Page 306 - The Toxicology of Fishes

286 The Toxicology of Fishes

Ceruloplasmin serves an analogous function for copper (Harris, 1995). Dietary copper entering the
bloodstream largely binds to albumin; this complex is taken up by the liver, where the copper is
incorporated into ceruloplasmin. Ceruloplasmin has a molecular weight of about 132,000 and tightly
binds six copper ions. The copper–ceruloplasmin complex is secreted by the liver into the plasma and
can contribute copper to cells requiring it following binding to cell surface receptors. Ceruloplasmin has
been found in several fish species, including carp, plaice, mullet, tilapia, and European eel (Grosell et
al., 1998).
Metallothioneins (MTs) are low-molecular-weight (about 6500) proteins, the primary role of which
is unclear, although evidence suggests that antioxidant activity may be at least an ancillary function
(Coyle et al., 2002). Most research has focused on their roles in metal metabolism and homeostasis,
including metal detoxification (Klaassen et al., 1999). They are very cysteine rich (about 30% of amino
acid residues), which underlies their high binding affinities for a number of metals, including copper,
cadmium, zinc, silver, bismuth, and mercury. Two isoforms, MT-1 and MT-2, appear to occur in all
animal tissues and are the most-studied MTs. They typically bind five to seven metal ions per molecule
and are inducible by some metals (including those mentioned above), stress hormones (such as gluco-
corticoids), oxidants, and inflammation. Considerable debate currently exists regarding whether their
primary role is regulation of essential metals, particularly copper and zinc, for use in metalloenzymes
vs. protection against metal toxicity, particularly from cadmium. Additionally, several lines of experi-
mental evidence support an antioxidant function for MTs, including inducibility by prooxidants such as
hydrogen peroxide and paraquat, protection against such chemicals in MT-enriched vs. MT-depleted
cells (via transfections and gene knock-out models, for example), and the ability of MT to scavenge
ROS and spare GSH (Klaassen et al., 1999). Whether or not these results have significant in vivo
ramifications remains unclear.

Reactive Oxygen Species and Gene Expression
Antioxidant defenses can be altered in response to exposure to oxidative stress in a variety of ways, and
the mechanisms by which such regulation occurs have been extensively studied in prokaryotic and
mammalian systems. Expression and activity of antioxidant, as well as prooxidant, gene products are
up- or downregulated at the levels of mRNA transcription, mRNA stabilization, and protein activation;
however, the mechanisms by which these alterations occur, as well as the genes involved, are much more
complex in eukaryotes than in prokaryotes.

ROS-Mediated Modulation of Gene Expression in Prokaryotes
In prokaryotes, well-defined redox-sensitive transcription factors first recognize ROS and then acti-
vate the transcription of antioxidant genes (Bauer et al., 1999). For example, the OxyR protein is
specifically oxidized by (recognizes) hydrogen peroxide; the oxidized form of the protein, in which
a disulfide bond has been created, drives transcription of genes, including MnSOD, catalase, glu-
tathione reductase, hydroperoxidase, heat shock proteins, and glutaredoxin (Bauer et al., 1999; Zheng
and Storz, 2000). Similarly, a [2Fe–2S] cluster in the SoxR protein is oxidized in the presence of
superoxide-generating chemicals, at which point SoxR drives the transcription of the soxS gene. The
SoxS protein, a transcription factor, acts to increase expression of genes including MnSOD, glucose-
6-phosphate dehydrogenase, NADPH:flavodoxin oxidoreductase, aconitase, and endonuclease IV
(Zheng and Storz, 2000). Interestingly, in some cases the proteins induced play an indirect rather
than direct role in conferring resistance to oxidative stress. For example, fumarase C is induced via
SoxS under conditions of oxidative stress, although fumarase, a citric acid cycle enzyme, is not itself
an antioxidant. Fumarase C induction is essential under conditions of oxidative stress because the
other forms of this enzyme, fumarases A and B, are inactivated by high oxygen conditions (Liochev
and Fridovich, 1992).