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Metabolism of selenomethionine by rainbow trout (Oncorhynchus mykiss) embryos can generate oxidative stress
ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 58 (2004) 17–21
Metabolism of selenomethionine by rainbow trout (Oncorhynchus mykiss) embryos can generate oxidative stress Vince P. Palace,a, Julian E. Spallholz,b Jodi Holm,b Kerry Wautier,a Robert E. Evans,a and Christopher L. Barona a
Department of Fisheries and Oceans, Habitat Impacts Research, Freshwater Institute, 501 University Crescent, Winnipeg, MB, Canada R3T 2N6 b Texas Tech University, Selenium Technologies Inc., Lubbock, TX, USA Received 2 December 2002; received in revised form 8 August 2003; accepted 25 August 2003
Abstract Although selenium is required by vertebrates, toxicity can arise at concentrations only slightly greater than those they require. The toxicity of Se is thought to arise from its ability to substitute for sulfur during the assembly of proteins. However, recent studies also indicate that some forms of selenium are capable of generating oxidative stress in an in vitro test system that includes glutathione. l-Selenomethionine, the predominant form of selenium in the eggs of oviparous vertebrates, does not generate oxidative radicals in this system, but lesions consistent with oxidative stress have been identified in fish and birds with high concentrations of Se. Here we report on the ability of rainbow trout embryos to transform l-Selenomethionine to a form capable of producing a superoxide radical. Oxidative stress appears to be generated by methioninase enzyme activity in the embryos that liberates methylselenol from l-Selenomethionine. Methylselenol redox cycles in the presence of glutathione producing superoxide and likely accounts for oxidative lesions present in fish and birds environmentally exposed to excessive loads of selenomethionine. r 2003 Elsevier Inc. All rights reserved. Keywords: Selenium; Rainbow trout; Oxidative stress; Superoxide radical; Teratogensis; Biotransformation
1. 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 severalfold 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
Corresponding author. Fax: +1-204-984-6587. E-mail address: [email protected] (V.P. Palace).
0147-6513/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2003.08.019
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)
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V.P. Palace et al. / Ecotoxicology and Environmental Safety 58 (2004) 17–21
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.
2. Materials and methods 2.1. 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 mL/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 traytype vertical incubator supplied with 6 L/min of dechlorinated Winnipeg city tap water (8 C, dissolved oxygen495% saturation). Subsamples of the incubating fertilized eggs or embryos were removed throughout their incubation period. Rainbow trout eggs require approximately 400 temperature units (TU=incubation temperature days held at that temperature) to reach the stage referred to as ‘‘swim-up,’’ when the yolk has been almost entirely absorbed and the fry begin to feed exogenously. Eggs and embryos were subsampled from 8 to 384 TU and frozen at 90 C until they could be prepared for analysis. To prepare for analysis, three eggs or embryos were homogenized in 5 vol of ice-cold 0.05 M phosphate buffer (pH, 7.0). Crude homogenates were centrifuged at 10,000g for 10 min and the supernatant was filtered through Supelco C-18 columns with 1 g of packing material (Sigma–Aldrich, Mississauga, Ont., Canada) to remove lipids. The filtrate from these columns was used directly in the assay for superoxide radical production from selenomethionine.
2.2. Assay for superoxide production The production of superoxide radical was measured in the presence of selenomethionine as previously described (Spallholz et al., 2001). Briefly, 100 mL of sample was introduced into 0.05 M sodium borate buffer (pH, 9.0) containing 1 mg/mL of reduced glutathione, 1 mg/mL of selenomethionine and 0.1 mg/mL of lucigenin (Sigma Chemical Co, St. Louis, MO, USA). The detection of chemiluminescence generated by the superoxide radical in the presence of lucigenin was performed using a Model 535 Los Alamos luminometer that integrated the signal for four consecutive 10-s intervals. Blanks for each sample were included by substituting 100 mL 0.05 M phosphate buffer (pH, 7.0) for the sample. Chemiluminescence not specific to metabolism of selenomethionine was corrected for by performing analyses of the sample using a buffer that had l-methionine instead of selenomethionine. Detection limits of the assay were established using known concentrations of methylselenol, the anticipated product of selenomethionine metabolism, in an identical assay procedure. The dependence of chemiluminescent activity on the production of superoxide radical was examined by the introduction of 5 mg of superoxide dismutase enzyme (SOD) (Sigma) to the reaction and the recording
ARTICLE IN PRESS V.P. Palace et al. / Ecotoxicology and Environmental Safety 58 (2004) 17–21
of chemiluminescence for a further four integration periods.
Each sample and blank was analyzed in duplicate and the means of four consecutive integration periods were used to quantify chemiluminescence. Data are presented as means7SEM.
2
R =0.99, p<0.05
250
Relative Light Units
60 40 20
Mean Se Blank Value
0
100
200
300
400
Temperature Units After Fertilization
Methylselenol, a metabolite of selenomethionine cleavage, produced chemiluminescence in a fashion linear (R2 ¼ 0:99; Po0:05) 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 mL. The detection limit for methylselenol in these studies is similar to previous detection levels published by Spallholz et al. (2001). Chemiluminescence from methylselenol was abolished to background levels by the addition of SOD, confirming that the emissions were dependent on the production of superoxide radical and its interaction with lucigenin. Fig. 2 shows relative light units emitted from the samples of fish eggs or embryos for the developmental stages that were sampled. Based on previous experiments and the range of light detection from analytical blanks, twice background light emission was accepted as a significant response for the assay. The superoxide radical, which is indicated by an increase in light emission, was not significantly detected in the rainbow trout embryos until 216 TU after fertilization, after
200 150 100 50 0 0
Selenomethionine L-Methionine
0
3. Results
300
80
Relative Light Units
2.3. Data analysis
19
20
40
60
80
100 120 140 160 180
Selenium in the Assay (ng) Fig. 1. Chemiluminescence produced by methylselenol in an in vitro assay system consisting of 0.05 M sodium borate buffer (pH, 9.0) with 1 mg/mL of reduced glutathione and 0.1 mg/mL of lucigenin. Data are shown as mean7SEM (n ¼ 5).
Fig. 2. Chemiluminescence produced by rainbow trout embryo extracts at various stages of development in an in vitro assay system for superoxide. Data are shown as mean7SEM (n ¼ 5).
which it remained elevated above background until 248 TU. The greatest response was obtained at 232 TU, when the rainbow trout embryos were at the eleuthroembryo stage with a functioning heart and circulatory system as well as liver (Ballard, 1973). Later stages (i.e., 4300 TU) of development did not produce chemiluminescence above background levels. Chemiluminescence from samples with the highest activity (232 TU) was reduced to background levels with the addition of 100 units of SOD to the assay volume, confirming the role of superoxide in the biologically mediated generation of chemiluminescence. Chemiluminescence was never above background for the sample extracts incubated in the same buffer when l-Methionine was substituted for selenomethionine.
4. 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 does not appear until later in development (Cowey et al., 1985), which accounts for the abolishment of activity in rainbow trout embryos after 300 TU in this study. In support of these observations for superoxide, the light emission from samples with the highest activity (232 TU) was reduced to background levels with the addition of 100 units of SOD to the assay volume. We have previously observed hemorrhages in the region of the heart in fry derived from adults at sites with elevated selenium concentrations (Holm, 2002). This hemorrhaging occurs at about the same time that
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V.P. Palace et al. / Ecotoxicology and Environmental Safety 58 (2004) 17–21
the livers of rainbow trout fry become functional. This liver development period coincides with the period during which superoxide production was most strongly observed from selenomethionine in the current experiments. Hemorrhaging in rainbow trout fry is followed closely by the development of yolk sac and pericardial edema (Fig. 3) (Holm, 2002). Increased permeability, inferred from hemorrhaging, and heart dysfunction could certainly be mediated by the production of the superoxide radical and the development of oxidative stress in myocardial tissue and the surrounding vasculature (Hill and Singal, 1996). At least one other group has shown similar pathologies in trout fry exposed to organic contaminats that induce biotransformation enzymes that elevate oxidative stress endpoints (Bauder, 2002). It has been shown previously that biotransformation of organoselenium compounds by P450 or flavin monoxygenase (FMO) enzymes can produce selenoxides. These selenoxides in turn may liberate seleninic acid (Chen and Zielger, 1994; Rooseboom et al., 2001), a potent redox cycling compound that can elevate oxidative stress in vivo (Spallholz and Hoffman, 2002).
Pericardial edema Yolk sac edema
Fig. 3. Rainbow trout embryos at 232 TU showing yolk sac and pericardial edema relative to normal development.
However, it is not likely that this activity accounts for the generation of chemiluminescence in the present studies. Biotransformation by both FMO and P450 enzymes requires the presence of NADPH, with declining rates of catalysis observed when this cofactor is depleted (Rooseboom et al., 2001). Our assay did not contain any added NADPH, and transformation rates remained elevated and stable for methylselenol standards and the egg extracts over long periods (440 min). Furthermore, selenocysteine, which was transformed by FMO and P450 activity in the previous studies, did not generate chemiluminescence in the presence of glutathione in our in vitro system (unpublished observations). The presence of selenomethionine in the assay system was essential for the production of the superoxide radical. This necessity was evident in the fact that light production was never above background for the sample extracts incubated in the same buffer when l-Methionine was substituted for selenomethionine. At least two independent groups have shown that some forms of selenium are able to form redox cycling species that oxidize reduced glutathione-generating superoxide radicals (Spallholz et al., 2001; Wang et al., 2002). Until recently, l-Selenomethionine has been regarded as one of the forms of selenium that does not generate superoxide radical in vitro. However, we have recently shown that l-Selenomethionine generates superoxide in the presence of the enzyme methioninase (Spallholz et al., 2003). It is likely, as shown by Wang et al. (2002), that methylselenol is the enzymatic product generating superoxide in this reaction (Fig. 4). Results from this series of experiments suggest that methioninase activity may be responsible for the teratogenesis observed in offspring of adult fish that have ingested sublethal but elevated concentrations of selenomethionine. Most notably, the stage of development after the liver becomes functional but before SOD enzyme activity is present appears particularly susceptible. Further investigations to characterize methioninase activity at various embryonic developmental stages of rainbow trout are ongoing in our laboratories.
Fig. 4. Proposed mechanism for the production of the superoxide radical from selenomethionine by methioninase enzyme activity in rainbow trout embryos. (GSH, reduced glutathione, GSSG, oxidized glutathione.)
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References Ballard, W.W., 1973. Normal embryonic stages for salmonid fishes, based on Salmo gairdneri Richardson and Salvelinus fontinalis (Mitchill). J. Exp. Zool. 184, 7–26. Bauder, M.B., 2002. Oxidative stress in juvenile and early life stages of rainbow trout (Oncorhynchus mykiss). M.Sc. Thesis, August 2002, Queens University, Kingston, Ont., Canada. Brinkworth, L.C., 2001. Occurrence of CYP1A activity and bluesac disease in relation to specific developmental stages in rainbow trout (Oncorhynchus mykiss). M.Sc. Thesis, November 2001. Queens University, Kingston, Ont., Canada. Chen, G.P., Zielger, D.M., 1994. Liver microsome and flavincontaining monooxygenase catalyzed oxidation of organic selenium compounds. Arch. Biochem. Biophys. 312, 566–572. Cowey, C.B., Bell, J.G., Knox, D., Fraser, A., Youngson, A., 1985. Lipids and antioxidant enzymes in developing eggs of salmon (Salmo salar). Lipids 20, 567–572. Hamilton, S.J., Buhl, K.J., Faerber, N.L., Wiedmeyer, R.H., Bullard, F.A., 1990. Toxicity of organic selenium in the diet of chinook salmon. Environ. Toxicol. Chem. 9, 347–358. Hill, M., Singal, P.K., 1996. Antioxidant and oxidative stress changes during heart failure subsequent to myocardial infarction in rats. Am. J. Pathol. 148, 291–303. Holm, J., 2002. Sublethal effects of selenium on rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis). M.Sc. Thesis, November 2002. Univesity of Manitoba, Winnipega, Man, Canada. Kroll, K.J., Doroshov, S.I., 1991. Vitellogenin: potential vehicle for selenium bioaccumulation in oocytes of the white sturgeon (Acipenser transmontanus). In: Williot, P. (Ed.), Acipenser (International Sturgeon Symposium). Camagret-Dicova, Bordeaux, France, pp. 99–106. Lemly, A.D., 1996. Selenium in aquatic organisms. In: Beyer, W.N., Heinz, G.H., Redmon-Norwood, A.W. (Eds.), Environmental Contaminants in Wildlife. CRC Press, Boca Raton, FL, USA, pp. 427–445. Lemly, A.D., 1997. Environmental implications of excessive selenium: a review. Biomed. Environ. Sci. 10, 415–435.
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Maier, K.J., Knight, A.W., 1994. Ecotoxicology of selenium in freshwater systems. Rev. Environ. Contam. Toxicol. 134, 31–48. Miki, K., Mingxu, X., Gupta, A., Ba, Y., Tan, Y., Al-Refaie, W., Bouvet, M., Makuuchi, M., Moosa, A.R., Hoffman, R.M., 2001. Methioninase cancer gene therapy with selenomethionine as suicide prodrug substrate. Cancer Res. 61, 6805–6810. Rooseboom, M., Commandeur, J.N.M., Floor, G.C., Rettie, A.E., Vermeulen, N.P.E., 2001. Selenooxidation by flavin-containing monooxygenases as a novel pathway for X-elimination of selenocyteine S-conjugates. Chem. Res. Toxicol. 14, 127–134. Rotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A.B., Haefeman, D.G., Hoejstra, W.G., 1973. Selenium: biochemical role as a component of glutathione peroxidase. Science 179, 588–590. Skorupa, J.P., 1998. Selenium poisoning of fish and wildlife in nature: lessons from twelve real-world examples. In: Frankenberger, W.T., Engberg, R.A. (Eds.), Environmental Chemistry of Selenium. Dekker, New York, pp. 315–354. Spallholz, J.E., 1994. On the nature of selenium toxicity and carcinostatis activity. Free Radical Biol. Med. 17, 45–64. Spallholz, J.E., Hoffman, D.J., 2002. Selenium toxicity: cause and effects in aquatic birds. Aquat. Toxicol. 57, 27–37. Spallholz, .J.E., Shriver, B.J., Reid, T.W., 2001. Dimthyldiselenide and methyseleninic acid generate superoxide in an in vitro chemiluminescence assay in the presence of glutathione: implications for the anti-carcinogenic activity of l-selenomethionine and Se-methylselnocysteine. Nutr. Cancer 40, 34–41. Spallholz, J.E., Palace, V.P., Reid, T.W., 2003. Methioninase and selnomethionine but not Se-methylselenocysteine generate methylselnol and superoxide in an in vitro assay. Biochem. Pharmacol., in press. Spallholz, J.E., Reid, T., Boylan, L.M., Shriver, B., 1998. Selenium free radical chemistry: applications for pharmaceuticals. Proceedings of the 6th International Symposium on Selenium and Tellurium (STDA). Scottsdale, AZ, pp. 47–56. Stadtman, T.C., 1979. Some selenium dependent biochemical processes. Adv. Enzymol. Relat. Areas Mol. Biol. 48, 1–28. Wang, Z., Jiang, C., Lu, J., 2002. Induction of caspase-mediated apoptosis and cell-cycle G1 arrest by selenium metabolite methylselenol. Mol. Carcinog. 34, 113–120.
Ecotoxicology and Environmental Safety 58 (2004) 17–21
Metabolism of selenomethionine by rainbow trout (Oncorhynchus mykiss) embryos can generate oxidative stress Vince P. Palace,a, Julian E. Spallholz,b Jodi Holm,b Kerry Wautier,a Robert E. Evans,a and Christopher L. Barona a
Department of Fisheries and Oceans, Habitat Impacts Research, Freshwater Institute, 501 University Crescent, Winnipeg, MB, Canada R3T 2N6 b Texas Tech University, Selenium Technologies Inc., Lubbock, TX, USA Received 2 December 2002; received in revised form 8 August 2003; accepted 25 August 2003
Abstract Although selenium is required by vertebrates, toxicity can arise at concentrations only slightly greater than those they require. The toxicity of Se is thought to arise from its ability to substitute for sulfur during the assembly of proteins. However, recent studies also indicate that some forms of selenium are capable of generating oxidative stress in an in vitro test system that includes glutathione. l-Selenomethionine, the predominant form of selenium in the eggs of oviparous vertebrates, does not generate oxidative radicals in this system, but lesions consistent with oxidative stress have been identified in fish and birds with high concentrations of Se. Here we report on the ability of rainbow trout embryos to transform l-Selenomethionine to a form capable of producing a superoxide radical. Oxidative stress appears to be generated by methioninase enzyme activity in the embryos that liberates methylselenol from l-Selenomethionine. Methylselenol redox cycles in the presence of glutathione producing superoxide and likely accounts for oxidative lesions present in fish and birds environmentally exposed to excessive loads of selenomethionine. r 2003 Elsevier Inc. All rights reserved. Keywords: Selenium; Rainbow trout; Oxidative stress; Superoxide radical; Teratogensis; Biotransformation
1. 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 severalfold 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
Corresponding author. Fax: +1-204-984-6587. E-mail address: [email protected] (V.P. Palace).
0147-6513/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2003.08.019
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)
ARTICLE IN PRESS 18
V.P. Palace et al. / Ecotoxicology and Environmental Safety 58 (2004) 17–21
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.
2. Materials and methods 2.1. 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 mL/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 traytype vertical incubator supplied with 6 L/min of dechlorinated Winnipeg city tap water (8 C, dissolved oxygen495% saturation). Subsamples of the incubating fertilized eggs or embryos were removed throughout their incubation period. Rainbow trout eggs require approximately 400 temperature units (TU=incubation temperature days held at that temperature) to reach the stage referred to as ‘‘swim-up,’’ when the yolk has been almost entirely absorbed and the fry begin to feed exogenously. Eggs and embryos were subsampled from 8 to 384 TU and frozen at 90 C until they could be prepared for analysis. To prepare for analysis, three eggs or embryos were homogenized in 5 vol of ice-cold 0.05 M phosphate buffer (pH, 7.0). Crude homogenates were centrifuged at 10,000g for 10 min and the supernatant was filtered through Supelco C-18 columns with 1 g of packing material (Sigma–Aldrich, Mississauga, Ont., Canada) to remove lipids. The filtrate from these columns was used directly in the assay for superoxide radical production from selenomethionine.
2.2. Assay for superoxide production The production of superoxide radical was measured in the presence of selenomethionine as previously described (Spallholz et al., 2001). Briefly, 100 mL of sample was introduced into 0.05 M sodium borate buffer (pH, 9.0) containing 1 mg/mL of reduced glutathione, 1 mg/mL of selenomethionine and 0.1 mg/mL of lucigenin (Sigma Chemical Co, St. Louis, MO, USA). The detection of chemiluminescence generated by the superoxide radical in the presence of lucigenin was performed using a Model 535 Los Alamos luminometer that integrated the signal for four consecutive 10-s intervals. Blanks for each sample were included by substituting 100 mL 0.05 M phosphate buffer (pH, 7.0) for the sample. Chemiluminescence not specific to metabolism of selenomethionine was corrected for by performing analyses of the sample using a buffer that had l-methionine instead of selenomethionine. Detection limits of the assay were established using known concentrations of methylselenol, the anticipated product of selenomethionine metabolism, in an identical assay procedure. The dependence of chemiluminescent activity on the production of superoxide radical was examined by the introduction of 5 mg of superoxide dismutase enzyme (SOD) (Sigma) to the reaction and the recording
ARTICLE IN PRESS V.P. Palace et al. / Ecotoxicology and Environmental Safety 58 (2004) 17–21
of chemiluminescence for a further four integration periods.
Each sample and blank was analyzed in duplicate and the means of four consecutive integration periods were used to quantify chemiluminescence. Data are presented as means7SEM.
2
R =0.99, p<0.05
250
Relative Light Units
60 40 20
Mean Se Blank Value
0
100
200
300
400
Temperature Units After Fertilization
Methylselenol, a metabolite of selenomethionine cleavage, produced chemiluminescence in a fashion linear (R2 ¼ 0:99; Po0:05) 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 mL. The detection limit for methylselenol in these studies is similar to previous detection levels published by Spallholz et al. (2001). Chemiluminescence from methylselenol was abolished to background levels by the addition of SOD, confirming that the emissions were dependent on the production of superoxide radical and its interaction with lucigenin. Fig. 2 shows relative light units emitted from the samples of fish eggs or embryos for the developmental stages that were sampled. Based on previous experiments and the range of light detection from analytical blanks, twice background light emission was accepted as a significant response for the assay. The superoxide radical, which is indicated by an increase in light emission, was not significantly detected in the rainbow trout embryos until 216 TU after fertilization, after
200 150 100 50 0 0
Selenomethionine L-Methionine
0
3. Results
300
80
Relative Light Units
2.3. Data analysis
19
20
40
60
80
100 120 140 160 180
Selenium in the Assay (ng) Fig. 1. Chemiluminescence produced by methylselenol in an in vitro assay system consisting of 0.05 M sodium borate buffer (pH, 9.0) with 1 mg/mL of reduced glutathione and 0.1 mg/mL of lucigenin. Data are shown as mean7SEM (n ¼ 5).
Fig. 2. Chemiluminescence produced by rainbow trout embryo extracts at various stages of development in an in vitro assay system for superoxide. Data are shown as mean7SEM (n ¼ 5).
which it remained elevated above background until 248 TU. The greatest response was obtained at 232 TU, when the rainbow trout embryos were at the eleuthroembryo stage with a functioning heart and circulatory system as well as liver (Ballard, 1973). Later stages (i.e., 4300 TU) of development did not produce chemiluminescence above background levels. Chemiluminescence from samples with the highest activity (232 TU) was reduced to background levels with the addition of 100 units of SOD to the assay volume, confirming the role of superoxide in the biologically mediated generation of chemiluminescence. Chemiluminescence was never above background for the sample extracts incubated in the same buffer when l-Methionine was substituted for selenomethionine.
4. 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 does not appear until later in development (Cowey et al., 1985), which accounts for the abolishment of activity in rainbow trout embryos after 300 TU in this study. In support of these observations for superoxide, the light emission from samples with the highest activity (232 TU) was reduced to background levels with the addition of 100 units of SOD to the assay volume. We have previously observed hemorrhages in the region of the heart in fry derived from adults at sites with elevated selenium concentrations (Holm, 2002). This hemorrhaging occurs at about the same time that
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the livers of rainbow trout fry become functional. This liver development period coincides with the period during which superoxide production was most strongly observed from selenomethionine in the current experiments. Hemorrhaging in rainbow trout fry is followed closely by the development of yolk sac and pericardial edema (Fig. 3) (Holm, 2002). Increased permeability, inferred from hemorrhaging, and heart dysfunction could certainly be mediated by the production of the superoxide radical and the development of oxidative stress in myocardial tissue and the surrounding vasculature (Hill and Singal, 1996). At least one other group has shown similar pathologies in trout fry exposed to organic contaminats that induce biotransformation enzymes that elevate oxidative stress endpoints (Bauder, 2002). It has been shown previously that biotransformation of organoselenium compounds by P450 or flavin monoxygenase (FMO) enzymes can produce selenoxides. These selenoxides in turn may liberate seleninic acid (Chen and Zielger, 1994; Rooseboom et al., 2001), a potent redox cycling compound that can elevate oxidative stress in vivo (Spallholz and Hoffman, 2002).
Pericardial edema Yolk sac edema
Fig. 3. Rainbow trout embryos at 232 TU showing yolk sac and pericardial edema relative to normal development.
However, it is not likely that this activity accounts for the generation of chemiluminescence in the present studies. Biotransformation by both FMO and P450 enzymes requires the presence of NADPH, with declining rates of catalysis observed when this cofactor is depleted (Rooseboom et al., 2001). Our assay did not contain any added NADPH, and transformation rates remained elevated and stable for methylselenol standards and the egg extracts over long periods (440 min). Furthermore, selenocysteine, which was transformed by FMO and P450 activity in the previous studies, did not generate chemiluminescence in the presence of glutathione in our in vitro system (unpublished observations). The presence of selenomethionine in the assay system was essential for the production of the superoxide radical. This necessity was evident in the fact that light production was never above background for the sample extracts incubated in the same buffer when l-Methionine was substituted for selenomethionine. At least two independent groups have shown that some forms of selenium are able to form redox cycling species that oxidize reduced glutathione-generating superoxide radicals (Spallholz et al., 2001; Wang et al., 2002). Until recently, l-Selenomethionine has been regarded as one of the forms of selenium that does not generate superoxide radical in vitro. However, we have recently shown that l-Selenomethionine generates superoxide in the presence of the enzyme methioninase (Spallholz et al., 2003). It is likely, as shown by Wang et al. (2002), that methylselenol is the enzymatic product generating superoxide in this reaction (Fig. 4). Results from this series of experiments suggest that methioninase activity may be responsible for the teratogenesis observed in offspring of adult fish that have ingested sublethal but elevated concentrations of selenomethionine. Most notably, the stage of development after the liver becomes functional but before SOD enzyme activity is present appears particularly susceptible. Further investigations to characterize methioninase activity at various embryonic developmental stages of rainbow trout are ongoing in our laboratories.
Fig. 4. Proposed mechanism for the production of the superoxide radical from selenomethionine by methioninase enzyme activity in rainbow trout embryos. (GSH, reduced glutathione, GSSG, oxidized glutathione.)
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