Fig 1.
Optical images of control and exposed cells to EMFs at 1.8 GHz.
Bright field optical images of controls (a, c, e, g) and respective 1 h, 5 h, 12 h and 20 h exposed (b, d, f, h) samples to the electric field strength of 200 V/m of microwave of 1.8 Ghz. The larger structures correspond to adherent PBLM cells, whereas the smaller ones are platelets which cannot be completely separated by the density gradient procedure to isolate PBLM cells. The scale bar corresponds to 15 μm. The images are representative of 3–4 independent experiments (i.e. biological replicates corresponding to samples from different donors for each condition), yielding comparable results. The plots at the bottom shows the percentage of cells whose size appeared altered after 1, 5, 12 and 20 h exposure (i) and the average number of platelets stuck on each single cell as a function of exposure time (m). For each sample, about 300 cells were analyzed from 20–30 randomly selected optical fields. The bar values are means ± SEM of 3–4 independent experiments.
Fig 2.
Optical images of stained control and exposed lympho-monocyte cells to EMFs at 1.8 GHz.
Representative May-Grunwald-Giemsa (MGG) images of controls (a, c, e) and respective 5 h, 12 h and 20 h exposed (b, d, f) samples to the electric field strength of 200 V/m of microwave at 1.8 GHz. The scale bar corresponds to 7 μm. Digital magnifications of selected cells are shown along with their rendering obtained by the software ImageJ 1.49 (http://imagej.nih.gov/ij) with the tool LUT-ICA. The plot g) shows the percentage of MGG stained cells whose size appeared altered after 1, 5, 12 and 20 h exposure. For each sample, about 300 cells were analyzed from 20–30 randomly selected optical fields. The bar values are means ± SEM. The images are representative of 3–4 independent experiments (i.e. biological replicates corresponding to samples from different donors for each condition), yielding comparable results.
Fig 3.
Average Raman spectra of control and exposed PBLM cells to EMFs at 1.8 GHz.
Raman spectra of control (continuous black line) and exposed (continuous red line) PBLM cells which were extracted from whole blood samples. The irradiated PBLM cells were exposed to the electric field strength of 200 V/m of microwave at 1.8 GHz inside a reverberation chamber for 1 (a), 5 (b), 12 (c) and 20 (d) h. The labeled Raman features are related to DNA (at 785, 1092 and 1578 cm-1) and protein (at 1003 and 1615 cm-1) content (see text). Each spectra is the average from 20 different cells under each condition and are representative of 3–4 independent experiments (i.e. biological replicates corresponding to samples from different donors for each condition). On the right hand side of each Raman spectrum is shown the corresponding comparison between mean values of the I785/I1003 (dots), I1092/I1003 (squares) and I1578/I1615 (triangles) intensity ratios of Raman peaks of control and 1 (e), 5 (f), 12 (g) and 20 (h) h EMFs exposed cells. The values are means ± SEM of 3–4 independent experiments (i.e. biological replicates corresponding to samples from different donors for each condition).
Table 1.
Attribution of Raman peaks.
Table 2.
t-test of area ratios of Raman peaks in exposed with respect to control samples.
Fig 4.
Area ratios of Raman peaks from spectra of control and exposed PBLM cells to EMFs at 1.8 GHz.
Difference of I785/I1003 (a), I1092/I1003 (b) and I1578/I1615 (c) area ratios of Raman peaks of the control with respect to the exposed lympho-monocyte samples for different exposure time of Fig 3. The values are means ± SEM of 3–4 independent experiments (i.e. biological replicates corresponding to samples from different donors for each condition).
Fig 5.
Mitochondrial respiratory analysis in intact PBLM cells.
PBLM cells isolated from human subjects were tested for oxygen consumption rates (OCRs) by high resolution respirometry as described under Materials and Methods. Respiratory activity expressed as nmol O2/min/106 PBLM cells. OCRr and OCRo are activities attained before and after addition of oligomycin and were corrected for rotenone-insensitive respiration. The bar values are means ± SEM of 3 independent experiment under each condition; t = 0, activity measured in freshly isolated PBLM cells; PBLM cells incubated for the indicated h in the absence (CTRL) or in the presence of EF (strength of 200 V/m).
Fig 6.
Mitochondrial respiratory analysis in intact PBLM cells.
(A) OCRr of EMFs-exposed PBMN cells normalized to the untreated cognate control samples incubated for the same time and under identical conditions. The bar values are means ± SEM of 3 independent experiment under each condition and were calculated from the absolute values shown as averages in Fig 5. (B) OCRATP of EMFs-exposed PBLM cells normalized to the untreated cognate control samples incubated for the same time and under identical conditions. The bar values are means ± SEM of 3 independent experiment under each condition and were calculated as OCRr-OCRo from the absolute values shown as averages in Fig 5. (C) OCRo was divided by the OCRr of the same assay and normalized to the relative CTRL value. The bar values are means ± SEM of 3 independent experiment under each condition and were calculated as from the absolute values shown as averages in Fig 5.
Fig 7.
Confocal microscopy analysis of ΔΨm in EMFs-exposed PBLM cells.
PBLM cells exposed to EMFs for the indicated time were incubated with TMRE and analyzed by confocal microscopy as detailed in Materials and Methods. (A) Representative images are shown with enlargements from each optical fields of selected nucleated cells and platelets. (B) Quantitative analysis of the TMRE-related fluorescence intensity/cell. The values are means ±SEM of 3 independent biological replicates; for each biological replicate the digitalized mean fluorescence/cell (ranging from 0 to 255) was averaged from 50 to 100 single cell analysis from at least three different optical fields for each condition. Image analysis was performed by ImageJ using the “particle analysis” tool on threshold adjusted optical fields. P < 0.05 of “12 h” vs all the other conditions.
Fig 8.
Confocal microscopy analysis of mitochondrial content in EMFs-exposed PBLM cells.
PBLM cells exposed to EMFs for the indicated time were incubated with MitoTracker® Green and analyzed by confocal microscopy as detailed in Materials and Methods. (A) Representative images of cell subsets pinpointed on the basis of the size; L-PBLM and S-PBLM indicate large and small lympho-monocytes respectively. The picture below each microscopy image was attained by rendering of the corresponding fluorescence signal using ImageJ after removal of the back-ground. (B) Quantitative analysis of the MitoTracker®-related fluorescence/cell; the top histogram shows the fluorescence intensity/cell, the bottom histogram shows the elongation parameter of the fluorescent intracellular signal defined as the reciprocal of the circularity value. The bars are means ±SEM of 3 independent biological replicates; for each biological replicate the digitalized mean fluorescence/cell (ranging from 0 to 255) and the circularity of the signal was averaged from 50 to 100 single cell-analysis from at least three different optical fields for each condition. Image analysis was performed by ImageJ using the “particle analysis” tool on threshold adjusted optical fields.
Fig 9.
Measurement of mitochondrial markers in EMFs-exposed PBLM cells.
The activity of citrate synthase (A) and of cytochrome c oxidase (B) was measured spectrophotometrically as detailed in Materials and Methods. The activity of citrate synthase was normalized to the cell number whereas that of cytochrome c oxidase to the citrate synthase. The values are means ±SEM of 3 biological replicates. P < 0.05 vs CTRL.
Fig 10.
Confocal microscopy analysis of ROS production in EMFs-exposed PBLM cells.
PBLM cells exposed to EMFs for the indicated time were incubated with 2,7-dichlorofluorescin diacetate and analyzed by confocal microscopy as detailed in Materials and Methods. (A) Representative images are shown with enlargement of selected cells. (B) DCF-stained PBLMs were clustered as a function of the calculated area and the fluorescence intensity/cell averaged (from 50 to 100 single cell-analysis under each condition). The values are means ±SEM of 3 biological replicates. The black bars in the graph denote the size range of the two major cell subsets indicated as small and large size PBLM (S-PBLM and L-PBLM respectively). Image analysis was performed in threshold adjusted optical fields using the “particle analysis” tool of ImageJ. Conditions for statistical significant difference (i.e. P < 0.05): “a”, vs CTRL of the same size cluster; “b”, 12 h vs 5 h of the same size cluster; “c”, 20 h vs 12 h of the same size cluster; “d”, L-PBLM vs S-PBLM subjected to the same EMF-exposure times.