title: De-intercalation of ethidium bromide and acridine orange by xanthine derivatives and their modulatory effect on anticancer agents: a study of DNA-directed toxicity enlightened by time correlated single photon counting.
abstract: Time Correlated Single Photon Counting (TCSPC) was used for the first time to analyze the effect/changes in the mode of intercalation of ethidium bromide (EtBr) and acridine orange (AO) to calf thymus DNA brought about due to interaction of naturally occurring methylxanthines such as theophylline (X1), theobromine (X2) and caffeine (X3). UV absorption and fluorescence studies were also carried to observe the behaviour of these xanthines on the modulation of the binding mode of anticancer agents (cisplatin, novantrone, and actinomycin D) and certain intercalating dyes (EtBr and AO) to DNA. In TCSPC analysis we found that when the concentration of the drugs (X1, X2 and X3) increased from 0.025 mM to 2 mM i.e. P/D 2.4 to P/D 0.03 reduction in intercalation of EtBr and AO was observed, suggesting that xanthine derivatives could play very important role in reducing the DNA-directed toxicity in a dose dependent manner. In TCSPC, the amplitude of smaller lifetime component A(1) and higher lifetime component A(2) are attributed to free and intercalated dye concentration and their variation could indicate the process of intercalation or reduced intercalation of EtBr and AO by xanthine derivatives. We found that at the maximum drug concentration the smaller lifetime component A(1) was increased by 7-8% and 17-37% in EtBr and AO intercalated complex respectively. Also the changes in lifetime and fluorescence decay profile were observed for the DNA-intercalated dyes before and after treatment with xanthines. Especially, at maximum P/D 0.03 the lifetime of DNA-intercalated EtBr and AO reduced by 1-2 ns. The present analysis reveals that xanthines are able to interact with free dyes and also with intercalated dyes, suggesting that when they interact with free dyes they might inhibit the further intercalation of dye molecules to DNA and the interaction with intercalated dyes might lead to displacement of the dyes resulting in de-intercalation. The results obtained from UV and fluorescence spectroscopy also support the present investigation of probable interaction of xanthines with the DNA damaging agents in modulating/reducing the DNA-directed toxicity.
authors.affiliation: Department of Genetics, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, 600 113, India.
authors.forename: I Maria
authors.initials: IM
authors.lastname: Johnson
authors.forename: S G Bhuvan
authors.initials: SG
authors.lastname: Kumar
authors.forename: R
authors.initials: R
authors.lastname: Malathi
mesh_terms: D000165:Acridine Orange / Q000378:metabolism* / Q000633:toxicity; D000970:Antineoplastic Agents / Q000378:metabolism*; D002110:Caffeine / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004996:Ethidium / Q000378:metabolism* / Q000633:toxicity; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D007364:Intercalating Agents / Q000378:metabolism* / Q000633:toxicity; D008954:Models, Biological; D017785:Photons; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013805:Theobromine / Q000494:pharmacology; D013806:Theophylline / Q000494:pharmacology; D014970:Xanthines / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D014970:Xanthines; D002110:Caffeine; D004247:DNA; C427203:calf thymus DNA; D013806:Theophylline; D004996:Ethidium; D000165:Acridine Orange; D013805:Theobromine