all complexes of the mitochondrial respiratory chain in middle-aged rat liver: V(max) (from 30 ± 1.6 to 17.9 ± 1.5; P < 0.001), V(succ) (from 33.9 ± 1.7 to 24.3 ± 1.0; P < 0.01), V(tmpd) (from 43.0 ± 1.6 to 26.3 ± 2.2 µmol O2/min/g protein; P < 0.001) using Fe3O4 350 µg/ml. Mitochondrial coupling also decreased. Interestingly, 350 μ g/ml Fe3O4 in the form of Fe(3+) solution did not impair liver mitochondrial function in middle-aged rats. Thus, IONPs showed a specific toxicity in middle-aged rats suggesting caution when using it in old age.

Figure 1

TEM image (a) and size histogram (b) of the Fe₃O₄ nanoparticles.

Figure 2

Effects of iron oxide nanoparticles (Fe₃O₄) on young liver mitochondrial respiratory chain complexes activities. (a) V _max⁡ reflects complexes I, III, and IV activities and is measured using glutamate and malate. (b) V _succ reflects complexes II, III, and IV activities and is measured using succinate. (c) V _tmpd reflects complex IV activity and is measured using TMPD and ascorbate as mitochondrial substrates. Data are means ± SEM

Figure 3

Effects of iron oxide nanoparticles (Fe₃O₄) on middle-aged liver mitochondrial respiratory chain complexes activities. (a) V _max⁡ reflects complexes I, III, and IV activities and is measured using glutamate and malate. (b) V _succ reflects complexes II, III, and IV activities and is measured using succinate. (c) V _tmpd reflects complex IV activity and is measured using TMPD and ascorbate as mitochondrial substrates. Data are means ± SEM (one-way ANOVA followed by Tukey). *P < 0.05; **P < 0.01; ***P < 0.001 compared to control. ^# P < 0.05 350 μg/mL compared to 300 μg/mL. ^§ P < 0.05 350 μg/mL compared to 250 μg/mL.

Figure 4

Effects of iron oxide nanoparticles (Fe₃O₄) on (a) young and (b) middle-aged liver mitochondrial coupling. Data are means ± SEM (one-way ANOVA followed by Tukey). *P < 0.05; **P < 0.01; ***P < 0.001 compared to control.

Figure 5

Effects of 350 μg/mL of Fe₃O₄ but not in its particulate form on middle-aged liver mitochondrial respiration and coupling. (a) V _max⁡ reflects complexes I, III, and IV activities and is measured using glutamate and malate. (b) V _succ reflects complexes II, III, and IV activities and is measured using succinate. (c) V _tmpd reflects complex IV activity and is measured using N, N, N′, N′-tetramethyl-p-phenylenediaminedihydrochloride (TMPD) and ascorbate as mitochondrial substrates. (d) Acceptor control ratio reflects the mitochondrial coupling. Data are means ± SEM.