The zebrafish gill model: Induction of CYP1A, EROD and PAH adduct formation
Introduction
Zebrafish (Danio rerio) is a common experimental species in fish toxicology. The small body size, short generation time, availability of suitable endpoints and techniques, and the extensive information on normal physiology and gene sequences make the zebrafish a useful model (Carney et al., 2006). However, in terms of susceptibility to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicity zebrafish is one of the least sensitive fish species (Elonen et al., 1998).
Exposure to aryl hydrocarbon receptor (AHR) agonists such as coplanar polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and polycyclic aromatic hydrocarbons (PAHs), leads to a variety of effects in zebrafish, including reproductive and developmental toxicity, histopathological changes, suppression of regenerative growth, and tumour induction (Spitsbergen et al., 2000, Zodrow and Tanguay, 2003, Zodrow et al., 2004, Antkiewicz et al., 2006, Incardona et al., 2006, Mathew et al., 2006, Jönsson et al., 2007a, Heiden et al., 2008). Associated with these effects is a changed expression of a large number of AHR-regulated genes, including those encoding cytochrome P450 1 (CYP1) enzymes, i.e., CYP1As, CYP1Bs and the recently discovered CYP1Cs (Godard et al., 2005, Handley-Goldstone et al., 2005, Jönsson et al., 2007a, Jönsson et al., 2007b).
Ethoxyresorufin-O-deethylase (EROD) activity is an established biomarker for exposure to AHR agonists in fish, birds and mammals. EROD activity is catalyzed by CYP1A and CYP1B enzymes in mammals (Chang and Waxman, 2006). The contributions of the different CYP1 enzymes to EROD activity vary among species. In humans for instance, CYP1A1 catalyzes the EROD reaction with a 10 times higher rate than CYP1B1 and with a 50 times higher rate than CYP1A2 (Shimada et al., 1997). Only CYP1A enzymes have so far been characterized with regard to EROD activity in fish (Gooneratne et al., 1997, Chung et al., 2004). Basal expression of the CYP1 genes shows considerable variation among organs in adult zebrafish. Liver expresses a higher level of CYP1A and a lower level of CYP1C1 than gill, whereas the two organs appear to express similar levels of CYP1B1 and 1C2 (Jönsson et al., 2007b). Exposure to PCB126 induces CYP1A, 1B1, and 1C1 in gill, and CYP1A, 1B1, 1C1 and 1C2 in liver (Jönsson et al., 2007b).
CYP1A and CYP1B enzymes metabolize PAHs, such as benzo[a]pyrene (BaP) and 7,12-dimethylbenz[a]anthracene (DMBA) to reactive intermediates, which may form DNA adducts and initiate carcinogenesis (Conney, 1982, Kleiner et al., 2004, Pottenger et al., 1991, Schober et al., 2006). In mammals DMBA is metabolized to 3,4-dihydroxy-3,4-dihydro-DMBA, a proposed precursor to a bay-region diol-epoxide (Slaga et al., 1979). Rainbow trout embryos exposed to DMBA were found to form various metabolites, including 3,4-dihydroxy-3,4-dihydro-DMBA, and within 9 months the fish had developed tumours, primarily localized to liver but also to stomach and kidney (Fong et al., 1993). Zebrafish exposed to DMBA as fry developed tumours within 6–12 months, and showed a greater diversity of tumour types than has been observed in rainbow trout (Spitsbergen et al., 2000). The primary site of tumour formation in zebrafish was the liver, and the second and third most frequently affected tissues were gills and blood vessels (Spitsbergen et al., 2000). In wild fish, tumours have been observed in benthic species inhabiting areas with high PAH contamination in the sediment (Baumann, 1998).
The gills are directly exposed to ambient water and filter considerable volumes of water in order to extract oxygen. This makes the gill epithelium a major route of uptake and a target for waterborne toxicants (Stagg and Shuttleworth, 1982, Miller et al., 1989, Wood et al., 1996, Arellano et al., 2001, Jönsson et al., 2004, Malins et al., 2004, Griffitt et al., 2007). The gill could consequently be a useful organ when screening for effects of waterborne toxic chemicals. EROD induction in fish liver is commonly used as a biomarker of exposure to AHR agonists in the aquatic environment. It seems most likely, however, that exposure of fish to waterborne AHR agonists is not always accurately reflected by hepatic EROD activity; rapidly metabolized chemicals such as PAHs may be biotransformed by the gills which limits liver exposure (Andersson and Pärt, 1989, Levine and Oris, 1999, Jönsson et al., 2006). We have developed a gill-based EROD assay, and proposed gill EROD activity as a sensitive and robust biomarker of exposure to waterborne AHR agonists in fish (Jönsson et al., 2002, Abrahamson et al., 2007). The gill EROD assay was characterized in rainbow trout, and has been applied in a number of other species (Jönsson et al., 2002, Jönsson et al., 2003, Mdegela et al., 2006, Andersson et al., 2007).
The main objective of this study was to modify and apply the gill filament EROD assay for use in zebrafish. We also compared EROD responses in gill and liver in zebrafish and rainbow trout following exposure to CYP1A inducers via ambient water at temperatures appropriate for these two species (23 and 12 °C, respectively). Finally, we localized sites of high DMBA adduct formation in zebrafish gills by means of microautoradiography and compared it to the immunohistochemical localization of CYP1A protein.
Section snippets
Fish husbandry
Zebrafish were purchased from Simontorp Säteri AB (Blentarp, Sweden) and rainbow trout from Näs fiskodling AB (By Kyrkby, Sweden). The fish were held in tanks with Uppsala tap water at the Evolutionary Biology Centre, Uppsala University. Zebrafish were kept in a glass tank containing 40 L aerated tap water (23 ± 1 °C; ≤0.5 g biomass L−1), and fed once daily with Amtra system premium flakes from Amtra Aquaristik GmbH (Rodgau, Germany). Rainbow trout were held in flow-through tanks continuously
Methodology
EROD analysis in whole gill arches worked successfully in both control and induced zebrafish. When EROD activity was expressed per milligram of protein the deviation of the duplicates from the duplicate mean was 17% (±14%) and when expressed per gill arch it was 7% (±6%). The high variation when referring to protein was likely due to the difficulty to completely homogenize the gill cartilage.
BaP-induced EROD activity
Firstly we examined the time course of EROD induction in gills of zebrafish following exposure to 100 nM
EROD activity in zebrafish gill and liver
The gill EROD assay based on gill filament tips excised from the gill arches of fairly large fish such as rainbow trout was successfully adapted to using intact gill arches in zebrafish. The results imply that very small fish can be used to assay gill CYP1 induction following exposure to waterborne chemicals in vivo.
The results revealed differences in EROD activity between zebrafish and rainbow trout both in the basal level and in the responsiveness to AHR agonists. Rainbow trout gills
Conclusion
This study shows that EROD activity can be readily measured in intact zebrafish gill arches ex vivo as a marker for exposure to AHR agonists. The results suggest that the rainbow trout gill assay is more suitable than the zebrafish gill assay in screening for AHR agonists in polluted water, since lower concentrations of CYP1 inducers are likely to be detected in rainbow trout than in zebrafish. Enzyme(s) induced by βNF catalyze bioactivation of DMBA, but kinetic factors largely determine the
Acknowledgements
We thank Margareta Mattsson for expert technical assistance. This study was financed by the Swedish Research Council Formas, and by MistraPharma, a research programme sponsored by the Foundation for Strategic Environmental Research (MISTRA).
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