Page 216 - The Toxicology of Fishes

196 The Toxicology of Fishes

less) to several mammalian GSTs, including the rat alpha-class GST form rGSTA4-4, which, as discussed,
has a high catalytic efficiency toward conjugation of GSH with 4HNE. The presence of this interesting
GST in several fish species suggests an important conservation of function that likely protects fish lipid
membranes against the deleterious effects of oxidative injury.

Tissue Distribution
With the exception of the mammalian theta-class enzymes, all GSTs conjugate the prototypical substrate
1-chloro-2,4-dinitrobenzene (CDNB) with greatest activity (Figure 4.11). CDNB conjugating activity
has been observed in all fish species examined to date (Table 4.14) and occurs in multiple tissues (George,
1994). Because CDNB is a general substrate and the rate of conjugation can vary between isoforms by
up to two orders of magnitude, comparison of total CDNB conjugating activity in livers of different
species, for example, is probably of little relevance in toxicological evaluation of environmentally relevant
xenobiotics, as it will bear little relationship to the rate of metabolism of other compounds which may
be isoform specific. Functional comparisons of activity toward individual toxicants is perhaps more
meaningful from a toxicological viewpoint. Whereas kidney is the most active toward styrene-7,8-oxide
and BaP-4,5-oxide relative to other tissues in the little skate (Raja erinacea) (Bend et al., 1978; Foureman
et al., 1987; Gill et al., 1982), this finding must be placed in the context of relevance to the animal as
compounds of this type may not be present systemically. They will more likely be taken up in the diet
or generated by metabolism in the intestine or liver and conjugated in these tissues.
In general, most in vitro laboratory investigations of GST–CDNB conjugating activities in fish tissues
use assay conditions and substrate concentrations similar to those proposed for mammals: 1 mM GSH
and 1 mM CDNB) (Habig and Jakoby, 1981). Kinetic studies, however, have demonstrated that these
substrate concentrations may not be at saturation with respect to initial rate kinetics for many isoforms
because the K values differ by up to two orders of magnitude. Because the enzymes contain both
m
electrophile and nucleophile binding sites, GST detoxification rates are determined by the concentration
of electrophilic substrate and by the concentrations of nucleophilic cosubstrate (GSH). Kinetic studies in
largemouth bass and brown bullheads in the presence of variable electrophile concentrations suggest that
in vitro saturation of hepatic GST–CDNB conjugation occurs at higher electrophile concentrations in
brown bullheads than in largemouth bass (Gallagher et al., 2000). Such an observation is consistent with
a higher capacity for brown bullheads to detoxify electrophilic GST substrates under conditions of high
environmental exposure. These observations are important under the prototypical conditions of environ-
mental chemical exposure when the amount of chemical reaching the liver is relatively low and the rate
of in vivo clearance is directly proportional to the amount of chemical concentrations in vivo. Also, as
alluded to earlier, it is important to consider that the rate of GST–CDNB activity may not be reflective
of rates of GST conjugation of environmentally relevant GST substrates, such as pesticides or epoxide
carcinogens. For example, the rates of hepatic GST–CDNB conjugation by starry flounder and English
sole are not correlated with the rates of hepatic GST conjugation of (+)-7β,8α-dihydroxy-9α,10α-oxy-
7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) in those species. Specifically, starry flounder catalyze the rate
of CDNB conjugation at initial rates threefold higher than is observed in English sole, whereas GST–BPDE
activities are threefold higher in English sole as compared to starry flounder (Gallagher et al., 1998).
Thus, specific GST conjugation of environmental chemicals in fish must be quantitated under assay
conditions where the conjugation of the chemical can be monitored directly.
Although glutathione conjugation is generally associated with detoxification, small halogenated alk-
enes, such as dibromoethane, are activated by glutathione conjugation. In mammals, theta-class GSTs
are particularly efficient in the conversion of dibromoethane to the carcinogenic sulfonium ion metabolite,
as shown in Figure 4.14 (Thier et al., 1996). It was shown that a small fish species, the medaka, was
quite susceptible to the development of liver cancer following exposure to ethylenedibromide and that
a form of GST in liver was increased in the exposed fish, suggesting the presence of a theta-like GST
in the medaka (Hawkins et al., 1998). This has been confirmed with cloning studies. Other fish species,
such as the pleuronectid flatfish, bass, and mullet, have been shown to have theta-like GST in their livers
(Gallagher et al., 2000; Leaver et al., 1993; Martinez-Lara, 1997). It is tempting to speculate that this
may account for the high tumor incidence observed in flatfish from some polluted environments.