Metabolism of selenomethionine by rainbow trout (Oncorhynchus mykiss) embryos can generate oxidative stress
Introduction
A requirement for the metalloid selenium (Se) in the diet of vertebrates has been recognized for several decades (Stadtman, 1979). Dietary deficiency results in tissue damage as a result of the inhibited activity of the enzyme glutathione peroxidase, which incorporates selenium in its structure as selenocysteine (Rotruck et al., 1973). Despite being an essential dietary factor, vertebrates exposed to levels of selenium only several-fold greater than those required exhibit toxicity (Skorupa, 1998; Lemly, 1997; Hamilton et al., 1990). In fact, toxic levels of selenium have been identified in several ecosystems, and Skorupa (1998) reviewed 12 examples in which elevated selenium exposure had adversely affected biota inhabiting those areas.
The toxicity of selenium has most often been attributed to its similar chemical properties to sulfur and its ability to substitute for that element during the assembly of proteins (Maier and Knight, 1994). Rapidly growing organisms appear to be most sensitive to the effects of elevated selenium. Oviparous vertebrates are the most sensitive, as these organisms efficiently transfer selenomethionine to their eggs (Kroll and Doroshov, 1991; Lemly, 1996). Toxic effects are manifested in the actively growing embryos when they have assimilated selenium from the yolk. In wild populations it is not unusual to find adults that appear healthy inhabiting areas of high selenium, while their offspring often exhibit elevated rates of mortality and characteristic deformities (Lemly, 1997). In fish, these characteristic deformities include overt spinal curvatures, shortened jaw structures, missing or deformed fins, and edema (Lemly, 1997), while in birds spinal, wing, and cranial deformities have been identified (Spallholz and Hoffman, 2002).
Over the last decade evidence has accumulated that not all of the toxic effect of selenium can be explained by the simple substitution of selenium for sulfur during protein assembly. In particular, the role of oxidative stress resulting from selenium toxicity has been receiving increased attention. Spallholz and Hoffman (2002) reviewed the literature relevant to birds and showed convincing evidence that oxygen radicals play a role in generating physical and biochemical lesions in birds exposed to high levels of selenium. Despite the fact that oxidative stress has been identified as a mechanism of selenium toxicity in birds, this has not been assessed in fish.
We began our investigations of oxidative stress as a mechanism of toxicity in fish exposed to Se after observing lesions consistent with oxidative damage. Specifically, rainbow trout collected where concentrations of Se in the water and biota (i.e., invertebrates and fish) are elevated have offspring that develop edema. This edema develops around the yolk sac and pericardial area subsequent to hemorrhage in the region of the developing heart and vasculature of the yolk sac soon after the development of these circulatory elements (Holm, 2002). The etiology of these embryonic alterations is similar to the development of edema in fish exposed to organochlorine contaminants that are known to generate oxidative stress in fish embryos (Bauder, 2002).
Selenium's prooxidant activity arises from its ability to oxidize thiols (Spallholz, 1994). Another cellular antioxidant and thiol, glutathione, appears to be particularly amenable to complexing with certain forms of selenium. In some of its forms, selenium may combine with glutathione to form a selenopersulfide anion that ultimately generates superoxide radicals, a potent oxidizing species that can damage cellular components (Spallholz et al., 1998). The chemical speciation of selenium is complex, and not all forms of selenium are capable of generating superoxide radicals by association with glutathione. In fact, selenomethionine, the dominant form of selenium in the eggs of fish and birds was not active in the generation of superoxide in an in vitro assay system (Spallholz et al., 2001). However, recently published studies have documented the ability of some cell types to catalyze the metabolism of selenomethionine to alternative forms that are capable of producing superoxide (Wang et al., 2002; Miki et al., 2001). Here we describe the results of experiments that examined the ability of fish embryos, at various stages of development, to produce a superoxide radical from selenomethionine in the presence of glutathione. Isolation of this enzymatic activity from the offspring of rainbow trout (Oncorhynchus mykiss) provides important information regarding the mechanism of toxicity in fish exposed to elevated selenium concentrations.
Section snippets
Fish
Eggs were obtained from rainbow trout brood stock held at the Freshwater Institute for approximately 1 year prior to spawning. The brood stock were originally obtained from Rainbow Springs hatchery (Thamesford, Ont., Canada). Eggs were fertilized with a consistent volume (10 μL/50 mL of eggs) of a composite milt sample obtained from five males of the same stock. After fertilization, the eggs were allowed to water harden and were then distributed into a Heath tray-type vertical incubator supplied
Results
Methylselenol, a metabolite of selenomethionine cleavage, produced chemiluminescence in a fashion linear (R2=0.99, P<0.05) to its concentration in the assay (Fig. 1). The detection limit of the assay was established at twice background light emissions in the blank assays and corresponded to 20 ng of selenium in the form of methylselenol in the final assay volume of 700 μL. The detection limit for methylselenol in these studies is similar to previous detection levels published by Spallholz et al.
Discussion
It is of particular interest that the peak of activity for generating superoxide from selenomethionine occurs after early liver development. The liver begins to be perfused with blood near 180 TU, and the appearance of bile in the gut can be detected at approximately 220 TU (Ballard, 1973). Metabolism of organic contaminants by the developing liver first appears in the same approximate time window (i.e., 160–210 TU) (Brinkworth, 2001). The appearance of superoxide dismutase activity in the liver
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