Photosensitized oxidation of 2',7'-dichlorofluorescin: singlet oxygen does not contribute to the formation of fluorescent oxidation product 2',7'-dichlorofluorescein

Abstract

2',7'-Dichlorofluorescin (DCFH) is often employed to assess oxidative stress in cells by monitoring the appearance of 2',7'-dichlorofluorescein (DCF), its highly fluorescent oxidation product. We have investigated the photosensitized oxidation of DCFH in solution and elucidated the role played by singlet molecular oxygen (1O(2)) in this reaction. We used rose bengal (RB), protoporphyrin, and DCF as photosensitizers. Irradiation (550 nm) of RB (20 microM) in 50 mM phosphate (pH 7.4) in the presence of DCFH (50 microM) resulted in the rapid formation of DCF, measured as an increase in its characteristic absorbance and fluorescence. The oxidation rate was faster in deoxygenated solution, did not increase in D(2)O, and even increased in the presence of sodium azide. The presence of antioxidants that react with 1O(2), thus removing oxygen, accelerated DCF formation. Such results eliminate any potential direct involvement of 1O(2) in DCF formation, even though DCFH is an efficient (physical) quencher of 1O(2) (k(q) = 1.4 x 10(8) M(-1)s(-1) in methanol). DCF is also a moderate photosensitizer of 1O(2) with a quantum yield of circa phi = 0.06 in D(2)O and phi = 0.08 in propylene carbonate, which unequivocally indicates that DCF can exist in a triplet state upon excitation with UV and visible light. This triplet can initiate photo-oxidization of DCFH via redox-and-radical mechanism(s) similar to those involving RB (vide supra). Our results show that, upon illumination, DCF can function as a moderate photosensitizer initiating DCFH oxidation, which may prime and accelerate the formation of DCF. We have also shown that, while 1O(2) does not contribute directly to DCF production, it can do so indirectly via reaction with cellular substrates yielding peroxy products and peroxyl radicals, which are able to oxidize DCFH in subsequent dark reactions. These findings suggest that DCFH should not be regarded as a probe sensitive to singlet molecular oxygen, and that care must be taken when using DCFH to measure oxidative stress in cells as a result of both visible and UV light exposure.