In vitro studies on radioprotective efficacy of silymarin against γ-irradiation.
Abstract Purpose: Silymarin has been widely exploited for its hepatoprotective activities. This study aimed to evaluate the protective efficacy of silymarin against γ-radiation.


METHODS
The radioprotective properties of silymarin were studied using different assays. Cytotoxicity of silymarin on Human embryonic kidney (HEK) cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Protective efficacy against γ-radiation was assessed by studying reduction in micronuclei frequency and free radical generation using 2',7'-dichlorodihydroflurescin diacetate (H2DCFDA). Radiation-induced apoptosis was estimated by Annexin V-PI (propidium iodide) analysis and cell cycle analysis. γ-radiation induced changes in mitochondrial membrane potential (MMP) and DNA damage was estimated employing flow-cytometry and comet assay respectively.


RESULTS
MTT assay and Annexin V-PI studies showed that pre-incubation of HEK cells with silymarin protected them from γ-irradiation. Significant reduction in apoptosis (76.36%) was observed. Silymarin also decreased the percentage of radiation-induced micronuclei (> 69%) (p < 0.05 ). Measurement of intracellular reactive oxygen species (ROS) by H2DCFDA revealed a reduction in ROS (21%) at 0.5 h. Cell cycle analysis revealed G1 block in the unirradiated control, which declined in the silymarin pretreated irradiated group (0.5 h). Silymarin treatment resulted in a significant increase in MMP (2 h) against the radiation control. Moreover, the presence of silymarin during irradiation significantly decreased the DNA damage (as measured by comet assay).


CONCLUSIONS
Protection against radiation-induced cell-death and DNA damage by silymarin could be attributed to a reduction in ROS induced by γ-radiation. In vitro experiments on HEK cells explicitly prove that silymarin is a promising, effective and safe radiation countermeasure agent.
Department of Radiation Biotechnology, Radiation Biosciences Division and Division of CBRN Defence, Institute of Nuclear Medicine and Allied Sciences, Brig SK Mazumdar Marg, Delhi, India.
Manish
M
Adhikari
Atlar
A
Dhaker
Jawahar
J
Adhikari
Veselin
V
Ivanov
Vijay
V
Singh
Raman
R
Chawla
Raj
R
Kumar
Rakesh
R
Sharma
Yana
Y
Karamalakova
Veselina
V
Gadjeva
Rajesh
R
Arora
D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D003114:Colony-Forming Units Assay; D020552:Comet Assay; D004249:DNA Damage; D005720:Gamma Rays / Q000009:adverse effects*; D057809:HEK293 Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000528:radiation effects; D015162:Micronucleus Tests; D011837:Radiation-Protective Agents / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D012838:Silymarin / Q000494:pharmacology*
D011837:Radiation-Protective Agents; D017382:Reactive Oxygen Species; D012838:Silymarin