Modification of radiation-induced DNA double strand break repair pathways by chemicals extracted from Podophyllum hexandrum: an in vitro study in human blood leukocytes.

Radiation exposure is a serious threat to biomolecules, particularly DNA, proteins and lipids. Various exogenous substances have been reported to protect these biomolecules. In this study we explored the effect of pre-treatment with G-002M, a mixture of three active derivatives isolated from the rhizomes of Podophyllum hexandrum, on DNA damage response in irradiated human blood leukocytes. Blood was collected from healthy male volunteers, preincubated with G-002M and then irradiated with various doses of radiation. Samples were analyzed using flow cytometry to quantify DNA double strand break (DSB) biomarkers including γ-H2AX, P53BP1 and levels of ligase IV. Blood samples were irradiated in vitro and processed to determine time and dose-dependent kinetics. Semiquantitative RT-PCR was performed at various time points to measure gene expression of DNA-PKcs, Ku80, ATM, and 53BP1; each of these genes is involved in DNA repair signaling. Pre-treatment of blood with G-002M resulted in reduction of γ-H2AX and P53BP1 biomarkers levels and elevated ligase IV levels relative to non-G-002M-treated irradiated cells. These results confirm suppression in radiation-induced DNA DSBs. Samples pre-treated with G-002M and then irradiated also showed significant up-regulation of DNA-PKcs and Ku80 and downregulation of ATM and 53BP1 gene expressions, suggesting that G-002M plays a protective role against DNA damage. The protective effect of G-002M may be due to its ability to scavange radiation-induced free radicals or assist in DNA repair. Further studies are needed to decipher the role of G-002M on signaling molecules involved in radiation-induced DNA damage repair pathways.

Podophyllum hexandrum-Mediated Survival Protection and Restoration of Other Cellular Injuries in Lethally Irradiated Mice.

This study aims at the development of a safe and effective formulation to counter the effects of lethal irradiation. The sub-fraction (G-001M), prepared from Podophyllum hexandrum has rendered high degree of survival (>90%) at a dose of 6 mg kg(-1) body weight (intramuscular) in lethally irradiated mice. Therapeutic dose of G-001M, at about 20 times lower concentration than its LD(100), has revealed a DRF of 1.62. Comet assay studies in peripheral blood leukocytes have reflected that, treatment of G-001M before irradiation has significantly reduced DNA tail length (P < .001) and DNA damage score (P < .001), as compared to radiation-only group. Spleen cell counts in irradiated animals had declined drastically at the very first day of exposure, and the fall continued till the 5th day (P < .001). In the treated irradiated groups, there was a steep reduction in the counts initially, but this phase did not prolong. More than 60% decline in thymocytes of irradiated group animals was registered at 5 h of irradiation when compared with controls, and the fall progressed further downwards with the similar pace till 5th day of exposure (P < .001). At later intervals, thymus was found fully regressed. In G-001M pre-treated irradiated groups also, thymocytes decreased till the 5th day but thereafter rejuvenated and within 30 days of treatment the values were close to normal. Current studies have explicitly indicated that, G-001M in very small doses has not only rendered high survivability in lethally irradiated mice, but also protected their cellular DNA, besides supporting fast replenishment of the immune system.

Whole body protection to lethally irradiated mice by oral administration of semipurified fraction of Podophyllum hexandrum and post irradiation treatment of Picrorhiza kurroa.

OBJECTIVE: to evaluate the radioprotective potential of alcoholic fraction of Podophyllum hexandrum rhizomes (REC-2001) individually as well as in combination with Picrorhiza kurroa administered orally in lethally irradiated Swiss albino mice. METHODS: The study was divided into different treatment groups. Whole body survival was observed upto 30 days in all the treatment groups. Besides survival, toxicity of REC-2001 was also evaluated. All the groups were studied for spleen endogenous colony forming units (CFUs), plasma antioxidant potential and hematological variables, using standard techniques. RESULTS: Animals in radiation alone group died with in 12 days of exposure. Single dose of REC-2001 which did not bring any toxic manifestation/mortality (MTD) was found to be 155 mg/kg b.w. On administration of 250 mg/kg b.w. (single dose) 50% of the animals died (LD50), while a dose of 350 mg/kg b.w. of REC-2001 brought 100% death. Oral administration of single dose of REC-2001 (25 mg /kg b.w. -1h) prior to irradiation (10 Gy) was observed rendering up to 48% protection. Survival enhanced to the level of 55% when the animals had pre- treatment of REC-2001 (25 mg /kg b.w. -1h) followed by irradiation (10 Gy) and post treatment with a single dose of Picrorhiza kurroa rhizome extract (pkre, 8 mg/kg b.w.+1h). Radiation induced plasma antioxidant status was significantly (P < 0.02) countered by REC-2001 administration. Post treatment of pkre elevated CFU counts (P < 0.05). Total leukocytes count and hemoglobin content in REC-2001 pretreated and pkre post treated group approached normal limits within 30 days of the study. CONCLUSION: REC-2001 in combination with pkre holds promise for further studies to achieve radioprotection against lethal radiation by oral administration.