Photogenotoxicity of Skin Phototumorigenic Fluoroquinolone Antibiotics Detected Using the Comet Assay

Abstract

The fluoroquinolone (FQ) antibiotics photosensitize human skin to solar UV radiation and are reported to photosensitize tumor formation in mouse skin. As tumor initiation will not occur without genotoxic insult, we examined the potential of ciprofloxacin, lomefloxacin, fleroxacin, BAYy3118 (a recently developed monofluorinated quinolone) and a nalidixic acid to photosensitize DNA damage in V79 hamster fibroblasts in vitro. Cells were exposed to 37.5 kJ/m2 UVA (320-400 nm; glass filtered Sylvania psoralen + UVA (PUVA) tubes; calibrated Waldmann radiometer) at 4 degrees C in the presence of FQ and immediately afterwards embedded in agarose, lysed and placed in an electrophoretic field at pH 12. Under these denaturing conditions, the presence of DNA single-strand breaks (SSB), alkali-labile sites (ALS) and double-strand breaks (DSB) can be visualized as DNA migrating away from the nucleus (characteristic "comet" appearance) after staining with a specific fluorochrome. At FQ concentrations that induced minimal loss of cell viability (neutral red uptake assay) the compounds tested induced comets with a rank order of BAYy3118 > norfloxacin > ciprofloxacin > lomefloxacin > fleroxacin > nalidixic acid. If cells were incubated after treatment for 1 h at 37 degrees C, the comet score decreased, suggesting efficient removal of SSB/ALS/DSB. Addition of the DNA polymerase(alpha) inhibitor, aphidicolin, to cells treated with either ciprofloxacin alone or ciprofloxacin + UVA resulted in an accumulation of SSB due to the endo/exonuclease steps of excision repair. We have demonstrated that the FQ are photogenotoxic in mammalian cells but the FQ-photosensitized SSB are efficiently repaired. Preliminary evidence that ciprofloxacin photosensitizes the formation of DNA lesions warranting excision repair may indicate production of more mutagenic lesions.

Synergistic activation of p53-dependent transcription by two cooperating damage recognition pathways

“…For the determination of b-galactosidase activity, cells were lysed by repeated rounds of freeze ± thaw, and lysates assayed as previously described (Blaydes et al, 1997). The COMET assay for the detection of single-and double-stranded breaks was performed as described in (Reavy et al, 1997) except that the COMET tails were scored using image analysis software (COMET 2.2, kinetic imaging, Liverpool, UK).…”

Induced and Photoinduced DNA Damage by Quinolones: Ciprofloxacin, Ofloxacin and Nalidixic Acid Determined by Comet Assay¶

“…Comparatively, nalidixic acid (5–500 μ M ) irradiated with a dose of 2 × 10 5 mJ/cm 2 in cells derived from human laringo carcinoma (Hep‐2) and in primary cultures of chick embryo fibroblasts (CEF) is also found to be phototoxic (17). With a UV‐A dose of 3750 mJ/cm 2 in V79 hamster fibroblasts, nalidixic acid produces smaller comets than fleroxacin, lomefloxacin or ciprofloxacin, demonstrating that the fluoroquinolones are more photogenotoxic in this cellular model (18). The damage to DNA upon UV‐A radiation of the plas‐mid pBR322 (the vector normally used in genetic engineering for cloning genes) is minor relative to that produced by fleroxacin and lomefloxacin, which possess two fluorine atoms in their structures, at positions 6 and 8, and generate carbenes at position 8, considerably increasing their genotoxic capacity relative to norfloxacin and enoxacin (19).…”

Photosensitization of Biomolecules by Phenothiazine Derivatives

“…Both reaction mechanism can be shared by the same molecule. As discussed in Chapter 2 photosensitizers may be transformed to reactive intermediates when they absorb UV radiation and/or may yield active oxygen species including singlet oxygen These UV mediated products have the potential to damage DNA and other cellular structures leading to phototoxic and/or photogenotoxic effects [6,[111][112][113]. If the cellular repair mechanisms are overloaded, primary DNA damage may lead to mutations or chromosomal damage and eventually cause tumors [114][115][116].…”