Modulation of chromatin organization by RH-3, a preparation of Hippophae rhamnoides, a possible role in radioprotection

The present study was aimed to understand the mode of action of alcoholic extract of whole berries of Hippophae rhamnoides (RH-3) which has already been reported to render more than 80% protection against radiation induced mortality in mice. Direct and indirect antioxidant action (free radical scavenging and metal chelating potential) were assayed using 2-deoxy ribose degradation and 2,2'-bipiridyl assays. Effect of RH-3 on radiation and chemical oxidant mediated DNA damage was evaluated using single cell gel electrophoresis (Comet assay) and alkaline halo assay. Ability of RH-3 to bind with calf thymus DNA was assayed through change in melting temperature (Tm) while toxicity was assayed in thymocytes by trypan blue exclusion. RH-3 inhibited 2-deoxy ribose degradation in a dose dependent manner (IC 50 approximate to 500 mug/ml). 2,2'-bipiridyl assay revealed the inability of RH-3 to chelate Fe2+ ions. RH-3 inhibited radiation and tertiary butyl hydroperoxide induced DNA strand breaks in a dose dependent manner and at concentrations of 100 and 120 mug/ml the length of comet tail was considerably reduced and became almost similar to that of untreated control. RH-3 at a concentration of 120 mug/ml or more induced a strong compaction of chromatin as was evident from lack of tail and appearance of intensely stained circular bodies. This made the nuclei resistant even to a radiation dose of 1000 Gy. The compaction of chromatin was not reversed even by relaxation buffer indicating that salt concentration had no role in RH-3 induced chromatin compaction. Alkaline halo assay also corroborated the results of comet assay. Lower DNA-RH-3 concentrations (1:0.5 and 1:1) induced a shift of Tm towards left by 2 and 5degreesC respectively; however higher concentrations (1:8 and 1:16) shifted the Tm towards right increasing it by 10 and 21degreesC correspondingly. RH-3, evinced only a mild free radical scavenging activity at concentrations used in the present study, therefore its ability to protect DNA could mainly be attributed to direct modulation of chromatin organization. Further work to unravel these facts would be necessary.