Induction of DNA-protein cross-links by Hippophae rhamnoides: implications in radioprotection and cytotoxicity.

Recently Hippophae rhamnoides has been reported to render chromatin compaction and significantly inhibit radiation induced DNA strand breaks. To investigate the mechanism of action of RH-3, a preparation of Hippophae rhamnoides, in this connection, present study was undertaken. Chromatin compaction induced by RH-3 (100 microg/ml or more) was maximum at alkaline pH but was completely negated by acidic pH (< 6) or presence of free radical scavengers like glycerol, DMSO etc. In a concentration dependent manner, RH-3 inhibited the intercalation of ethidium ions from Et Br into calf thymus DNA and also increased the precipitation of DNA-protein cross-links (DPC) in thymocytes. Chromatin compaction caused by RH-3 treatment did not permit the separation of proteins from DNA even after treatment with 2 M NaCl solution. SDS-PAGE profiles also revealed that RH-3 in a dose dependent manner compacted the chromatin organization, induced DPC and inhibited the extraction of both histone and non-histone matrix proteins from chromatin maximally at 80 microg/ml. More than 80 microg/ml of RH-3, though extracted low molecular weight histones but did not separate non-histone proteins. The RH-3 mediated DPCs were resistant even to 1% SDS, 4 M NaCl and 3.8 M hydroxyl amine hydrochloride but were prone to both urea (8 M) and guanidine hydrochloride (6 M) indicating covalent bonding between DNA and proteins (serine/threonine). RH-3 in a concentration dependent manner induced superoxide anions and the phenomenon was dependent upon nature of medium, presence of metal ions and pH. RH-3 at concentrations up to 100 microg/ml in presence of 50 microM copper sulfate inflicted significant damage to extraneously added 2-deoxyribose molecules and maximum TBARS were formed at a concentration of 100 microg/ml. Higher concentrations of RH-3 more than 100 microg/ml quenched free radicals and inhibited 2-deoxyribose degradation. RH-3 also induced strand breaks in plasmid DNA at concentrations lower than 100 microg/ml but completely inhibited at concentrations higher than 250 microg/ml, indicating bimodal function. Strand breaks induced by lower concentrations of RH-3 (up to 100 microg/ml) were inhibited by antioxidants like GSH, DFR etc. RH-3, in a concentration dependent mode also inhibited the relaxation of supercoiled plasmid DNA (PBR322) by topoisomerase I. Present study indicated that RH-3 caused compaction of reversible (< 100 micrpg/ml) and irreversible (> 100 microg/ml) nature which was related to the magnitude of DNA-protein cross-links formed. Maintenance of chromatin organization, induction of hypoxia, hydrogen atom donation, free radical scavenging and blocking of cell cycle at G2-M phase by interfering with topoisomerase I activity seem to contribute towards the radioprotective efficacy of RH-3.