A representative transmission electron microscope (TEM) image (a) and the elemental composition of the interesting spots on it (b), as investigated with an energy dispersive X-ray (EDX) spectrometer, for a cell slice of <i>C. reinhardtii</i> exposed to 100 mg/l TiO₂-ENs but without any addition of Cd²⁺.

<p>A representative transmission electron microscope (TEM) image (a) and the elemental composition of the interesting spots on it (b), as investigated with an energy dispersive X-ray (EDX) spectrometer, for a cell slice of <i>C. reinhardtii</i> exposed to 100 mg/l TiO₂-ENs but without any addition of Cd²⁺.</p

Relative changes of the cell specific growth rate (μ) (a–b) and intracellular Cd²⁺ concentration ([Cd²⁺]_intra, pg/cell) (c–d) with either the total dissolved ([Cd²⁺]_T, mg/l) (a, c) or free Cd²⁺ ([Cd²⁺]_F, mg/l) concentrations (b, d) at the beginning of the three toxicity experiments where 0, 100, and 1–100 mg/l TiO₂-ENs were applied, respectively.

<p>Dashed lines represent the simulated curves for the relative changes of μ (a–b) and [Cd²⁺]_intra (c–d) at different [Cd²⁺]_T (a, c) and [Cd²⁺]_F (b, d) by the Logistic dose-response and Freundlich models, respectively. Data are mean ± standard deviation (n = 2).</p

Adsorption of Cd²⁺ (q_t, mg/g) on TiO₂-ENs in the kinetics (a) and 4-h equilibrium isotherm (b–d) experiments, respectively.

<p>There were five treatments with different concentrations of TiO₂-ENs (1.0, 3.0, 10.0. 30.0, and 100.0 mg/l) but the same concentration of total Cd²⁺ (1 mg/l) in the kinetics experiment. Various concentrations of Cd²⁺ (0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, and 10.0 mg/l initially) were used in each equilibrium isotherm experiment with different TiO₂-EN concentrations (b–d: 1, 10, 100 mg/l, respectively). Since Cd²⁺ adsorption got saturated when its concentration approached 0.3 mg/l with 1 mg/l TiO₂-ENs, the two highest Cd²⁺ concentrations (3.0 and 10.0 mg/l) were not used for this EN concentration treatment. Dashed lines represent the simulated curves of Cd²⁺ adsorption kinetics and equilibrium isotherm by the pseudo-first order (a) and Langmuir (b–d) models. Data are mean ± standard deviation (n = 2).</p

Accumulation of TiO₂-ENs ([TiO₂-ENs]_cell, pg/cell) by <i>Chlamydomonas reinhardtii</i> in the different treatments of the second (Exp_2) and third (Exp_3) toxicity experiments.

<p>In Exp_2, treatment A–G indicates different initial concentrations of Cd²⁺ (0, 0.1, 0.3, 0.5, 0.8, 1.0, and 3.0 mg/l) with the TiO₂-EN concentration fixed at 100 mg/l. In Exp_3, the initial Cd²⁺ concentration was fixed at 1 mg/l and various concentrations (0, 1, 3, 10, 30, and 100 mg/l) TiO₂-ENs were used for treatments A–F. Data are mean ± standard deviation (n = 2).</p

The transmission electron microscope image of TiO₂-ENs dispersed in the modified WC medium (WC_m) (a) and their zeta potentials (mV) at different pH (b).

<p>The transmission electron microscope image of TiO₂-ENs dispersed in the modified WC medium (WC_m) (a) and their zeta potentials (mV) at different pH (b).</p

Pre-exposure to Fe₂O₃ or TiO₂ Nanoparticles Inhibits Subsequent Biological Uptake of ⁵⁵Fe-Labeled Fe₂O₃ Nanoparticles

Aquatic organisms are frequently exposed to various nanoparticles (NPs) in the natural environment. Thus, studies of NP bioaccumulation should include organisms that have been previously exposed to NPs. Our study investigated the effects of pre-exposure of Tetrahymena thermophila (T. thermophila) to Fe2O3 or TiO2 NPs on the protozoan’s subsequent uptake of 55Fe-labeled Fe2O3 (55Fe2O3) NPs. Molecular mechanisms underlying the pre-exposure effects were explored in transcriptomic and metabolomic experiments. Pre-exposure to either NPs inhibited the subsequent uptake of 55Fe2O3 NPs. The results of the transcriptomic experiment indicated that NP pre-exposure influenced the expression of genes related to phagosomes and lysosomes and physiological processes such as glutathione and lipid metabolism, which are closely associated with the endocytosis of 55Fe2O3 NPs. The differentially expressed metabolites obtained from the metabolomic experiments showed an enrichment of energy metabolism and antioxidation pathways in T. thermophila pre-exposed to NPs. Together, these results demonstrate that the pre-exposure of T. thermophila to Fe2O3 or TiO2 NPs inhibited the protozoan’s subsequent uptake of 55Fe2O3 NPs, possibly by mechanisms involving the alteration of endocytosis-related organelles, the induction of oxidative stress, and a lowering of the intracellular energy supply. Thus, NP pre-exposure represents a scenario which can inform increasingly realistic estimates of NP bioaccumulation

Pre-exposure to Fe₂O₃ or TiO₂ Nanoparticles Inhibits Subsequent Biological Uptake of ⁵⁵Fe-Labeled Fe₂O₃ Nanoparticles

Aquatic organisms are frequently exposed to various nanoparticles (NPs) in the natural environment. Thus, studies of NP bioaccumulation should include organisms that have been previously exposed to NPs. Our study investigated the effects of pre-exposure of Tetrahymena thermophila (T. thermophila) to Fe2O3 or TiO2 NPs on the protozoan’s subsequent uptake of 55Fe-labeled Fe2O3 (55Fe2O3) NPs. Molecular mechanisms underlying the pre-exposure effects were explored in transcriptomic and metabolomic experiments. Pre-exposure to either NPs inhibited the subsequent uptake of 55Fe2O3 NPs. The results of the transcriptomic experiment indicated that NP pre-exposure influenced the expression of genes related to phagosomes and lysosomes and physiological processes such as glutathione and lipid metabolism, which are closely associated with the endocytosis of 55Fe2O3 NPs. The differentially expressed metabolites obtained from the metabolomic experiments showed an enrichment of energy metabolism and antioxidation pathways in T. thermophila pre-exposed to NPs. Together, these results demonstrate that the pre-exposure of T. thermophila to Fe2O3 or TiO2 NPs inhibited the protozoan’s subsequent uptake of 55Fe2O3 NPs, possibly by mechanisms involving the alteration of endocytosis-related organelles, the induction of oxidative stress, and a lowering of the intracellular energy supply. Thus, NP pre-exposure represents a scenario which can inform increasingly realistic estimates of NP bioaccumulation

Cellular concentration of glutathione ([GSH]_cell) in treatments A-H of the (a) nutrient-enriched (+NP), (b) phosphorus-limited (-P), and (c) nitrogen-limited (-N) toxicity tests for <i>Microcystis aeruginosa</i> PCC 7806 (WT, black bar) and its MC-lacking mutant (MT, white bar).

<p>All values were normalized to levels (100% as represented by the dashed lines) detected in the WT strain at the lowest respective Cd concentration (Treatment A). Cd concentration in treatments A-H ([Cd]_T, 1.00×10^-8—9.95×10^-6 M; [Cd²⁺]_F, 1.00×10^-13—1.21×10^-8 M) is listed in Table B of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116659#pone.0116659.s001" target="_blank">S1 File</a>. Data are mean ± standard error (n = 2)</p

Cell-volume-normalized concentration of (a, d, g) monosaccharide and (b, e, h) polysaccharide excreted by the cells as well as (c, f, i) cellular concentration of carbohydrates retained inside the cells in the (a-c) nutrient-enriched (+NP), (d-f) phosphorus-limited (-P), and (g-i) nitrogen-limited (-N) toxicity tests for <i>Microcystis aeruginosa</i> PCC 7806 (WT, black bar) and its MC-lacking mutant (MT, white bar).

<p>All values were normalized to levels (100% as represented by the dashed lines) detected in the WT strain at the lowest respective Cd concentration (Treatment A). Cd concentration in treatments A-H ([Cd]_T, 1.00×10^-8—9.95×10^-6 M; [Cd²⁺]_F, 1.00×10^-13—1.21×10^-8 M) is listed in Table B of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116659#pone.0116659.s001" target="_blank">S1 File</a>. Data are mean ± standard error (n = 2)</p

[Cd²⁺]F-based median effect concentration (EC50) in the nutrient-enriched (+NP), phosphorus-limited (-P), and nitrogen-limited (-N) toxicity tests for <i>Microcystis aeruginosa</i> PCC 7806 (WT) and its MC-lacking mutant (MT).

<p>* No significant difference (<i>p</i> > 0.05) between these EC50s.</p><p>Data are mean ± standard error (n = 2)</p