Palmitoyl ascorbic acid 2-glucoside has the potential to protect mammalian cells from high-LET carbon-ion radiation

Abstract

DMSO, glycerol, and ascorbic acid (AA) are used in pharmaceuticals and known to display radioprotective effects. The present study investigates radioprotective properties of novel glyceryl glucoside, ascorbic acid 2-glucoside, glyceryl ascorbate, and palmitoyl ascorbic acid 2-glucoside (PA). Gamma-rays or high-LET carbon-ions were irradiated in the presence of tested chemicals. Lambda DNA damage, cell survival, and micronuclei formation of CHO cells were analyzed to evaluate radioprotective properties. Radiation-induced Lambda DNA damage was reduced with chemical pre-treatment in a concentration-dependent manner. This confirmed tested chemicals were radical scavengers. For gamma-irradiation, enhanced cell survival and reduction of micronuclei formation were observed for all chemicals. For carbon-ion irradiation, DMSO, glycerol, and PA displayed radioprotection for cell survival. Based on cell survival curves, protection levels by PA were confirmed and comparable between gamma-rays and high-LET carbon-ions. Micronuclei formation was only decreased with AA and a high concentration of glycerol treatment, and not decreased with PA treatment. This suggests that mechanisms of protection against high-LET carbon-ions by PA can differ from normal radical scavenging effects that protect DNA from damage.

Figure 1

Chemical structures of (A) dimethyl sulfoxide, (B) glycerol, (C) ascorbic acid, (D) glyceryl glucoside, (E) ascorbic acid 2-glucoside, (F) glyceryl ascorbate, and (G) palmitoyl ascorbic acid 2-glucoside.

Figure 2

Gel electrophoresis of Lambda DNA. (A) Radiation dose-dependent DNA damage. (B left, and C left) Effects of tested chemicals on DNA without irradiation. (B right) Radioprotective effects of pre-treated DMSO, glycerol, and GG. Triangles indicate concentration gradient. Lane A: control water without irradiation; lane B and C water with irradiation; lane D–G DMSO at 14 mM, 1.4 mM, 0.14 mM, and 0.014 mM; lane H-K: glycerol at 13.7 mM, 1.37 mM, 0.137 mM, and 0.0137 mM; lane L–O: GG at 4 mM, 0.4 mM, 0.04 mM, and 0.004 mM. (C right) Radioprotective effects of AA, 2G, GA, and PA. Triangles indicate concentration gradient. Lane A: control water without irradiation; lane B and C: water with irradiation; lane D–F AA at 0.56 mM, 0.056 mM, and 0.0056 mM; lane G–I: 2G at 0.3 mM, 0.03 mM, and 0.003 mM; lane J–L: GA at 0.4 mM, 0.04 mM, and 0.004 mM; lane M–O: PA at 0.17 mM, 0.017 mM, and 0.0017 mM. (D–G) Fraction of intact irradiated Lambda DNA with chemicals. Error bars indicate standard error of the means. At least three independent experiments were carried out. The cropped gel images were used in the figure, and full-length gel images are presented in Supplementary Fig. S1.

Figure 3

DMSO, glycerol, GG, AA and its derivatives on radioprotective effects for cell survival of CHO cells. (A) Under 12 Gy gamma-ray irradiation, cells irradiated without chemicals induced a 0.0086 cell survival fraction. (B) Under 6 Gy high-LET carbon-ion irradiation, cells irradiated without chemicals stimulated a 0.018 cell survival rate. * indicate statistical significance (P < 0.05). Error bars indicate standard error of the means. At least three independent experiments were carried out.

Figure 4

Radioprotective effects of DMSO, glycerol, GG, AA, 2G, GA, and PA by micronuclei assay. (A) On the left is a normal binucleated cell. On the right is binucleated cell with micronuclei formation indicated by red arrows. (B) Micronuclei formation per binucleated CHO cell with pre-treated chemicals after gamma-ray irradiation. Horizontal dashed line indicates non-irradiated control (less than 0.1 micronuclei per binucleated cells for each condition). Horizontal dotted line indicates the control (0.91 micronuclei per binucleated cells). (C) Micronuclei formation after high-LET carbon-ion irradiation. Horizontal dashed line indicates non-irradiated control (less than 0.1 micronuclei per binucleated cells for each condition). Horizontal dotted line indicates the control (0.93 micronuclei per binucleated cells). Error bars indicate standard error of the means. At least three independent experiments were carried out.

Figure 5

Cell survival curves with and without 0.2 mM palmitoyl ascorbic acid 2-glucoside (PA). (A) gamma-ray irradiation. (B) Carbon-ions irradiation. Closed circles indicate control; open circles indicate PA. Curves were fitted to liner-quadratic model. Error bars indicate standard error of the means. At least three independent experiments were carried out.