Effects of North Sea oil and alkylphenols on biomarker responses in juvenile Atlantic cod (Gadus morhua)
Introduction
A major part of the oil consumed globally derives from off shore oil fields. A consequence of oil drilling at sea is the formation of produced water, and this formation increases considerably with age of the oil field. The produced water contains large amounts of pollutants including polycyclic aromatic hydrocarbons (PAH) and alkylphenols (Brendehaug et al., 1992, Stephens et al., 2000). Several PAH have been found to have cytotoxic, immunotoxic, mutagenic and/or carcinogenic effects in aquatic organisms (Aas et al., 2000, Reichert et al., 1998, Vogelbein, 2003). PAH are primarily metabolized, and thereby detoxified, by enzymes in the cytochrome P450 system, mainly CYP1A (Goksøyr and Förlin, 1992). CYP1A levels are strongly up regulated by planar aromatic compounds, such as PAH and planar polychlorinated biphenyls and this is demonstrated by an increase in ethoxyresorufin-O-deethylase (EROD) activities (Förlin et al., 1994). It has been shown that CYP1A activity is inhibited by alkylphenols in several fish species (Arukwe et al., 1997, Hasselberg et al., 2004b, Navas and Segner, 2001). This inhibition may have consequences in clearance of xenobiotics. This may result in increased accumulation of harmful compounds including PAH (Levine et al., 1997).
In addition to detoxifying function, CYP1A mediated metabolism of PAH can also lead to the formation of reactive oxygen species (ROS) through the formation of redox labile metabolites. Benzo[a]pyrene, for example, can be metabolized by CYP1A to benzo[a]pyrene diones that have the ability to form ROS through the process of redox cycling (Lemaire et al., 1994). Exposure to several PAH can lead to a state of oxidative stress in aquatic organisms (Livingstone, 2001, Winston and Di Giulio, 1991). It has also been shown in Atlantic cod that exposure to alkylphenols results in elevated glutathione reductase activities and total glutathione levels, possibly as a result of oxidative stress (Hasselberg et al., 2004a).
Although acute toxicity for produced water from oil drilling is low, mainly due to the high dilution (Stephens et al., 2000) it is still of great interest to study sublethal effects in marine organisms since very little is known about the fate of alkylphenols in the marine environment (Roe, 1998). The aim of the present study was to investigate the effects of alkylphenols and North Sea oil, alone or in combination, on CYP1A and CYP3A protein expression in juvenile Atlantic cod (Gadus morhua). The composition of the alkylphenol mixture reflects the composition that can be observed in produced water. One group of fish were also exposed to nonylphenol. In addition, the effects on the phase II enzyme glutathione S-transferase (GST), the antioxidant enzymes glutathione reductase (GR) and catalase (CAT) and levels of total and oxidized glutathione (GSH and GSSG) were analysed. Oxidative damage was estimated as levels of lipid peroxidation measured as thiobarbituric acid reactive substances (TBARS).
Section snippets
Chemicals
GSH, GSSG, GR (3664), 5-sulfosalicylic acid, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), β-nicotinamide adenine di-nucleotide phosphate (NADPH), 1-chloro-2,4-dinitrobenzene (CDNB), 2-thiobarbituric acid, butylated hydroxytoluene, phosphoric acid, 2-vinylpyridine, 7-ethoxyresorufin and bovine serum albumin were obtained from Sigma (St. Louis, USA). Ethylenedinitrilotetraacetic acid, rodamin B and hydrogen peroxide were purchased from Merck. Ready gels (12% continuous acrylamide in Tris–HCl) and
EROD activity
Exposure to nonylphenol led to a significant decrease in EROD activities in juvenile Atlantic cod (Fig. 1). North Sea oil exposure, on the other hand, resulted in a significant increase in EROD activities (Fig. 1). An increase in EROD activity was also observed in the fish exposed to a combination of the oil and alkylphenols, although the increase was not significant compared to control fish (Fig. 1).
CYP1A and CYP3A protein expression
Exposure to nonylphenol decreased both CYP1A- and CYP3A-protein expression in juvenile cod (
Discussion
The present study demonstrates that ecological relevant concentrations of North Sea oil and alkylphenols lead to biochemical changes in juvenile Atlantic cod. Exposure to this oil resulted in an increase in the levels of CYP1A and also CYP1A mediated EROD activities. This confirms previous studies that have reported increased EROD activities in Atlantic cod and turbot (Scophthalmus maximus) exposed to North Sea oil (Aas et al., 2000, Stephens et al., 2000). Exposure to nonylphenol, on the other
Acknowledgements
This study was supported by the European Union BEEP project (Biological Effects of Environmental Pollution in marine coastal ecosystems). The authors also wish to express their gratitude to Dr. Odd Ketil Andersen and the people at IRIS—International Research Institute of Stavanger AS and Akvamiljø a/s, for organizing the experiment and the exposure of the fish. Linda Hasselberg and Malin Celander were supported by grants from the Swedish EPA (Reprosafe).
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