Phloxine B (PhB) (2',4',5',7'-tetrabromo-4,5,6,7-tetrachlorofluorescein; D&C Red No. 28) is a red
dye found in drugs,
cosmetics and foods; it is also currently being evaluated as a phototoxin for the potential control of fruit flies. Previous studies have shown that PhB is an efficient
photosensitizer of damage to cellular membranes; thus, exposure of the skin to the
dye and sunlight or artificial light may result in
phototoxicity. Therefore, we have studied the
phototoxicity of PhB and its structural analogue
2',7'-dichlorofluorescein (DCF) to HaCaT keratinocytes. Anaerobic visible irradiation (>400 nm) of PhB generated a semiquinone type radical, as detected by direct electron paramagnetic resonance. Aerobic visible irradiation of a reaction mixture containing PhB, the spin trap 5,5-dimethyl-1-pyrroline N-
oxide (DMPO) and
nicotinamide adenine dinucleotide (reduced) generated a
superoxide dismutase-sensitive DMPO/O(2)(.-) adduct. Irradiation of PhB and DCF in D(2)O generated
singlet oxygen with quantum yields of 0.59 and 0.06, respectively. PhB was much more phototoxic than DCF when cell viability was measured using the
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Visible irradiation of HaCaT keratinocytes in the presence of PhB (5 micro M) resulted in a 90% decrease in cell viability. 3beta-Hydroxy-5alpha-cholest-6-ene-5-hydroperoxide, a
singlet oxygen photoproduct of
cholesterol, was isolated from HaCaT keratinocytes irradiated in the presence of PhB. Furthermore, PhB
phototoxicity was inhibited by
histidine and
cysteine, quenchers of
singlet oxygen. PhB (0.5 microM) and light irradiation also resulted in DNA damage, as measured by the Comet assay. The
phototoxicity mechanism of PhB most probably initially involves a Type-II reaction with
free radicals playing a minor role. However, secondary oxidative species such as radicals generated as a result of lipid peroxidation may serve to further promote oxidative damage. Our findings suggest that concern is warranted about the use of this
dye in cosmetic products, as a
food additive and in insecticidal sprays.