Ultrafast Fluorescence Spectroscopy and Cell Imaging Techniques in Exploring the Caffeine Mediated Dissociation of Mutagen from Biomimetics, Nucleic Acids and Cells

Abstract

We report a systematic investigation of caffeine induced dissociation of ethidium (Et) cation, a potential mutagen from nucleic acids and biomimetic systems. Time-resolved fluorescence studies are consistent with a mechanism where caffeine-Et complex formation in bulk solution drives the dissociation of DNA-bound Et. Caffeine induced extraction of Et from whole cells were also performed on squamous epithelial cells collected from the inner lining of the human mouth, A549 (lung carcinoma), A375 (human skin), RAW (macrophage) and Vero (African green monkey kidney epithelium) cell lines. Interestingly, the efficiency of caffeine in extracting Et has been found to be dependent on cell types. Our steady state and picosecond resolved spectroscopic studies on the detachment of Et from various biomimicking micelles of different charges reveal the specificity of caffeine molecule for carrying out such dissociation. The picosecond resolved Forster resonance energy transfer (FRET) studies between a DNA minor groove binder dye Hoeschst 33258 (H258, donor) and Et (acceptor) have been employed to investigate the alteration in their association in presence of caffeine in the molecular level. Finally, our fluorescence micrographs of epithelial cells validate the alteration of FRET efficiency between the donor and the acceptor due to the caffeine mediated release of the latter. Our results both in-vitro as well as ex-vivo provide important clues about efficiency and mechanism of caffeine as a potential anti-mutagenic therapeutic agent.