title: Induction of DNA-protein cross-links by Hippophae rhamnoides: implications in radioprotection and cytotoxicity.
abstract: 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.
authors.affiliation: Department of Radiation Biology, Institute of Nuclear Medicine and Allied Sciences, Brig S.K. Majumdar Marg, Delhi, India. radbiol@nda.vsnl.net.in
authors.forename: H C
authors.initials: HC
authors.lastname: Goel
authors.forename: I Prem
authors.initials: IP
authors.lastname: Kumar
authors.forename: Namita
authors.initials: N
authors.lastname: Samanta
authors.forename: S V S
authors.initials: SV
authors.lastname: Rana
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000008:administration & dosage / Q000494:pharmacology; D015687:Cell Hypoxia / Q000187:drug effects; D002478:Cells, Cultured; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016166:Free Radical Scavengers / Q000378:metabolism / Q000494:pharmacology; D005720:Gamma Rays / Q000009:adverse effects; D031281:Hippophae / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D017665:Hydroxyl Radical / Q000378:metabolism; D008297:Male; D051379:Mice; D008805:Mice, Inbred A; D010936:Plant Extracts / Q000008:administration & dosage / Q000494:pharmacology*; D011837:Radiation-Protective Agents / Q000008:administration & dosage / Q000494:pharmacology*; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D013950:Thymus Gland / Q000166:cytology; D059004:Topoisomerase I Inhibitors
chemical_list: D000975:Antioxidants; D002843:Chromatin; D003432:Cross-Linking Reagents; D016166:Free Radical Scavengers; D010936:Plant Extracts; D011837:Radiation-Protective Agents; D017392:Thiobarbituric Acid Reactive Substances; D059004:Topoisomerase I Inhibitors; D017665:Hydroxyl Radical; D004247:DNA; D004264:DNA Topoisomerases, Type I