Modulation of radiation-induced cytotoxicity in U 87 cells by RH-3 (a preparation of Hippophae rhamnoides).

Background & objectives: Hippophae rhamnoides L. has been widely exploited for medicinal purposes and an extract of its whole berries coded as RH-3 has been found to render radioprotection. Effect of pre-irradiation treatment of up to 10 μg/ml RH-3 was studied in U 87 cells using MTT assay. This study aims at unraveling the mechanism of action of RH-3 in amelioration of radiation-induced cytotoxicity in vitro. Methods: Most effective doses selected were studied further for the elucidation of radiomodifying properties of RH-3, especially with respect to early and late events of apoptosis. Results: RH-3 at concentrations of 7.5 and 10 μg/ml (-15 min) were found most effective in protecting against 2 Gy induced cytotoxicity in terms of MTT reducing ability in U 87 cells. RH-3 was observed to mitigate radiation-induced cellular and mitochondrial free radicals. Mitochondrial membrane potential depletion (studied up to 12 h) was prevented by RH-3 pre-irradiation administration. It could also restore the level of antiapoptotic protein Bcl-2 at 24 and 48 h comparable to the control value. RH-3 also prevented radiation-induced increase in mitochondrial mass at 48 and 72 h post-treatment and the values were comparable to that of control cells. Annexin-V-FITC assay at 12 and 24 h time intervals indicated significant protection against radiation-induced apoptosis by RH-3 pre-irradiation treatment. Interpretation & conclusion: Our findings showed that probably RH-3 acts as an antioxidant preventing cellular and mitochondrial free radical generation that could contribute to its ability to inhibit radiationinduced apoptosis and cytotoxicity.

Protective effect of RH-3 with special reference to radiation induced micronuclei in mouse bone marrow.

Effect of pre-irradiation administration of different doses of RH-3, the herbal preparation of an Indian medicinal plant Hippophae rhamnoides, 30 min before 10 Gy whole body gamma irradiation was studied. Doses between 25 to 35 mg/kg body wt. were found to render > 80 % survival in mice. In order to investigate whether RH-3 protected against radiation induced genotoxicity, mice were administered different doses of RH-3, 30 min before 2 Gy dose and compared with untreated, RH-3 treated and irradiated controls. The bone marrow cells were collected at different time intervals following various treatments and processed for scoring micronuclei (MN). Administration of RH-3 alone did not enhance the MN frequency as compared to the control, and radiation dose of 2 Gy significantly enhanced the MN frequency (3.1 %, P < 0.01). Pre-irradiation treatment with RH-3, however, reduced the radiation induced MN frequency in a drug dose dependent manner suggesting its radioprotective efficacy. The protective effect of RH-3 on radiation induced perturbations in cell cycle progression was studied flowcytometrically in mouse bone marrow cells. RH-3 treatment (30 mg/kg body wt.) enhanced DNA synthesis (S-phase) in unirradiated controls and also countered radiation induced depression of S-phase to facilitate replenishment of cells lost due to radiation injury.