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Ethoxyresorufin-O-deethylase (EROD) activity, a catalytic function of the cytochrome P450 1A (CYP1A) microsomal oxygenase subfamily, is a popular biomarker for exposure to xenobiotics, polyhalogenated aromatic hydrocarbons (PHAHs) in particular. It has found wide use in aquatic pollution assessment both in vivo and in vitro. In such studies, subjects are often exposed to complex mixtures where various constituents can interfere with EROD-activity, possibly resulting in inadequate estimation of toxic hazard or biological response. The present study investigates the effects of polybrominated diphenyl ethers (PBDEs), a relatively new and increasingly detected group of environmental contaminants, on the validity of EROD activity as exposure marker in carp (Cyprinus carpio) hepatocytes. Freshly isolated hepatocytes of a genetically uniform strain of male carp were co-exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at concentrations of 0, 1, 3, 10, 30, and 100 pM, and one of the highly purified PBDE/PCB congeners (at concentrations of 0, 0.25, and 2.5 microM) or cleaned-up and untreated DE-71 samples (0, 0.1, and 1 microM). PBDEs were selected from the 209 possible congeners based on their relative abundance in environmental samples: BDE-47, BDE-99, BDE-100, and BDE-153. A tentative metabolite of BDE-47, 6OH-BDE-47, was also included. In addition, a commercial pentabrominated dipenylether mixture (DE-71) was tested for interference with EROD activity both with and without clean-up by carbon fractionating which removed possible planar contaminants. Polychlorinated biphenyl (PCB)-153, a reported inhibitor of EROD activity in flounder, was included for comparison. Cells were cultured for a total period of 8 days; exposure started at day 3 after cell isolation. After 5 days of exposure, cell pellets were frozen before EROD activity was determined. Upon exposure to TCDD, the cells responded with increased EROD activity as expected. Significant reduction of TCDD-induced EROD activity was found in the presence of BDE-47, BDE-99, and BDE-153, but not with BDE-100 and 6-hydroxylated BDE-47. Of these PBDE congeners, the most abundant congener in environmental samples, BDE-47, exhibited the strongest inhibition (down to 6% of the TCDD control value). The cleaned-up fraction of commercial penta-BDE (DE-71) mixture proved an even more potent inhibitor, resulting in reduction of EROD activity to 4% of the control values observed at 1.0 microM. BDE-47 and BDE-153 did not reduce TCDD-induced EROD activity when added shortly prior to measurement, suggesting possible interaction with TCDD at the level of CYP1A biosynthesis. PCB-153 did not show significant effects on EROD activity in carp in this study. The present results indicate that environmentally relevant PBDEs can interfere with determination of EROD activity in vitro, at levels reported earlier for PCBs. The observation that detected PBDE levels are rising, stresses the need for caution when interpreting EROD data on environmental samples.

A series of experiments was set up to elucidate the effects of pollution on marine and estuarine fish health, since the European flounder (Platichthys flesus) has shown a relatively high prevalence of (pre)neoplastic liver lesions and lymphocystis virus disease in Dutch coastal and estuarine waters. The hypothesis of a causal relationship between pollution and the above-mentioned diseases was supported by results from semi-field experiments. Therefore several laboratory experiments were carried out to substantiate causality further and to identify the xenobiotics that may play a major role in the field. The present study focuses on polychlorinated biphenyls (PCBs). European flounders (Platichthys flesus) were orally exposed to a single dose of 0, 0.5, 5 or 50 mg PCB-126/kg body weight under controlled laboratory conditions. The effects on liver, gills, gastrointestinal tract, gonads, spleen and mesonephros were examined histologically after 16 days. Induction and localization of cytochrome P4501A (CYP1A) immunoreactivity, and effects on hepatocyte proliferation were visualized immunohistochemically. Effects on thymus size were examined by morphometric analysis of serial sections. Three out of five animals of the highest dose group showed haemorrhages in the fins and tail after 16 days. All animals showed reduced activity in the later stages of the experiment, and some animals of the highest dose group discontinued feeding 14 days after exposure. Strong and exposure-related induction of CYP1A immunoreactivity was noted in hepatocytes, endothelium in all organs examined, and epithelium of the digestive tract and mesonephros at PCB-126 levels of 0.5, 5 and 50 mg/kg. In addition, the strong induction of CYP1A immunoreactivity in a distinct population of haematopoietic cells in the mesonephros and in circulating blood is remarkable, and has not been described previously in other fish species. Furthermore, a morphometrically determined significant reduction in relative thymus size was noted in animals exposed to 50 mg PCB-126/kg. Although the functional implications for the immune system of this reduction need to be further investigated, an impact on the specific resistance against infectious diseases as observed in the field, e.g. viral lymphocystis disease, is not implausible. In addition, a significant increase in absolute liver weight, in hepatosomatic index, and in number of proliferating hepatocytes [measured as immunoreactivity against proliferating cell nuclear antigen (PCNA)] was noted in animals of the highest dose group. From these findings we suppose that PCB-126 (and related chemicals) may play a role in the promotion of tumour development in the liver of European flounders as observed in the field. The results of the present experiment show relatively stronger effects than effects previously reported from experiments with TCDD, suggesting that the TEF of 0.005 assigned to PCB-126 from early life stage mortality experiments in rainbow trout (Oncorhynchus mykiss), underestimates the toxic potential of PCB-126.

The present study is part of a project that focuses on the relationship between environmental pollution and fish diseases. Field studies in various polluted coastal areas in Europe and the United States of America clearly indicate a relationship between pollution and the increase in prevalence of tumours and infectious diseases in fish. Research under controlled laboratory conditions is necessary to prove causal links between specific xenobiotics and disease prevalence. One of the chemicals of interest in the myriad of xenobiotics found in polluted waters and sediments is the organotin compound tributyltin (TBT), originating mainly from antifouling paints used on the hulls of ships. This report describes a study in which flounders (Platichthys flesus) were exposed to bis(tri-n-butyltin)oxide (TBTO) in the water under controlled laboratory conditions. The effects on several organs (gills, skin, eye, liver, mesonephros, ovary/testis, spleen, and gastrointestinal tract) were examined using histopathology, and morphometric analysis of the thymus was performed to assess the target organ(s) for TBTO in this fish species. Also the function of the non-specific and specific resistance was studied using ex vivo/in vitro immune function tests. Exposure of flounder to TBTO, in concentrations which were in the same order of magnitude as maximum TBT levels measured in the field (experiment: 17.3 μg TBT/l; field: 7.2 μg TBT/l), caused mortality after 7–12 days, resulted in gill lesions, and induced significant reduction of the non-specific resistance. A significant decrease of the relative thymus volume, but no marked effects on the specific immune system were noted after exposure to TBTO.