Effects of alkylphenols on redox status in first spawning Atlantic cod (Gadus morhua)
Introduction
Oil production in the Norwegian sector of the North Sea results in large disposal of produced water into the ocean. The amount of produced water is increasing rapidly with the age of the oil field, and the discharge of produced water from the Norwegian sector reached a level of more than 120 million m3 in year 2001 (SFT; OLF). The produced water is contaminated with a wide spectrum of organic and inorganic compounds originating from the oil, formation water or production additives. Alkylated phenols with an alkyl chain length ranging from C4 to C7 have been reported in concentrations of 2–237 ppb in produced water from platforms outside the coast of Norway (Brendehaug et al., 1992).
The acute toxicity of produced water to marine organisms is low. The dilution factor is high in offshore discharge, and thus the concentration of alkylphenols in the water column will be low. However, relatively little is known about the fate and long-term effects of alkylphenols in the marine environment (Røe, 1998). The biodegradation rate of phenols dramatically decreases with increasing length of alkyl chain, and their bioavailability increases due to their lipophilic nature. The bioconcentration factors in fish range from 118 to 578 for butyl- to heptylphenol (McLeese et al., 1981, Freitag et al., 1985, Tollefsen et al., 1998).
The estrogenic activity of alkylphenols has been well established both in vitro and in vivo (Nimrod and Benson, 1996, Arukwe et al., 2000, Arukwe et al., 2001). The alkylphenols are shown to interact with the estrogen receptor, although weaker than 17β-estradiol. The estrogenicity of the alkylphenols depends on position (para>meta>ortho) and branching (tertiary>secondary=primary) of the alkyl chain. The highest estrogenic activity is found in C6–C8 para substituted tertiary alkylphenols (1000- to 6000-fold less potent than E2), but also C5, C4 and C3 phenols are estrogenic (100 000- to 20 000 000-fold less potent than E2) (Routledge and Sumpter, 1997).
The effects of alkylphenols on redox status and detoxification enzymes in Atlantic cod are unclear. Estrogenic environmental compounds such as para-nonylphenol and bisphenol A were found to stimulate hydroxyl radical formation in the rat striatum (Obata and Kubota, 2000). Another study concluded that phenols showed a double action, acting as antioxidants at lower doses, but acting as pro-oxidants at higher doses under certain circumstances (Fujisawa et al., 2002). Nonylphenol is also found to inhibit cell growth and cellular oxygen consumption in Saccharomyces cerevisiae, suggesting nonylphenol-induced oxygen radical generation in yeast mitochondria. Nonylphenol-induced cell growth inhibition was efficiently protected by the lipophilic antioxidants α-tocopherol and β-carotene (Okai et al., 2000).
The phenol group makes the alkylphenols directly available to phase II enzymes, and thus the alkylphenols are relatively rapid metabolized. In fish, organic compounds usually are conjugated to either glutathione or glucuronic acid (Ankley and Agosin, 1987). Glutathione S-transferase (GST) is a family of enzymes catalysing conjugation of a large variety of foreign compounds to glutathione. Glutathione (GSH) also functions as an antioxidant (DeLeve and Kaplowitz, 1991). For example, hydrogen peroxide can be reduced by GSH in the presence of selenium-dependent glutathione peroxidase. As a consequence, GSH is oxidized to GSSG, which rapidly is reduced back to GSH by glutathione reductase (GR) at the expense of NADPH (DeLeve and Kaplowitz, 1991). NADPH is regenerated by oxidation of glucose-6-phosphate to 6-phosphogluconate within the pentose phosphate pathway. The key enzyme for this pathway is glucose-6-phosphate dehydrogenase (G6PDH) (Eggleston and Krebs, 1974).
The aim of this investigation was to study the effects of alkylphenols on the redox status in first spawning Atlantic cod. A computer simulation estimated the discharge of alkylphenols in produced water from the Halten Bank area outside the Norwegian West coast, and the probable uptake by pelagic fish species was calculated (Rye et al., 1996). The calculations estimated the body burden of alkylphenols in fish to be in the 0–10 ppb range and the lowest exposure dose was chosen on the basis of this calculation and the bioconcentration factor 600 for the alkylphenol mixture. Oxidative stress response was determined by measuring total and reduced amount of glutathione as well as GR and G6PDH activities in the liver. In addition, the activity of GST was determined. Our results, together with a study of CYP1A and CYP3A expression and activities (Hasselberg et al., 2004) and a study on endocrinology changes (Meier et al., 2002), will hopefully facilitate the evaluation in environmental risk assessment studies of alkylphenols.
Section snippets
Chemicals
Bakers yeast glutathione reductase, 1-chloro-2,4-dinitrobenzene (CDNB), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), dimethyl sulfoxide (DMSO), glucose-6-phosphate, glutathione (reduced and oxidized form), NADP+ and NADPH were all purchased from Sigma–Aldrich Sweden AB (Stockholm, Sweden). 4-tert-Butylphenol (C4) and 4-n-hexylphenol (C6) were purchased from Sigma–Aldrich Norway AS (Oslo, Norway). 4n-Pentylphenol (C5) was obtained from Acros (Gell, Belgium) and 4-n-heptylphenol (C7) from Avocado
Glutathione
Both tGSH and GSH increased in female Atlantic cod liver after 1 week of exposure to alkylphenols compared to controls (Fig. 1A). The ratio of tGSH versus GSH was relatively constant in all treatment groups (Fig. 1A–C). Control fish had close to 100% of all glutathione in its reduced form after 1 week. The corresponding levels for female and male fish after 4 weeks were 95 and 91%, respectively. After 4 weeks of exposure to alkylphenols, there was a decrease in tGSH levels (nearly 50%) in the 2
Glutathione
Intracellular glutathione is normally in its reduced form, and in mammals, oxidized glutathione constitute only a few percent or less of the total glutathione pool (Meister and Anderson, 1983). Thus, measuring total hepatic glutathione often reflects the reduced levels. In fish, the GSSG portion often is higher than in mammals, although the relationship between GSH and GSSG varies with species and tissue. In our study, we measured the level of tGSH and GSH and the ratio was calculated. The
Acknowledgements
This work was supported by the Faculty of Science at the Göteborg University and grants from Nordisk Forskerutdanningsakademi (NorFA) and Helge Ax:son Johnson to L. Hasselberg, Norwegian Research Council and the Norwegian Oil Industry Association to A. Svardal and S. Meier. The authors are very grateful to Torunn Eide for excellent technical assistance, Eirı́kur Stephensen, Malin Celander and Tove Hegelund for valuable comments on the manuscript.
References (48)
- et al.
Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure
Biochem. Biophys. Acta
(2000) - et al.
Comparative aspects of hepatic UDP-glucuronosyltransferases and glutathione S-transferases in bluegill and channel catfish
Comp. Biochem. Physiol. [B]
(1987) - et al.
In vivo and in vitro metabolism and organ distribution of nonylphenol in Atlantic salmon (Salmo salar)
Aquat. Toxicol.
(2000) - et al.
Differential biomarker gene and protein expressions in nonylphenol and estradiol-17β treated juvenile rainbow trout (Oncorhynchus mykiss)
Comp. Biochem. Physiol.
(2001) - et al.
Oxidative stress in gill, erythrocytes, liver and kidney of Nile tilapia (Oreochromis niloticus) from a polluted site
Aquat. Toxicol.
(1996) - et al.
Glucuronidation in fish
Aquat. Toxicol.
(1991) - et al.
Use of a microplate reader in an assay of glutathione reductase using 5,5′-dithiobis(2-nitrobenzoic acid)
Anal. Biochem.
(1989) - et al.
Glutathione metabolism and its role in hepatotoxicity
Pharmacol. Ther.
(1991) - et al.
Effects of Black Rock Harbour sediments on indices of biotransformation, oxidative stress, and DNA integrity in channel catfish
Aquat. Toxicol.
(1993) - et al.
Bioconcentration and distribution of 4-tert-octylphenol residues in tissues of the rainbow trout (Oncorhynchus mykiss)
Mar. Environ. Res.
(2001)