Page 321 - The Toxicology of Fishes

Reactive Oxygen Species and Oxidative Stress 301

Studies with Fishes
A large number of studies of oxidative stress in fish have been carried out in the last 25 years or so,
with different motivations (Di Giulio et al., 1989). These motivations might be summarized as: (1)
increasing the understanding of basic biochemical and molecular mechanisms related to oxidative stress
in fish, (2) developing biomarkers related to oxidative stress fish, and (3) investigating the relationships
between oxidative stress and the health of wild populations of fish. Some of the results of the studies
carried out are presented below with respect to these three concerns.

Basic Biochemical and Molecular Mechanisms of Oxidative Stress in Fish
The study of the toxicity of prooxidant xenobiotics in fish serves both to inform risk assessment with
regard to fish populations and to inform our understanding of the toxicity of such chemicals in other
organisms, including humans (Bailey et al., 1996; Di Giulio et al., 1989; Kelly et al., 1998; Winston,
1991). Mechanistic studies in fish can be further categorized as characterizing the ability of a xenobiotic
to generate ROS in fish cells or living fish, as well as characterizing the antioxidant defenses present in
different species of fish, either with or without prior xenobiotic exposure. It is often useful to employ
comparative studies involving different species (piscine and nonpiscine) to better understand the impact
of prooxidant chemicals in the environment.

In Vitro Biochemical Generation of ROS
In vitro studies have clearly demonstrated that ROS are generated in subcellular fractions (microsomal,
mitochondrial, and cytosolic) of fish tissues. Generation of ROS in fish, as in mammals, is sometimes
involved in normal physiological processes such as immune function (Anderson, 1994); moreover, release
of ROS by phagocytic cells can be stimulated after pollutant exposure in fish (Fatima et al., 2000). In
addition, laboratory and field exposures to prooxidant chemicals have resulted in increased ROS pro-
duction in subcellular fractions of various tissues, particularly liver, gill, and kidney (Lemaire et al.,
1994; Livingstone, 2001; Livingstone et al., 2000; Peters et al., 1996; Schmieder et al., 2003; Washburn
and Di Giulio, 1988). As discussed previously in this chapter, xenobiotics have been shown in vitro to
enhance ROS production and oxidative stress by diverse mechanisms, including redox cycling, depletion
of glutathione or other antioxidants, facilitation of Fenton chemistry, uncoupling of mitochondrial
electron transport, and uncoupling of cytochrome P4501A monooxygenation reactions.

Enzymatic Antioxidant Defenses in Fish
Studies aimed at understanding the biochemistry and, to a much lesser degree, the molecular biology
of fish antioxidant systems have also been carried out. Many if not all of the basic antioxidant defenses
(enzymatic and nonenzymatic) characterized in mammals are also present in fish. Representative studies
describing antioxidant enzymatic activities, reactivity of mammalian antibodies to presumably homol-
ogous fish antioxidant enzymes, and sequences for antioxidant genes in fish are presented in Table 6.3.
In addition, similar evidence supports the existence of many of the phase II enzymes that can be viewed
as playing an antioxidant role or that are regulated by an ARE in mammals (discussed earlier in the
section on the ARE and in Chapter 4). Certain GST isoforms in mammals, for example, may play an
antioxidant role both by conjugating electrophilic compounds in general (Chapter 4) and by metabolizing
4-hydroxy-2-nonenal (HNE) (Eaton and Bammler, 1999). HNE is a lipid peroxidation product that is
both highly toxic and involved in ROS-related signaling (Poli and Schaur, 2000); recent evidence
indicates that fish species also express a GST isoform capable of metabolizing HNE (Leaver and George,
1998; Pham et al., 2002; 2004). Similarly, there is evidence that in fish, as in mammals, some GST
isoforms possess a glutathione peroxidase activity (Martínez-Lara et al., 2002).
Unfortunately, the exact isoforms of antioxidant enzymes present in different fish species have rarely
been carefully identified and characterized. In most cases, enzyme activities have been measured with
slight adaptations of assays developed with mammalian species and may reflect the activities of multiple