Photochemical binding of fluoroquinolones to DNA. Free

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A variety of compounds, including the fluoroquinolone (FQ) antibiotics, can be photoactivated to genotoxic and photochemically carcinogenic products. The experimental FQ, BAY y3118 (3118), photochemically generates oxidative damage and is photochemically mutagenic (Arch.Toxicol., 74, 555, 2000). We have investigated the photochemically generated DNA adducts from 3118. It was photoactivated by 345 nm UVA light in the presence of calf thymus DNA and the DNA was purified by ethanol precipitation. Photoactivation of 3118 yielded DNA that exhibited substantial fluorescence (Ex 350 nm, Em 450 nm). Such modification did not occur when 3118 was irradiated prior to mixing with the DNA suggesting the formation of a short lived species during the photochemical reaction. Using ¹⁴C-3118, photoactivation resulted in radioactivity bound to DNA corresponding to about 70 adducts in 10⁶ normal nucleotides. Oxygen was not required for the reaction since, under anaerobic conditions the fluorescence associated with the purified DNA was even higher. ³²P-Postlabeling of these DNA samples either directly, using nuclease P₁ or column (Water Oasis HLB) enrichment of the samples showed only very low levels of DNA adducts inconsistent with the ¹⁴C experiments. These results suggested that some of the fluorescence and ¹⁴C-label may have been associated with intercalated compound. Monoclonal antibodies have been prepared (J. Agric. Food Chem. 45, 1984, 1997) against a related FQ, sarafloxacin (SF). These were tested and free SF>ciprofloxacin>3118>lomofloxacin were found to inhibit an ELISA using SF-photochemically modified DNA (SF-DNA), which itself was inhibitory while 3118-DNA resulted in very weak inhibition. In gel shift experiments using ³²P-labeled either single or double stranded 20 base pair SF-oligomers shifts only occurred when the oligomer was irradiated in the presence of SF. The experiments suggest, despite the difficulty in directly isolating a FQ-DNA adduct, that the FQs are photochemically covalently linked to the DNA. (Supported in part by NCI Grant RO1-CA86056)