Assessment of cytochrome P450 1A in dab as biomarker of exposure to polychlorinated biphenyls and related compounds

The objective of this thesis was to investigate whether the CYP1A response in dab could be used as a biological marker of exposure to PCBs and related compounds in monitoring programmes in the southern North Sea. Only because of its wide occurrence in the North Sea, dab is already playing an important role in international contamination monitoring programmes, but no attempt has been made to investigate the suitability of the organism for monitoring purposes so far. During the field studies and laboratory experiments, emphasis has been laid on the study of PCBs, since they are by far the most important class of compounds capable of inducing CYP I A in the southern North Sea.In chapter 2 the effects of maturity and sex on CYP1A expression in the field were investigated. Mature and juvenile dab of both sexes were collected from different areas of the southern North Sea with varying levels of PCB contamination. In all cases, muscle PCB concentrations were highest near the Dutch coast. The highest CYP1A levels, measured as EROD activity and CYP1A protein, were also found in this area for mature fish of both sexes in autumn. The same was found during the spawning season in winter for juvenile females and mature males. During this season gravid females showed significantly lowered contents of CYP1A protein and EROD activity compared to mature males and juveniles. Muscle PCB concentrations and both biochemical parameters were positively correlated for mature males, and not for other groups of dab, during both seasons. It was concluded that the sensitivity of CYP1A induction in dab as a biomarker for PCBs and related compounds was highest in mature males.In chapter 3 it was shown that bottom water temperature differences of up to 10°C, occuring between stratified and vertically mixed areas during spring and summer, have a strong effect on CYP1A expression in dab. Highly elevated CYP1A levels were observed in mature male dab collected from off-shore stations with low bottom water temperatures due to stratification whereas considerably lower CYP1A levels were observed at stations with higher water temperatures in vertically mixed areas, including coastal stations. Statistical analyses of the data indicated that water temperature was inversely related to CYP1A levels, whereas PCB concentrations showed a positive correlation with CYP1A levels. The effect of water temperature, however, dominated over the effect of PCB contamination. A laboratory study confirmed that EROD activity was inversely proportional to water temperature. Furthermore, it was shown that differences in the nutritional status of dab, reflected by the condition factor, also obscured the effects of PCB contamination on CYP1A levels during these seasons.Because it was demonstrated that spring and summer are less suitable seasons for monitoring purposes and next to this, dab in the southern North Sea show considerably more migration during the spawning period in winter, it was investigated whether the most suitable period for annual monitoring would be the late autumn. Chapter 4 describes a final field study during which differences in interfering natural factors, like water temperature, condition factor of the fish and percentage lipid in muscle tissue proved to be minimal. The results showed that the CYP1A induction response in male dab was strong enough to separate the Dutch coastal area from three more pristine offshore areas of the North Sea. Thus, the general conclusion of all field studies is that the late autumn indeed offers the best conditions to investigate the relation between environmental contamination with PCBs and related compounds, since disturbing influences of factors other than PHAH contamination are minimal.The induction capacity of CYP1A by PCBs has been studied in laboratory experiments. Chapter 5 describes an experiment in which mature female dab were dosed with 1 mg of the technical PCB mixture Clophen A40 every 6 weeks, with a maximum of three doses per fish. Induction of CYP1A was observed following this Clophen A40 exposure. Biochemical effects were related to increases in concentrations of total PCBs and specific congeners and consequently to the corresponding toxicity equivalencies of these PCBs (CB-TEQs) in muscle. In all treated groups the EROD activity, CYP 1 A protein and total CYP 1 A levels were higher than those of the control groups. The maximum for these biochemical parameters was already reached after the first single dose, although the CB concentrations in muscle tissue increased further after administration of a second and third dose. It was concluded that the CYP1A system of dab is sensitive towards PCBs.Since PCB congeners differ in potency to induce CYP1A the biochemical effects of several non- and mono- ortho substituted CB congeners were investigated. Exposure to the non- ortho chlorinated CB77 resulted in induction of CYP1A (chapter 6). On the contrary, exposure to three different mono- ortho chlorinated CB congeners and the non- ortho chlorinated CB126 did not result in increases of CYP1A levels (chapter 7). For CB126 this is in sharp contrast with earlier studies in fish where this congener was found to be a potent inducer of CYP1A. A large inter-individual variation in especially the reference group was held partly responsible for this. Exposure to CB 126 even caused a dose-dependent inhibition of EROD activity at higher concentrations.In chapter 6 the influence of temperature on the temporal induction pattern of CYP1 A was investigated. Mature males were exposed to a single dose of a non- ortho chlorinated CB congener (CB77). The fish were acclimated at two different temperatures (10 and 16°C) and kept at these temperatures for a period of 40 days. At both temperatures, CYP1A protein and EROD activity were induced 40 days after dosing. Maximum responses of both EROD activity and CYP1A protein for the warm-acclimated fish were observed 5 days after treatment. For the cold-acclimated fish a slow, progressive elevation for both biochemical parameters was observed and maximum responses were measured 40 days after treatment. In the control groups EROD activity and CYP1A protein levels were higher in the cold-acclimated fish, which is in accordance with the study described in chapter 3. In the dosed groups, however, absolute biochemical levels were equally high at 40 days after treatment. It is therefore concluded that the magnitude of induction was higher in warm- acclimated fish.Since CYP1A is a biomarker of exposure it does not provide knowledge of adverse (toxic) effects. The question can thus be raised whether enzyme induction reflects deleterious effects on dab. In the study where dab were exposed to Clophen A40 (chapter 5), no effects on reproductive parameters were found (Fonds et al. , 1995). It is very likely that the concentrations of contaminants needed for effects at higher levels of biological organisation will seldom be reached in the marine environment.From the present research, it has become clear that dab is a suitable organism to monitor hepatic CYP1A indicating exposure to PHAHs, in the North Sea. Dab has shown to be sensitive towards PCBs. However, when applying the CYP1A measurement in dab, several interfering co- factors, like seawater temperature, maturity and sex, have to be taken into account. As a consequence, monitoring with mature males in the autumn seems to be preferable. With respect to monitoring programmes, the CYP1A measurement could very well serve as a quick and cheap screening method for contamination of the aquatic environment. The joint impact of a whole group of chemicals, the PHAHs, on CYP1A can be assessed. Although there is ample evidence for the existence of a dose-response relationship between PHAHs and CYP1A direct linear correlations will not always be found in the environment. For instance, concentrations of PAHs in the North Sea are too low to induce CYP1A in dab. Therefore measurement of CYP1A can never fully replace chemical analyses, but it can give complementary information about inducing PHAH compounds. Application of CYP1A seems to be useful to discrimate between estuarine and/or coastal areas and the open North Sea, which implies that increased concentrations of PHAHs reaching the marine environment via the outflow of contaminated rivers can be detected.