Page 214 - The Toxicology of Fishes

194 The Toxicology of Fishes

three fish species, including cod, plaice, and trout. The relative hepatic activity ratios are 40 for trout,
4 for cod, and 1 for plaice. Although this would correctly indicate the predominance of a pi-class enzyme
in trout and not in plaice (despite the presence of a significant amount of a GSTA4 homolog), it does
not reflect immunological data showing that the concentration of GST pi in cod liver is very little lower
than that of trout. Thus, the usefulness of diagnostic substrates is limited, and this approach is less
reliable than immunochemical investigations.
In lower vertebrates, such as elasmobranchs and teleosts, both the covalent and noncovalent binding
activities of the GSTs are very much lower as compared to rodents (Foureman et al., 1987; George and
Buchanan, 1990). This may be attributed to the lower abundance of a GSTA1 homolog or an evolutionary
adaptation of the enzyme in terrestrial vertebrates, as it has been postulated that terrestrial plant phyto-
alexins are bound by this protein. Teleost species contain stores of polyunsaturated fatty acids that are
readily oxidized by free-radical attack, and this may explain the high constitutive levels of isoforms that
detoxify lipid peroxidation products such as the alkenals and hydroxynonenals (e.g., GSTA class and
GSTA4 homologs) as they will be better protected against xenobiotic-induced oxidative damage. This
is particularly relevant in fish such as the cod and plaice where the fat is stored in droplets within the
hepatocytes and not in adipose tissue as in the salmonids.
The substrate specificities, primarily with prototypical and endogenous substrates, have been deter-
mined with a number of highly purified preparations or recombinant GSTs from several fish species. In
common with mammalian GSTs, they show greatest activity with CDNB as the substrate. The alpha-
class enzyme from sea bass conjugates the alkenal trans-non-2-enal (N2E) at a higher rate than the
prototypical xenobiotic substrates, and of these the highest rates were observed with ethacrynic acid
(ETHA) and nitrobutyl chloride (NBC) (Angelucci et al., 2000). This is in agreement with an assignment
of the sea bass enzyme as a GSTA4 homolog. The pi-class enzymes from salmon, trout, and catfish all
exhibit relatively high rates of conjugation of ETHA, again following the pattern observed with a
mammalian GST. The catfish enzyme has high activity with (±)-benzo(a)pyrene-4,5-oxide and anti-
benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide as substrates, showing that it is an effective detoxicant of
the active carcinogenic metabolite of BaP (Gallagher et al., 1996).

Fish GST and Oxidative Stress
As mentioned above, in addition to their protective activities toward electrophilic chemicals, certain GST
isozymes can catalyze the reduction of cellular peroxides to their corresponding alcohols, as well as
conjugate endogenous genotoxic unsaturated aldehydes formed during the peroxidation of membrane
lipids (Alin et al., 1985; Hubatsch et al., 1998). Accordingly, the GST pathway in some species is an
integral component of the cellular antioxidant defense system. Of the reactive intermediates produced
during oxidative stress, 4-hydroxynonenal (4HNE) is a particularly reactive α,β-unsaturated aldehyde that
is generated during lipid peroxidation as a result of the degradation of ω-6 polyunsaturated fatty acids
(Esterbauer et al., 1991). 4HNE production is accelerated during exposure to a variety of prooxidant
environmental pollutants (Figure 4.13). Because of its high reactivity, 4HNE rapidly forms covalent
adducts with biomolecules containing nucleophilic sites, such as sulfhydryl groups of glutathione, cysteine,
lysine, and histidine residues of proteins, and nucleophilic sites of nucleic acids. In rodents and humans,
the alpha-class GSTA4 subclass displays uniquely high catalytic activity toward 4HNE and other α,β-
unsaturated aldehydes, suggesting that these enzymes may have distinctively evolved as a secondary line
of defense against oxidative injury (Hubatsch et al., 1998). As discussed previously, studies with the
marine fish plaice (Pleuronectes platessa) have revealed the presence of a GST enzyme (GSTR1) that is
a relatively efficient catalyst for the conjugation of a series of unsaturated alkenals and hydroxyalkenals,
including 4HNE, but displaying little or no activity toward model substrates for mammalian GST. The
recombinant Rho class of enzymes in plaice displays higher rates of conjugation of the natural substrates
trans-oct-2-enal (O2E), N2E, and 4-hydroxy-2,3-trans-nonenal (4HNE) than prototypical substrates (apart
from CDNB) (Leaver and George, 1998; Martinez-Lara et al., 2002). They also exhibit a high glutathione-
dependent peroxidase activity with cumene hydroperoxide; substrate activity with phospholipid hydro-
peroxides has not been studied. Although both isoforms conjugate 4HNE at the same rate, GSTR1 displays
a two- to tenfold higher activity toward O2E and N2E and also shows low but measurable activity with