Electrochemical evaluation of proton beam radiation effect on the B16 cell culture

Abstract

The interaction of radiation with matter takes place through energy transfer and is accomplished especially by ionized atoms or molecules. The effect of radiation on biological systems involves multiple physical, chemical and biological steps. Direct effects result in a large number of reactive oxygen species (ROS) within and outside and inside of the cells as well, which are responsible for oxidative stress. Indirect effects are defined as alteration of normal biological processes and cellular components (DNA, protein, lipids, etc.) caused by the reactive oxygen species directly induced by radiation. In this work, a classical design of an electrochemical (EC) three-electrodes system was employed for analyzing the effects of proton beam radiation on melanoma B16 cell line. In order to investigate the effect of proton radiation on the B16 cells, the cells were grown on the EC surface and irradiated. After optimization of the experimental set-up and dosimetry, the radiobiological experiments were performed at doses ranging between 0 and 2 Gy and the effect of proton beam irradiation on the cells was evaluated by the means of cyclic voltammetry and measuring the open circuit potential between working and reference electrodes.

Figure 1

Fluorescence microscopy images of B16 cells grown 48 h on (A) biocompatible glass and (B) gold surface; scale bars: 100 µm.

Figure 2

Scanning electron microscopy images of B16 cells grown 24 h on gold surface obtained at different magnifications and using signals from different detectors (SE2 and InLens): (A) 300×; (B) and (C) 500×.

Figure 3

Normalized values to control cells (cells not irradiated) of (A) mitochondrial superoxide and (B) ROS recorded for B16 cells at 2, 4 and 24 h after proton irradiation at dose rate of 1 Gy/min. Data are reported as mean ± SD. *p < 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

Figure 4

Cyclic voltammograms recorded at gold electrode for (grey line) culture medium and (black line) B16 cells (A) before and (B) after proton irradiation at a dose of 1 Gy.

Figure 5

Cyclic voltammograms results recorded at gold working electrodes after 30 min incubation with different concentrations of H₂O₂ for (A) culture medium with the corresponding (B) plot of oxidation current vs. H₂O₂ concentration and (C) B16 cells grown at the electrode surfaces.

Figure 6

(A) Fluorescence microscopy and (B) FESEM images obtained for B16 cells at gold surface after interaction with H₂O₂; scale bars: 100 µm.

Figure 7

OCP recorded at gold electrodes for culture medium and B16 cells before and after incubation with H₂O₂ and proton irradiation (1 Gy at 1 Gy/min).

Figure 8

Main reactive species generated by water radiolysis.

Figure 9

The workflow chart of the experimental steps.