Gamma-Linolenic Acid (GLA) Protects against Ionizing Radiation-Induced Damage: An In Vitro and In Vivo Study

Abstract

Radiation is pro-inflammatory in nature in view of its ability to induce the generation of reactive oxygen species (ROS), cytokines, chemokines, and growth factors with associated inflammatory cells. Cells are efficient in repairing radiation-induced DNA damage; however, exactly how this happens is not clear. In the present study, GLA reduced DNA damage (as evidenced by micronuclei formation) and enhanced metabolic viability, which led to an increase in the number of surviving RAW 264.7 cells in vitro by reducing ROS generation, and restoring the activities of desaturases, COX-1, COX-2, and 5-LOX enzymes, TNF-α/TGF-β, NF-kB/IkB, and Bcl-2/Bax ratios, and iNOS, AIM-2, and caspases 1 and 3, to near normal. These in vitro beneficial actions were confirmed by in vivo studies, which revealed that the survival of female C57BL/6J mice exposed to lethal radiation (survival~20%) is significantly enhanced (to ~80%) by GLA treatment by restoring altered levels of duodenal HMGB1, IL-6, TNF-α, and IL-10 concentrations, as well as the expression of NF-kB, IkB, Bcl-2, Bax, delta-6-desaturase, COX-2, and 5-LOX genes, and pro- and anti-oxidant enzymes (SOD, catalase, glutathione), to near normal. These in vitro and in vivo studies suggest that GLA protects cells/tissues from lethal doses of radiation by producing appropriate changes in inflammation and its resolution in a timely fashion.

Keywords: cytokines; inflammation; lipoxin A4; prostaglandins; radiation; survival.

Figure 1

(A) Metabolism of essential fatty acids. (B) Metabolism of arachidonic acid.

Figure 1

(A) Metabolism of essential fatty acids. (B) Metabolism of arachidonic acid.

Figure 2

(A). Effect of ionizing radiation on the viability of RAW 264.7 cells as assessed by trypan blue dye exclusion method. Values (n = 6) expressed as mean ± SEM. ^a (p < 0.05) represents significant decrease in %Survival when compared with control at 24 h. ^b (p < 0.05) represents significant decrease in %Survival when compared with control at 48 h. (B). Study of effect of PUFAs, GLA, AA, EPA, and DHA, on the viability of RAW 264.7 cells by MTT assay. Values (n = 6) expressed as mean ± SEM. ^a (p < 0.05) represents significant decrease in %Survival when compared with control at 24 h. GLA, gamma-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.

Figure 3

Effect of various doses of different PUFAs on radiation-induced cytotoxic action on RAW 264.7 cells in vitro at 24 and 48 h post-radiation, as assessed by MTT assay. Doses of PUFAs used were 250 ng/mL, 500 ng/mL, and 1 μg/mL). (A) = GLA; (B) = AA; (C) = EPA; and (D) = DHA; (E) = Effect of various PUFAs (GLA, AA, EPA, and DHA) on the growth kinetics of RAW 264.7 cells following irradiation. All values (n = 6) are expressed as mean ± SEM. Significance (p < 0.05) represented by ^a, ^b when compared with control and irradiation, respectively. C, control; GLA, gamma-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; IR, irradiation (2 Gy).

Figure 4

Effect of various PUFAs on radiation-induced reactive oxygen species (ROS) in RAW 264.7 cells using DCFH₂DA. Values (n = 6) expressed as mean ± SEM. Significance (p < 0.001) represented by ^a, ^b when compared with control, PUFAs, and irradiation, respectively. C, control; GLA, gamma-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; IR, irradiation.

Figure 5

Estimation of DNA damage in RAW 264.7 cells through micronuclei estimation using DAPI (A). Values (n = 6) expressed as mean ± SEM. Significance (p <0.01) represented by ^a, ^b when compared with control and irradiation, respectively. C, control; GLA, gamma-linolenic acid; IR, irradiation (2 Gy). Pictorial representation of RAW 264.7 cells stained with DAPI under fluorescence microscope (B), with nucleus visible at 100× resolution. A. normal nucleus, B. nucleus with single micronucleus, C. nucleus with two micronuclei, D. nucleus with multiple micronuclei, and E. nucleus of apoptotic cell.

Figure 6

Effect of GLA and irradiation on genes of EFA (essential fatty acid) metabolic pathways (desaturases, COX-1, COX-2, and 5-LOX) in RAW 264.7 cells at 24 and 48 h post-irradiation (A,B). Values (n = 3) expressed as mean ± SEM. ^a, ^b, ^c Significant (p < 0.01) when compared with control, GLA, and irradiation, respectively. 1, C; 2, GLA; 3, IR; and 4, GLA + IR. C, control; GLA, gamma-linolenic acid; IR, irradiation (2 Gy).

Figure 7

Effect of GLA and IR on genes regulating the inflammatory pathway (TNF, TGF, NF-kB, IkB, iNOS) in RAW 264.7 cells at 24 and 48 h post-irradiation (A,B). Values (n = 3) expressed as mean ± SEM. ^a, ^b, ^c Significant (p < 0.01) when compared with control, GLA, and irradiation, respectively. 1, C; 2, GLA; 3, IR; and 4, GLA + IR. C, control; GLA, gamma-linolenic acid; IR, irradiation (2 Gy).

Figure 8

Effect of GLA and irradiation on genes associated with apoptotic pathways in RAW 264.7 cells at 24 and 48 h post-irradiation (A,B). Values (n = 3) expressed as mean ± SEM. ^a, ^b, ^c Significant (p < 0.01) when compared with control, GLA, and irradiation, respectively. 1, C; 2, GLA; 3, IR; and 4, GLA + IR. C, control; GLA, gamma-linolenic acid; IR, irradiation (2 Gy).

Figure 9

(A). Protocol of the in vivo study with radiation and GLA treatment. Mice were pre-acclimatized and treated with GLA (10, 50, and 100 μg/kg body weight at 48, 24, and 1 h prior to irradiation (7.5 Gy) at a dose rate of 1 Gy/min. (B). Protocol for mechanistic studies. Mice were pre-acclimatized and treated with GLA (100 μg/kg body weight at 48, 24, and 1 h prior to irradiation (7.5 Gy) at a dose rate of 1 Gy/min. Samples were collected from mice on days 1, 3, 7, and 14 for further analysis.

Figure 10

Effect of various PUFAS and LXA4 on radiation-induced mortality in female mice. Mice were pre-treated with GLA (10, 50, and 100 μg/kg) at 48, 24, and 1 h prior to irradiation and were subjected to total-body irradiation of 7.5 Gy (at 1 Gy/min). Values (n = 12) expressed as %Survival (A) and variation in body weight (B). Mice were pre-treated with 100 μg/kg PUFA (DGLA, AA, EPA, and DHA) at 48, 24, and 1 h prior to irradiation, and were subjected to total-body irradiation of 7.5 Gy (at 1 Gy/min). Values (n = 6) expressed as %Survival (C) and variation in body weight (D). Mice were pre-treated with 10, 50, 100 ng/kg of LXA4 at 48, 24, and 1 h prior to irradiation, and were subjected to total-body irradiation of 7.5 Gy (at 1 Gy/min). Values (n = 6) expressed aISurvival (E). %Survival with LXA4 50 ng/kg post-treatment (F) on day 7, 14, 21, and 28, respectively. DGLA, di-homo-gamma-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; IR, irradiation; LXA4, lipoxin A4.

Figure 10

Effect of various PUFAS and LXA4 on radiation-induced mortality in female mice. Mice were pre-treated with GLA (10, 50, and 100 μg/kg) at 48, 24, and 1 h prior to irradiation and were subjected to total-body irradiation of 7.5 Gy (at 1 Gy/min). Values (n = 12) expressed as %Survival (A) and variation in body weight (B). Mice were pre-treated with 100 μg/kg PUFA (DGLA, AA, EPA, and DHA) at 48, 24, and 1 h prior to irradiation, and were subjected to total-body irradiation of 7.5 Gy (at 1 Gy/min). Values (n = 6) expressed as %Survival (C) and variation in body weight (D). Mice were pre-treated with 10, 50, 100 ng/kg of LXA4 at 48, 24, and 1 h prior to irradiation, and were subjected to total-body irradiation of 7.5 Gy (at 1 Gy/min). Values (n = 6) expressed aISurvival (E). %Survival with LXA4 50 ng/kg post-treatment (F) on day 7, 14, 21, and 28, respectively. DGLA, di-homo-gamma-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; IR, irradiation; LXA4, lipoxin A4.

Figure 10

Effect of various PUFAS and LXA4 on radiation-induced mortality in female mice. Mice were pre-treated with GLA (10, 50, and 100 μg/kg) at 48, 24, and 1 h prior to irradiation and were subjected to total-body irradiation of 7.5 Gy (at 1 Gy/min). Values (n = 12) expressed as %Survival (A) and variation in body weight (B). Mice were pre-treated with 100 μg/kg PUFA (DGLA, AA, EPA, and DHA) at 48, 24, and 1 h prior to irradiation, and were subjected to total-body irradiation of 7.5 Gy (at 1 Gy/min). Values (n = 6) expressed as %Survival (C) and variation in body weight (D). Mice were pre-treated with 10, 50, 100 ng/kg of LXA4 at 48, 24, and 1 h prior to irradiation, and were subjected to total-body irradiation of 7.5 Gy (at 1 Gy/min). Values (n = 6) expressed aISurvival (E). %Survival with LXA4 50 ng/kg post-treatment (F) on day 7, 14, 21, and 28, respectively. DGLA, di-homo-gamma-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; IR, irradiation; LXA4, lipoxin A4.

Figure 11

Estimation of duodenal concentrations of cytokines HMGB1 (A), TNF-α (B), IL-6 (C), and IL-10 (D) by ELISA method. Mice were pre-treated with 100 μg/kg GLA at 48, 24, and 1 h prior to irradiation, and were subjected to total-body irradiation of 7.5 Gy (at 1 Gy/min). Duodenum samples were obtained on day 1, day 3, day 7, and day 14 post-irradiation. All values are expressed as mean ± SEM (n = 3). Statistical significance was calculated using t-tests, and p-values < 0.05 are represented by ^a, ^b, ^c when compared to control, GLA, and irradiation, respectively. C, control; GLA, gamma-linolenic acid; IR, irradiation (7.5 Gy).

Figure 12

Effect of GLA and irradiation on genes associated with PUFA metabolism in duodenum tissue on day 1, day 3, day 7, and day 14. Values (n = 3) expressed as mean ± SEM. ^a, ^b, ^c Significant (p < 0.01) when compared with control, GLA, and irradiation, respectively. 1, C; 2, GLA; 3, IR; and 4, GLA + IR. C, control; GLA, gamma-linolenic acid; IR, irradiation (7.5 Gy).

Figure 13

Estimation of PUFA metabolite PG E₂ (A), LT E₄ (B), and LXA4 (C) levels by ELISA method. Mice were pre-treated with 100 μg/kg GLA at 48, 24, and 1 h prior to irradiation, and were subjected to total-body irradiation of 7.5 Gy (at 1 Gy/min). Duodenum samples were obtained on day 1, day 3, day 7, and day 14 post-irradiation. All the values are expressed as mean ± SEM (n = 3). Statistical significance was calculated using t-tests, and p-values < 0.001 are represented by ^a, ^b, ^c when compared to control, GLA, and IR, respectively. C, control; GLA, gamma-linolenic acid; IR, irradiation (7.5 Gy).

Figure 14

Effect of GLA and radiation on the expression of NF-kB/IkB in duodenal tissue of mice on days 1, 3, 7, and 14. Values (n = 3) are expressed as mean ± SEM. ^a, ^b, ^c Significant (p < 0.01) when compared with control, GLA, and IR, respectively. 1, C; 2, GLA; 3, IR; and 4, GLA + IR. C, control; GLA, gamma-linolenic acid; IR, irradiation (7.5 Gy).

Figure 15

Effect of GLA and irradiation on genes associated with apoptotic pathways (BCL-2 and Bax) in duodenum tissue on day 1, day 3, day 7, and day 14. Values (n = 3) expressed as mean ± SEM. ^a, ^b, ^c Significant (p < 0.01) when compared with control, GLA, and IR, respectively. 1, C; 2, GLA; 3, IR; and 4, GLA + IR. C, control; GLA, gamma-linolenic acid; IR, irradiation (7.5 Gy).

Figure 16

Effect of GLA and irradiation on protein expression (Bcl-2, Bax, Nf-κB, IκB, 5-LOX, and COX-2) in duodenum tissue on day 1 and day 14 post-irradiation by Western blotting. GAPDH was used as a loading control. Values (n = 3) expressed as mean ± SEM. ^a, ^b, ^c Significant (p < 0.05) when compared with control, GLA, and irradiation, respectively. 1, C; 2, GLA; 3, IR; and 4, GLA + IR. C, control; GLA, gamma-linolenic acid; IR, irradiation (7.5 Gy).

Figure 17

Histology of duodenum of surviving mice (A). Control, non-irradiated mice; GLA, mice treated with 100 μg/kg GLA; IR, irradiated mice (7.5 Gy); GLA + IR, GLA-pre-treated and irradiated mice. On day 1, day 3, day 7, and day 14, cells were stained with hematoxylin and eosin (10× magnification). Villi length and width (in μm) were measured (B). Values (n = 3) expressed as mean ± SEM. ^a, ^b, ^c Significant (p < 0.05) when compared with control, GLA, and irradiation, respectively. GLA, gamma-linolenic acid; IR, irradiation (7.5 Gy).

Figure 18

Scheme showing potential mechanisms involved in the beneficial actions of GLA and its products (PGE2/LTE4/LXA4 derived from AA and AA, in turn, is derived from GLA by the action of delta-5-desaturase).