Page 312 - The Toxicology of Fishes

292 The Toxicology of Fishes

antioxidant capacity, at which point detrimental cellular impacts may ensue. This corresponds to the
classic definition first introduced by Sies and Cadenas (1985) of oxidative stress as “a disturbance in
the prooxidant–antioxidant balance in favor of the former, leading to potential damage.”
A major difficulty in the study of oxidative stress is the variety of ways at which it can manifest; that
is, it is far more difficult to determine if a particular chemical is acting, as a principal mechanism of
toxic action, as a prooxidant causing oxidative stress vs. determining, for example, if it is an acetylcho-
linesterase inhibitor, an aryl hydrocarbon receptor agonist, or an estrogen mimic. A lack of effect as
measured by any particular assay (or two or three) cannot be used to rule out the occurrence of oxidative
stress. A similar caution applies to the measure of antioxidants as markers for exposure or adaptation
to prooxidants, with the expectation that exposures below the threshold for toxicity will result in
upregulations of antioxidants. Again, the complexity of mechanisms by which prooxidants may or may
not augment various antioxidant system components presents major challenges to this approach. These
are issues that have plagued the development of biomarkers for oxidative stress in fish, as described later.
Although oxidative stress is a complex and often difficult subject of inquiry, it remains an important
underlying mechanism of cellular toxicity. Given the high reactivity of many ROS, such as ·OH, it is
not surprising that they are often indiscriminant in terms of cellular targets. Principal cellular constituents,
including lipids, proteins, and DNA, are subject to attack, as are cellular functions such as redox status
and energetics and cellular signaling. Tools have been developed to study all of these phenomena. The
following is not an exhaustive list of cellular effects but does address major ones that have received
considerable research attention in a variety of organisms, including fishes. (Note that oxidative DNA
damage is discussed in detail in Chapter 12 and hence is excluded here.)

Lipid Peroxidation
Perhaps the most studied targets of ROS are polyunsaturated fatty acids (PUFAs), which are fatty acids
containing two or more carbon–carbon double bonds. Of particular interest are those associated with
the membranes of cells, including organelles such as mitochondria, lysosomes, and endoplasmic reticula.
These membranes are complex and diverse structures, typically comprised of various phospholipids
(such as phosphatidylcholine, or lecithin), cholesterol, lipoproteins, and proteins that serve various
functions, particularly with respect to signal transduction and transport of materials across membranes.
In PUFAs, the hydrogen atoms on saturated carbons (allylic hydrogens) adjacent to carbons partici-
pating in double bonds are most prone to hydrogen abstraction by oxygen radicals such as ·OH, RO·,
and ROO·. These allylic hydrogens form less stable bonds to carbon due to the adjacent carbon–carbon
double bonds; an example within a hydrocarbon chain is denoted here in bold:

−
−CH = CH CH 2 − CH = CH –
Abstraction of an allylic hydrogen by an oxygen radical comprises the initiation step of lipid peroxi-
dation (Porter et al., 1995; Wagner et al., 1994), which is included in the summary schematic of lipid
peroxidation (Figure 6.5); susceptibility of different PUFAs to lipid peroxidation increases with increasing
number of unsaturated carbon double bonds. Initiation results in a lipid radical (R·), which then undergoes
rearrangement to a conjugated diene radical; under typical aerobic conditions, this lipid radical will readily
react with O , yielding a lipid peroxyl radical (ROO·). The peroxyl radical can react with another PUFA,
2
thereby abstracting hydrogen, becoming a lipid peroxide (LOOH), and generating another R·. This second
R· can also react with O to yield ROO·, and this process can be repeated many times, constituting a
2
free-radical chain reaction termed propagation of lipid peroxidation; thus, initiation by one molecule of
an oxygen radical can potentially result in the peroxidation of many PUFA molecules. Propagation is an
important feature of many free-radical reactions whereby one radical can stimulate a cascade of potentially
deleterious reactions in biological systems; this phenomenon is addressed again later.
A number of other important reactions are associated with the process of lipid peroxidation; for
example, the peroxyl radical can react with other membrane lipids (e.g., cholesterol) or proteins, in
addition to PUFA, thus altering these molecules while forming ROOH. Transition metals such as iron
and copper, in addition to enhancing production of the powerful initiator ·OH through Fenton chemistry,