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

• Published in: Molecular and cellular biochemistry 2003-03, Vol.245 (1-2), p.57-67
• Main Authors: Goel, H C, Kumar, I Prem, Samanta, Namita, Rana, S V S
• Format: Article
• Language: English
• Subjects: Animals; Anions; Antioxidants - administration & dosage; Antioxidants - pharmacology; Cell cycle; Cell Hypoxia - drug effects; Cells, Cultured; Chromatin - drug effects; Chromatin - metabolism; Chromatin - radiation effects; Compaction; Cross-Linking Reagents - chemistry; Cytotoxicity; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - metabolism; DNA - radiation effects; DNA Damage - drug effects; DNA Damage - radiation effects; DNA Topoisomerases, Type I - metabolism; Dose-Response Relationship, Drug; Free Radical Scavengers - metabolism; Free Radical Scavengers - pharmacology; Free radicals; Gamma Rays - adverse effects; Hippophae - chemistry; Hippophae rhamnoides; Hydrogen-Ion Concentration; Hydroxyl Radical - metabolism; Hypoxia; Male; Metal concentrations; Mice; Mice, Inbred A; Molecular weight; Plant Extracts - administration & dosage; Plant Extracts - pharmacology; Proteins; Radiation-Protective Agents - administration & dosage; Radiation-Protective Agents - pharmacology; Sodium chloride; Thiobarbituric Acid Reactive Substances - metabolism; Thymus Gland - cytology; Topoisomerase I Inhibitors; Urea
• ISSN: 0300-8177; 1573-4919
• DOI: 10.1023/A:1022809625826

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