Page 301 - The Toxicology of Fishes

Reactive Oxygen Species and Oxidative Stress 281

O O
C
H C H Gly
N H
Gly Gly
O C
H C CH 2 SH
N H Cys Cys S S Cys

O C
CH Glu Glu
2
Glutathione disulfide
CH 2
H N C H Glu
3
C
O O
Reduced glutathione
FIGURE 6.1 Structures of reduced glutathione (GSH) and glutathione disulfide (GSSG).

Glutamate cysteine ligase is the rate-limiting enzyme of GSH synthesis; it is sensitive to inhibition
by buthionine sulfoximine (BSO), which can be used experimentally to deplete cellular GSH (Meister,
1995). GCL has been purified from a number of eukaryotes, and all are heterodimers composed of a
heavy and a light subunit (molecular weights of about 73,000 and 31,000, respectively) (Griffith, 1999;
Griffith and Mulcahy, 1999). The heavy subunit (GCL or GCLC, where C stands for “catalytic”) is
h
responsible for catalytic activity; the light subunit (GCL or GCLM, where M stands for “modifier”)
l
plays a regulatory function. In mammals, genes for the two are on separate chromosomes, and their
transcription does not appear to be highly coordinated, although both have an antioxidant response
element (ARE) in the promoter region (discussed below). Factors affecting GCL activity include avail-
ability of substrates (glutamate and cysteine) and feedback inhibition by GSH. GCLM modulates activity
of the complete enzyme essentially by reducing K values for substrate concentrations and increasing
m
the K for GSH.
i
As described above, GPX couples the reduction of H O and LOOH to corresponding alcohols with
2
2
•–
the oxidation of GSH to GSSG. GSH can also directly scavenge ROS, including O , ·OH, RO·, ROO·,
2
−
and ONOO (Griffith, 1999; Halliwell and Gutteridge, 1999), with the thiol (–SH) group provided by
cysteine as the active moiety that undergoes oxidation. In these cases as well, GSSG arises from GSH
oxidation as a consequence. GSSG contains the backbones of two GSH molecules, covalently linked
through a disulfide bond (Figure 6.1). Normal healthy cells not experiencing undue oxidative pressure
typically exhibit GSH:GSSG ratios approaching or greater than 100:1, and declines in this ratio have
been used as a marker of oxidative stress. This glutathione redox balance is maintained by GSH synthesis,
export of oxidized and conjugated glutathione, and activity of glutathione reductase (GR), which cata-
lyzes the reduction of GSSG to GSH:
+
+
+
+
GSSG NADPH H → 2 GSH NADP + (6.19)
Glutathione reductase contains two subunits, each with fatty acid desaturase (FAD) at its active site
(Thienne et al., 1981). Electrons provided by NADPH (produced by the pentose phosphate pathway)
are apparently passed through FAD en route to their reduction of GSSG. In addition to the direct effects
of ROS described below, the energetic costs of antioxidant defense activity are significant, as illustrated
by the energy required to produce, maintain, and utilize GSH for this role.