The Hydroxyl Radical - Free-Radical-Induced DNA Damage and Its Repair - page 60

many f fluctuations in the reported data may be partly due to the fact that these
systems are very sensitive to variations in the reaction conditions. This, how-
ever, is not be very helpful for their use as • OH probes.
It is seen from Table 3.5 that in the 2-hydroxybenzoate system, for example,
the yields of the 2,3- and 2,5-dihydroxybenzoic acids are close to 1:1 when the
precursor radicals are oxidized by O 2 , but in the absence of an oxidant this ratio
is around 9:1. For the detection of • OH in cellular or in in v ivo systems, it is hence
not sufficient that these products are formed, but the second requirement is that
an adequate oxidant is present (for example, a sufficiently high O 2 tension must
be maintained) in order to guarantee that they are formed in a 1:1 ratio. Experi-
ments have been carried out with rats that were given high doses of salicylic acid
as a probe for • OH formation induced by the drug (Ste-Marie et al. 1996). Much
more 2,5- than 2,3-dihydroxybenzoic acid was detected (cf. Table 3.8). The au-
thors realized that the second requirement of this probe was not fulfilled and
stated that there was still the possibility that the drug had induced a metabolic
oxidation of salicylic acid yielding mainly 2,5-dihydroxybenzoic acid. Never-
theless, when this paper was later cited in the literature, it was taken as a proof
for • OH formation under these conditions. We would like to emphasize here that
both requirements, formation of the 2,3- and 2,5-dihydroxybenzoic acids and
their 1:1 ratio, is necessary in order to ascertain • OH formation. It is noted that
2,5-dihydroxybenzoic acid is formed in the reaction of salicylic acid with sin-
glet dioxygen (O 2 1

∆ g ) (Kalyanaraman et al. 1993). More importantly, it may also
result from enzyme reactions (Ingelman-Sundberg et al. 1991). Especially the
latter makes it difficult if not impossible to ever observe a 1:1 ratio. Whether
the suggestion to only concentrate on 2,3-dihydroxybenzoic acid formation for
• OH detection (Ingelman-Sundberg et al. 1991) is an adequate solution remains
debatable.

3.5.2
Spin Traps

Hydroxyl-radical spin trapping (and detection by EPR) would be a direct detec-
tion method [e.g., reaction (67); k = 2 10 9 dm 3 mol −1 s −1 ]. Besides reacting with

• OH, spin traps also react with O 2 • − [e.g., reaction (66); k = 10 dm 3 mol −1 s −1 ] and
the HO 2 • adduct to DMPO has only a short lifetime of about 8 min [reaction (68);
Pou et al. 1989; Rosen et al. 1994].
Although the difference in rate between • OH and O 2 • − scavenging by the
spin trap is eight orders of magnitude, the yield of the O 2 • − -spin-adduct may be
considerably higher, because of the usually much higher steady-state concentra-
tion of O 2 • − . In vivo, steady-state concentrations of O 2 • − have been estimated at
around 10 −11 to 10 −10 mol dm −3 (Boveris and Cadenas 1997) and those of • OH at
some 10 −20 mol dm −3 (the latter using too low a scavenger capacity in our opin-
ion, i.e., the • OH steady-state concentration would be at least an order of magni-
tude lower). As a consequence, its eight orders of magnitude lower rate is more
than compensated by the ten (or more) orders of magnitude higher steady-state
concentration of O 2 • − . Thus, in a biological system, spin trapping of O 2 • − will
strongly dominate, and the decay of its adduct, reaction (68), may lead to the

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