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

doi:10.1016/S0891-5849(02)00982-6

Free Radical Biology and Medicine

Volume 33, Issue 7, 1 October 2002, Pages 938-946

https://doi.org/10.1016/S0891-5849(02)00982-6 Get rights and content

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 (¹O₂) in this reaction. We used rose bengal (RB), protoporphyrin, and DCF as photosensitizers. Irradiation (550 nm) of RB (20 μM) in 50 mM phosphate (pH 7.4) in the presence of DCFH (50 μM) 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 D₂O, and even increased in the presence of sodium azide. The presence of antioxidants that react with ¹O₂, thus removing oxygen, accelerated DCF formation. Such results eliminate any potential direct involvement of ¹O₂ in DCF formation, even though DCFH is an efficient (physical) quencher of ¹O₂ (k_q = 1.4 × 10⁸ M⁻¹s⁻¹ in methanol). DCF is also a moderate photosensitizer of ¹O₂ with a quantum yield of circa φ = 0.06 in D₂O and φ = 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 ¹O₂ 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.

Introduction

Fluorogenic probes are a convenient and sensitive means to monitor oxidative activity in cells [1]. The nonfluorescent 2′-7′-dichlorodihydrofluorescin (DCFH) has been used extensively to detect oxidizing species in cells by monitoring the formation of 2′-7′-dichlorofluorescein (DCF), an oxidation product that shows strong fluorescence. To facilitate cell penetration, DCFH is usually used as its diacetate that is able to cross the cell membrane. After hydrolysis of the ester by intracellular esterases, DCFH is liberated where it may react with oxidizing species, such as H₂O₂ and peroxynitrite, and with certain peroxidases [2].

Singlet oxygen is a highly oxidizing species that may be generated in cells as a result of photosensitization or from the decomposition of the tetra-oxygen intermediate formed by the reaction of two peroxyl radicals (Russell mechanism). Although it has been suggested that singlet oxygen (¹O₂) could oxidize DCFH (e.g., [3], [4]), to our knowledge, the reaction of ¹O₂ with the probe has not been examined in detail. DCFH and similar fluorescence probes based on the xanthene chromophore (rhodamines) are often used to investigate photosensitization processes that may occur in the skin and eyes. Thus it becomes important to understand basic physicochemical principles that operate during the photosensitized oxidation of DCFH.

We have investigated the mechanism of photosensitized oxidation of DCFH using rose bengal (RB) and DCF as photosensitizers together with several antioxidants and ¹O₂ quenchers. We mostly focus our attention on the roles of oxygen and ¹O₂ in the formation of a fluorescent product. Our findings show that although DCFH is converted to DCF by irradiation in the presence of photosensitizers RB or porphyrin, ¹O₂ is not involved. Furthermore, ¹O₂ generation is a channel of physical deactivation of the photosensitizer triplet, which competes with the redox and radical processes that are responsible for the DCF production and fluorescence increase during photochemical oxidation. To the best of our knowledge, this is the first study to examine experimentally the potential oxidation of the DCFH probe by singlet molecular oxygen.

Section snippets

Materials and methods

The following chemicals were used as received. Rose bengal, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), sodium azide, ascorbic acid, gallic acid, acetonitrile, propylene carbonate (1,2-propanediol cyclic carbonate), methanol, ethanol, N,N-diethylhydroxylamine (DEHA), protoporphyrin IX were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Deuterium oxide was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Chelex 100 was from Bio-Rad Laboratories (Richmond, CA, USA). All

Production of ¹O₂ by DCF

While DCF has previously been established to be a weak photosensitizer [12], its ability to photogenerate ¹O₂ has neither been spectrally shown nor quantified by any means. We directly observed ¹O₂ by its phosphorescence (Fig. 2), and calculated the quantum yield of ¹O₂ photosensitization by DCF in selected solvents using RB as a standard (Fig. 2, table inset). DCF appeared to be a weak ¹O₂ photogenerator in both aqueous and organic phases. This means that, independent of its cellular

Conclusions

We have found that, although ¹O₂ is efficiently quenched by DCFH, this process is physical in nature and does not contribute to fluorescence production. Unexpectedly, ¹O₂ acted as a classical quencher of the reaction(s) leading to DCF fluorescence. In agreement with this postulate, we have found that oxygen removal from the solution increased the intensity of photochemically induced fluorescence. Oxygen removal could be accomplished by the use of antioxidants such as ascorbic acid, DEHA, gallic

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Original contributionPhotosensitized oxidation of 2′,7′-dichlorofluorescin: singlet oxygen does not contribute to the formation of fluorescent oxidation product 2′,7′-dichlorofluorescein

Abstract

Introduction

Section snippets

Materials and methods

Production of 1O2 by DCF

Conclusions

Free Radic. Biol. Med.

J. Photochem. Photobiol. B-Biol.

Arch. Biochem. Biophys.

J. Biochem. Biophys. Meth.

J. Invest. Dermatol.

Free Radic. Biol. Med.

Photoreduction of 2’-7’-dichlorofluorescein. An ESR study with implications for oxidative stress measurements

Free Radic. Biol. Med.

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

Production of ¹O₂ by DCF