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Reactive Oxygen Species and Oxidative Stress 283

referred to as quinone reductase type 1 (QR1), which is DT diaphorase, and quinone reductase type 2
(QR2). Major differences between QR1 and QR2 include functional electron donors (QR1 employs
NADH and NADPH, but QR2 employs nonphosphorylated nicotinamides), inhibition (QR1 but not QR2
is very sensitive to dicoumarol), and regulation. QR1 is highly inducible through both xenobiotic response
elements and antioxidant response elements (described below), but QR2 is apparently not inducible.
Fish clearly express QR1 (see below), but we are unaware of reports concerning QR2 in this vertebrate
class.
The proteins discussed here are generally considered major players as antioxidant enzymes; however,
they do not include all enzymes known to have some antioxidant function. Peroxiredoxin (Georgiou and
Masip, 2003), thioredoxin and glutaredoxin (Holmgren, 2000; Watson et al., 2004), heme oxygenase
(Kvam et al., 1999), and other enzymes may play important roles at well, at least under some circum-
stances. For additional information, see Halliwell and Gutteridge (1999).

Low-Molecular-Weight Antioxidants
A number of biomolecules can directly scavenge ROS nonenzymatically. GSH, discussed above, is a
very important component of this group, although it is also essential to GPX activity. Several other
prominent antioxidants are vitamin related and are obtained through the diet by most animals. These
include ascorbic acid (vitamin C), tocopherols (vitamin E components), and carotenoids (vitamin A, or
retinol, precursors). A number of other compounds synthesized by animals exhibit antioxidant capacity
in vitro, but their antioxidant functions in vivo generally remain unclear.

Ascorbic Acid (Vitamin C)
Plants and many animals can synthesize ascorbic acid from glucose, although humans and other primates,
bats, and passerine birds cannot (Moreau and Dabrowski, 2003; Nishikimi and Yagi, 1996). Among
fishes, teleosts cannot perform this synthesis, but those retaining more ancestral characteristics such as
lampreys, sharks, rays, lungfishes, sturgeons, paddlefishes, and bowfin can (Moreau and Dabrowski,
1998). Those animals that cannot synthesize ascorbic acid lack the enzyme that catalyzes the terminal
step in biosynthesis, gulonolactone oxidase. A DNA sequence resembling the gene for this enzyme in
other animals has been identified in humans and guinea pigs, but it is extensively mutated and inactive
(Nishikimi and Yagi, 1996).
At physiological pH, ascorbic acid exists largely as ascorbate (Figure 6.3) and is highly water soluble.
Ascorbate serves as a cofactor for a number of enzymes, including proline hydroxylase and lysine
hydroxylase, that are involved in collagen synthesis, as well as for dopamine-β-hydroxylase, which
catalyzes the conversion of dopamine into norepinephrine (Nishikimi and Yagi, 1996). The hallmark
human disease associated with vitamin C deficiency is scurvy, a common disease among sailors in earlier
times (before the importance of fresh fruit was recognized); it is characterized by muscle weakness,
blood vessel fragility, and bleeding from gums and other mucous membranes.
Ascorbate is a powerful reducing agent and has been shown with numerous in vitro studies to scavenge
•–
a number of ROS, including O , ·OH, ROO·, and HOCl (Halliwell, 1996). Although unequivocally
2
proving the significance of these reactions in vivo is difficult, the function of ascorbate as an antioxidant
is widely accepted based on considerations of its in vitro reactivity, concentrations in tissues, experimental
manipulations of its levels in animals and accompanying markers of oxidative stress, and related
consequences in human deficiencies (Halliwell and Gutteridge, 1999).
Upon reduction of an oxygen radical, ascorbate is oxidized to the ascorbyl radical (also known as
semidehydroascorbate) (Bendich et al., 1986). This radical is relatively stable and unreactive, a feature
central to its antioxidant function; that is, a much more reactive free radical reacts with ascorbate,
yielding a less reactive and hence more innocuous ascorbyl radical. The ascorbyl radical is readily
oxidized again, yielding dehydroascorbate (DHA, the two-electron oxidation metabolite of ascorbate).
DHA has little antioxidant activity and can break down to several products (notably oxalic acid and
threonic acid) or be reduced back to ascorbate by an apparently nonspecific GSH-dependent reductase
(Wells et al., 1990).