Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?

Abstract

Free radicals and other reactive species (RS) are thought to play an important role in many human diseases. Establishing their precise role requires the ability to measure them and the oxidative damage that they cause. This article first reviews what is meant by the terms free radical, RS, antioxidant, oxidative damage and oxidative stress. It then critically examines methods used to trap RS, including spin trapping and aromatic hydroxylation, with a particular emphasis on those methods applicable to human studies. Methods used to measure oxidative damage to DNA, lipids and proteins and methods used to detect RS in cell culture, especially the various fluorescent "probes" of RS, are also critically reviewed. The emphasis throughout is on the caution that is needed in applying these methods in view of possible errors and artifacts in interpreting the results.

Figure 1

Conversion of DCFDA to a fluorescent product DCFDA is dichlorofluorescin (sometimes written as dichlorofluorescein) diacetate, perhaps better described by its proper chemical name as 2′,7′-dichlorodihydrofluorescein diacetate. It is hydrolyzed by cellular esterases to dichlorofluorescin (2′,7′-dichlorodihydrofluorescein), whose oxidation by several RS yields florescent DCF (dichlorofluorescein, more correctly called 2′7′-dichlorofluorescein) via an intermediate radial, DCF^•−. Peroxidases can also convert it into a phenoxyl radical that can interact with antioxidants such as ascorbate (AH⁻), reducing the phenoxyl radical and oxidizing ascorbate, or with GSH. GS^• resulting from the latter reaction can lead to O₂^•− generation. The phenoxyl radical can also be recycled by NADH (not shown), producing NAD^• radical, which reacts rapidly with O₂ to produce O₂^•−:

Figure 2

Structures of some common probes used for the detection of RS: (a) HPF and APF, (b) luminol, (c) LC and (d) MCLA. (e) Cis-parinaric acid, (f) 4,4-difluoro-5-(4-phenyl-1,3,-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY ^581/591), (g) coelenterazine and (h) 8-amino-5-chloro-7-phenylpyridazo [3,4-d] pyridazine-1,4-(2H, 3H) dione (L-O12).

Figure 3

Conversion of dihydrorhodamine 123 to rhodamine 123.

Figure 4

Conversion of DHE to ethidium.

Figure 5

Conversion of diphenyl-1-pyrenylphosphine to fluorescent product by peroxides.