Table 1.
Characterization of the two nanoparticles used in this study.
Fig 1.
Determination of the cell viability after treatment with copper oxide nanoparticles (A), and copper acetate (B).
Experiments were carried out in quadruplicate on independent cultures. Insignificant toxicity was induced by titanium dioxide up to 100μg/ml and by copper oxide microparticles up to 50μg/ml. Grey bars show the absence of toxicity of PVP at the concentrations present in the copper oxide dispersions (respectively1, 2 and 4 μg/ml). In C, surviving cell numbers after a combined treatment by copper acetate alone (blue curve, circles), copper acetate + 25 μg/ml titanium dioxide (red curve, squares), copper acetate + 10μg/ml 250nm latex beads (green curve, diamonds), copper acetate + 25μg/ml 1μm latex beads (purple curve, triangles). None of the observed variation was significant (Mann Whitney U test). In D and E, Transmission electron microscopy of J774 cells exposed to 50μg/ml titanium dioxide nanoparticles for 18 hours (D) or to 10μg/ml copper oxide (E).
Table 2.
Copper dissolution in cells.
Fig 2.
Damages to DNA on J774 cells after treatment with copper oxide nanoparticles, copper ion and titanium dioxide nanoparticles were evaluated using the alkaline version of the comet assay, taking into account both single and double strand breaks. The results are expressed in percentage of DNA in the tail. Butylhydroperoxide (BHP) was used as a positive control. Measurements were carried out in triplicate (except for BHP where n = 1). Statistical confidence (Student T-test) is indicated as follows: *: p ≤ 0.05; ** p ≤ 0.01; *** p≤ 0.001
Fig 3.
Proteomic analysis of total cell extracts by 2D electrophoresis.
Total cell extracts of J774 cells were separated by two-dimensional gel electrophoresis. The first dimensions covered a 4–8 pH range and the second dimension a 15–200 kDa range. Total cellular proteins (150 μg) were loaded on the first dimension gel. A: gel obtained from control cells. B: gel obtained from cells treated with titanium oxide (100μg/ml, 24 hours). C: gel obtained from cells treated with copper oxide (10μg/ml, 24 hours). D: gel obtained from cells treated with copper oxide (125μM, 24 hours). The arrows point to spots that show reproducible and statistically significant changes between the control and NP-treated cells (p≤ 0.05). Spot numbering according to Table 3.
Table 3.
Differentially-expressed proteins identified in the proteomic screen.
Fig 4.
Phagocytic activity and cytokine production of J774 cells upon treatment with particles or copper ion.
The phagocytic activity index of the cells is indicated. The results are expressed in percentage of the activity of control cells at 37°C. Negative control: proportion of fluorescent cells when the experiment is carried out without fluorescent beads. Measurements were carried out in triplicate. Statistical confidence (Student T-test) is indicated as follows: *: p ≤ 0.05; ** p ≤ 0.01; *** p≤ 0.001. Grey bar: phagocytic index of cells treated with PVP only (4μg/ml). In panels B and C, the production of respectively IL6 and TNF alpha was assessed. Cells were pretreated for 8 hours with copper ion or with the indicated particles at the indicated concentrations. LPS was then added or not (1μg/ml) and the cells left for an additional 18 hours prior to supernatant harvesting and cytokine measurement. Black bars: cytokine production without LPS stimulation. White bars: cytokine production with LPS stimulation. Grey bars: cytokine production with LPS stimulation and treatment with PVP only (2μg/ml light grey, 4μg/ml dark grey). Hatched bar: cytokine production upon treatment with PVP only (4μg/ml) and without LPS stimulation.
Fig 5.
Importance of glutathione in cell survival after treatment with copper.
In panel A, the intracellular free glutathione concentration was measured by the monochlorobimane conjugation assay after a 24 hour treatment with copper oxide nanoparticles or copper ion or diethylmaleate (DEM). The results are expressed in % of positive cells compared to the control population. DEM adds to the SH group of glutathione and is used as a control for GSH depletion. In panel B, J774 cells were first treated with 100μM buthionine sulfoximine for 6 hours (black bars) or with vehicle (white bars) Copper oxide nanoparticles or copper ion or titanium dioxide nanoparticles were then added at the indicated concentrations for the remaining 18 hours prior to viability measurement. Grey bar: cells treated with buthionine sulfoximine and PVP (4μg/ml). Experiments were carried out in quadruplicate. Statistical confidence (Student T-test) is indicated as follows: *: p ≤ 0.05; ** p ≤ 0.01; *** p≤ 0.001.
Fig 6.
Importance of heme oxygenase induction in cell survival after treatment with copper.
J774 cells were first treated with 2μM lovastatin for 6 hours (black bars) or with vehicle (white bars). Copper oxide nanoparticles were then added at the indicated concentrations for the remaining 18 hours prior to viability measurement. Experiments were carried out in quadruplicate. Statistical confidence (Student T-test) is indicated as follows: *: p ≤ 0.05; ** p ≤ 0.01; *** p≤ 0.001.
Fig 7.
The NO production was evaluated after treatment with particles or copper ion. Black bars, after further stimulation with 1μg/ml of lipopolysaccharide (LPS). White bars, without stimulation with LPS. Hatched bar: stimulation with PVP (4 μg/ml) and LPS. The results are expressed in percentage of the activity of control cells after LPS stimulation. Both measurements were carried out in triplicate on independent cultures and the statistical confidence in the Student T-test is indicated as follows: *: p ≤ 0.05; ** p ≤ 0.01; *** p≤ 0.001.
Fig 8.
Interplay between DOPA and copper in cell survival.
J774 cells were first treated with 100μM DOPA for 6 hours (black bars) or with vehicle (white bars). Copper oxide nanoparticles or copper ion or titanium dioxide nanoparticles were then added at the indicated concentrations for the remaining 18 hours prior to viability measurement. Hatched bar: cells treated with 100μM oxidized DOPA. Grey bar cells treated with PVP only (4μg/ml). Experiments were carried out in quadruplicate. Statistical confidence (Student T-test) is indicated as follows: *: p ≤ 0.05; ** p ≤ 0.01; *** p≤ 0.0011.