Page 297 - The Toxicology of Fishes

Reactive Oxygen Species and Oxidative Stress 277

the cellular generation of ROS is an unavoidable cost of aerobic life—the price exacted for making use
of the energetic efficiency of O as an electron acceptor.
2
Perhaps quantitatively the most important sources of ROS during normal cellular metabolism are the
electron transport chains of mitochondria, the endoplasmic reticulum, and chloroplasts. Photosynthesis
within chloroplasts poses unique problems for plants in terms of oxidative damage, which can be
exacerbated by many common air pollutants (Hippeli and Ernstner, 1996). In animals, mitochondrial
respiration is likely the most important source of ROS in vivo. It is estimated that under normal cellular
levels of O consumption, about 0.1% of the O utilized by mitochondria forms O (Beckman and Ames,
•–
2
2
2
1998; Fridovich, 2004). Although the precise location of this leakage is not completely resolved and
may depend on conditions, complex I and III constituents are often involved (Finkel and Holbrook,
2000; Turrens, 1997). Endoplasmic reticula (microsomes in subcellular fractions) are another important
source of ROS production. Oxidative metabolism of endogenous compounds and xenobiotics by the
cytochrome P450 enzyme systems that function in this fraction were discussed in detail in Chapter 4.
As described, P450s catalyze oxidations by cleaving O , with one oxygen atom ultimately added to the
2
substrate and the other ultimately giving rise to H O. In this process, two electrons provided by
2
NADPH–P450 reductase or cytochrome b are sequentially added to drive catalysis. This cycle can be
5
•−
uncoupled, resulting in the diversion of electrons to give rise to O and H O (Goeptar et al., 1995).
2
2
2
Some xenobiotics can greatly enhance this uncoupling, as described later in this chapter.
Several oxidative enzymes can also generate ROS during catalysis, including xanthine oxidase, tryptophan
dioxygenase, diamine oxidase, guanyl cyclase, and glucose oxidase (Fridovich, 1978; Halliwell and Gut-
teridge, 1999). Additionally, a number of molecules with key roles in cellular function can auto-oxidize in
the presence of O , yielding O ; these include glyceraldehyde, FMNH , FADH , epinephrine and norepi-
•–
2
2
2
2
nephrine, dopamine, tetrahydropteridines, and thiols such as cysteine (Halliwell and Gutteridge, 1999). These
autoxidations are oftentimes substantially accelerated in the presence of transition metal ions such as iron
•–
and copper. The O -carrying proteins hemoglobin and myoglobin can also be sources of O (Balago-
2
2
palakrishna et al., 1996; Brantley et al., 1993). In these proteins, O binding requires the reduced state of
2
•–
iron, Fe(II), in the heme group. Occasionally, oxygen will release as O and concomitantly yield Fe(III) in
2
the heme. These forms of hemoglobin and myoglobin are referred to as methemoglobin and metmyoglobin,
respectively, and are unable to bind O . Approximately 3% of human hemoglobin is thought to undergo
2
conversion to methemoglobin every day, suggesting a substantial basal flux of ROS in erythrocytes (Win-
terbourn, 1985). As discussed later, many chemicals can significantly increase methemoglobin formation.
In the context of oxidative stress, ROS are cast as bad actors, but it is important to note that ROS
play positive roles as well. An important example of this is the production of ROS by neutrophils and
macrophages by many vertebrates wherein ROS are employed in the phagocytic activity of these cells
(Babior, 2000). Upon stimulation, these cells increase O consumption up to 20 times resting levels; this
2
phenomenon is referred to as the respiratory burst, a misnomer because it is unrelated to mitochondrial
respiration. Stimulants for this burst include opsonized bacteria and zymosan (from yeast cell walls),
bacterial peptides such as N-formylmethionylleucylphenylalanine (FMet-Leu-Phe), the lectin concanava-
lin A, and phorbol esters, such as phorbol myristate acetate (derived from oil of the seeds of Croton
tiglium, a plant native to southeastern Asia). During the respiratory burst, NADPH provided by the
pentose phosphate pathway is oxidized to NADP+ by NADPH oxidase, an enzyme complex associated
with the plasma membrane (Henderson and Chappell, 1996); the two electrons abstracted from each
•–
NADPH are transferred to O , yielding O as indicated in Equation 6.11:
2
2
+
+
NADPH + 2 O → NADP + H + 2 O 2 •– (6.11)
2
•–
•–
Because O is not highly reactive in aqueous solution, it is considered unlikely that O itself is the
2
2
ultimate phagocytic agent employed by neutrophils and macrophages (Halliwell and Gutteridge, 1999);
•–
however, O can readily react with itself and dismutate into hydrogen peroxide (Equation 6.12) or react
2
with NO·, which is sometimes produced by these cells, to produce peroxynitrite (see Equation 6.10).
Both products are bactericidal.
+
2O + 2H → H 2 O + O 2 (6.12)
•–
2
2