Page 300 - The Toxicology of Fishes

280 The Toxicology of Fishes

classical GPX (GPX1), gastrointestinal GPX (GPX2), plasma GPX (GPX3), and phospholipid hydrop-
eroxide GPX (GPX4 or PHGPX). All contain selenocysteine at the active site. Selenocysteine is an
amino acid analogous to cysteine in which the sulfur atom has been replaced by selenium. It is inserted
by a specific tRNA into selenoproteins, including GPXs. Most GPXs characterized in vertebrates contain
four subunits, each containing one selenium residue; PHGPX, however, is monomeric.
Little work has been done verifying the nature of GPXs in fish, although based on tissues examined
and substrates used, it is likely that most selenium-dependent GPX activity measured in fish thus far
corresponds to GPX1. Kryukov and Gladyshev (2000) detected 18 genes in zebrafish that contained
selenocysteine. Two appeared very similar to one another and resembled both human GPX1 and
GPX2; the other two were also very similar to one another and resembled human GPX4. The
occurrence of highly matched pairs probably arose from a gene duplication event in many fishes,
including zebrafish.
The reactions catalyzed by GPXs involve the reduction of a peroxide substrate to its corresponding
alcohol, coupled with the oxidation of reduced glutathione (GSH) to glutathione disulfide (GSSG)
(Chance et al., 1979). In the case of H O , the corresponding alcohol is water (Equation 6.15); with lipid
2
2
peroxides, it is the corresponding lipid alcohol (LOH, Equation 6.16):
+
HO 2 + 2 GSH → GSSG HO (6.15)
2
2
+
LOOH → 2 GSH → GSSG LOH (6.16)
An important distinction between catalase and GPX is the ability of only the latter to reduce lipid
peroxides; however, most GPXs, including classical GPX, cannot metabolize lipid peroxides that are
esterified to lipid molecules in membranes, for example. Such peroxides must first be released by lipase
activity. An exception to this is PHGPX, which can directly reduce lipid peroxides associated with
membranes (and also free LOOH), as well as thymine hydroperoxide, a form of oxidative DNA damage
(Bao and Williamson, 2000).
Some glutathione S-transferases (GSTs; extensively discussed in Chapter 4) exhibit peroxidase activity,
which is specific for LOOH (they are unable to act on H O ). This activity is sometimes referred to as
2
2
selenium-independent or non-selenium GPX activity, as GSTs contain no selenium. Although it can
account for an appreciable portion of total GPX activity measured in tissue preparations with standard
substrates such as cumene hydroperoxide, its significance in vivo is unclear (Halliwell and Gutteridge,
1999). Quantification of real GPX activity using H O as substrate is sometimes preferred. Some GSTs
2
2
play another role in antioxidant defense by conjugating breakdown products from lipid peroxidation
such as 4-hydroxynonenal (Hayes et al., 2005).

Glutathione: Synthesis and Maintenance
As should be clear from earlier discussions of GSTs (Chapter 4) and discussions in this chapter,
glutathione (GSH) plays critical roles in the protection of cells from chemical insult. It is also plays
additional roles in metabolism, biosynthesis, transport, and cellular communication; thus, a brief descrip-
tion of this molecule with particular reference to oxidative stress is warranted at this juncture. GSH is
the tripeptide γ-glutamyl-cysteinyl-glycine (Figure 6.1); concentrations vary widely among species and
tissue but are generally far higher in mammals than amino acid concentrations, typically occurring in
the low millimolar range (Griffith and Mulcahy, 1999); similar levels have been reported in fishes (see
below). GSH is synthesized through two reactions, the first catalyzed by glutamate cysteine ligase (GCL;
previously termed γ-glutamylcysteine synthase, or GCS) and the second by GSH synthetase (Equation
6.17 and Equation 6.18, respectively):

L-glutamate + -cysteine + ATP → L- -glutamylccysteine + ADP + P i (6.17)
γ
L
+
+
γ
L--glutamyl- -cysteine + L-glycine + ATP → GSSH ADP P i (6.18)
L