A bioassay approach to determine the dioxin-like activity in sediment extracts from the Danube River: ethoxyresorufin-O-deethylase induction in gill filaments and liver of three-spined sticklebacks (Gasterosteus aculeatus L.).

Sediment samples from the upper Danube River in Germany have previously been characterized as ecotoxicologically hazardous and contaminants in these sediments may contribute to the observed decline of fish populations in this river section. For the investigation of sediment toxicity there is a need for development, standardization and implementation of in vivo test systems using vertebrates. Therefore, the main objective of this study was to apply and evaluate a recently established fish gill EROD assay as a biomarker in sediment toxicity assessment by using extracts of well characterised sediment samples from the upper Danube River. This to our knowledge is the first application of this novel assay to sediment extracts. Sediments from four different sites along the upper Danube River were Soxhlet-extracted with acetone and dissolved in DMSO. Three-spined sticklebacks (Gasterosteus aculeatus L.) were exposed for 48 h to various concentrations of the extracts, to the positive control beta-naphthoflavone or to the solvent. Measurements of EROD activity in gill filaments and liver microsomes followed the exposure. Concentration-dependent induction of EROD in both gill and liver was found for all sediment extracts. The highest EROD-inducing potency was determined for extracts of sediments from the sites "Opfinger See" and "Sigmaringen" and the EROD activities in gill and liver correlated well. The results from the gill and liver assays were in accordance with in vitro results of previous investigations. The EROD activities measured in the present study corresponded with the concentrations of PAHs, PCBs and PCDD/Fs in the sediment samples derived in a previous study. The sticklebacks in this study were in the reproductive phase and a stronger EROD induction was obtained in the females than in the males. Implementation of the EROD assay in testing of sediment extracts gave highly reliable results which make this assay an ecotoxicologically relevant method for assessment of contamination with Ah receptor agonists in sediments.

Monitoring contaminants from oil production at sea by measuring gill EROD activity in Atlantic cod (Gadus morhua).

An ex vivo gill EROD assay was applied in Atlantic cod (Gadus morhua) as a biomarker for waterborne CYP1A-inducing compounds derived from oil production at sea. Exposure to nominal concentrations of 1 ppm or 10 ppm North Sea crude oil in a static water system for 24 h caused a concentration-dependent gill EROD induction. Further, exposure of cod for 14 days to environmentally relevant concentrations of produced water (PW, diluted 1:200 or 1:1000) from a platform in the North Sea using a flow-through system resulted in a concentration-dependent induction of gill EROD. Crude oil (0.2 ppm) from the same oil field also proved to induce EROD. Finally, gill EROD activity in cod caged for 6 weeks at 500-10 000 m from two platforms outside Norway was measured. The activities in these fish were very low and did not differ from those in fish caged at reference sites.

Gill EROD in monitoring of CYP1A inducers in fish: a study in rainbow trout (Oncorhynchus mykiss) caged in Stockholm and Uppsala waters.

The gill filament 7-ethoxyresorufin O-deethylase (EROD) assay was evaluated as a monitoring tool for waterborne cytochrome P4501A (CYP1A) inducers using rainbow trout (Oncorhynchus mykiss) caged in urban area waters in Sweden. To compare the CYP1A induction response in different tissues, EROD activity was also analyzed in liver and kidney microsomes. Immunohistochemistry was used to localize CYP1A protein in gill and kidney. In two separate experiments fish were caged at sites with fairly high expected polyaromatic hydrocarbon (PAH) contamination. In the first experiment, gill EROD activities were analyzed in fish exposed for 1-21 days in a river running through Uppsala. The reference site was upstream of Uppsala. In the second, gill, liver and kidney EROD activities were analyzed in fish exposed for 1-5 days in fresh or brackish waters of Stockholm and in a reference lake 60km north of Stockholm. Fish exposed for 5 days followed by 2 days of recovery in tap water in the laboratory were also examined. The gill consistently showed a higher EROD induction compared with the liver and the kidney. After 1 day of caging, gill EROD activity was markedly induced (6-17-fold) at all sites examined. Induction in gill was pronounced (5-7-fold) also in fish caged at the reference sites. In the 21-day exposure study gill EROD activity remained highly induced throughout the experiment (26-fold at most) and the induced CYP1A protein was exclusively confined to the gill secondary lamellae. In the 5-day exposure experiment, EROD activity peaked after 1 day and then declined in both gill and liver, while CYP1A immunostaining in the gill remained intense over the 5-day period. In the kidney, CYP1A staining was weak or absent. We conclude that gill EROD activity is a more sensitive biomarker of exposure to waterborne CYP1A inducers than EROD activity in liver and kidney.

Cytochrome P4501A induction in rainbow trout gills and liver following exposure to waterborne indigo, benzo[a]pyrene and 3,3',4,4',5-pentachlorobiphenyl.

We have developed a gill-filament based ethoxyresorufin O-deethylase (EROD) assay to be used as a tool to monitor cytochrome P4501A (CYP1A) induction in caged fish. The present study aimed to compare temporal patterns of EROD induction in gills and liver of rainbow trout (Oncorhynchus mykiss) exposed in the laboratory to readily metabolized and persistent CYP1A inducers, i.e. indigo, benzo[a]pyrene (BaP), and 3,3',4,4',5-pentachlorobiphenyl (PCB#126). Branchial and hepatic EROD activities were examined in fish exposed for 6, 12, or 24h and in fish exposed for 24h and then held in clean water for 2 or 14 days. Furthermore, branchial CYP1A protein expression was localized by immunohistochemistry. All compounds strongly induced branchial EROD activity within 6h. The highest EROD inductions observed for indigo, BaP, and PCB#126 were roughly similar in gills (52-, 76-, and 74-fold), but differed considerably in liver (11-, 78-, and 200-fold). In indigo- and BaP-exposed fish, both hepatic and branchial EROD activities decreased rapidly in clean water. In PCB#126-exposed fish, decreased branchial and increased hepatic EROD activities were observed following transfer to clean water. The substances gave rise to immunostaining for CYP1A at different cellular sites. All inducers increased the CYP1A-immunostaining in the gill filament secondary lamellae, but PCB#126 also induced a pronounced CYP1A immunoreactivity in cells near the basal membrane of the epithelium of the primary lamellae. The observation that the low BaP and indigo concentrations induced EROD activity markedly in the gills but only slightly or not at all in the liver, supports the contention that readily metabolized AhR agonists may escape detection when hepatic EROD activity is used for environmental monitoring. The results show that gill filament EROD activity is a sensitive biomarker both for persistent and readily metabolized AhR agonists in polluted water.

Impact of humic substances on EROD activity in gill and liver of three-spined sticklebacks (Gasterosteus aculeatus).

Humic substances (HS) are ubiquitous in the environment and have been found to influence physiological functions of aquatic organisms. In the present study, three-spined sticklebacks (Gasterosteus aculeatus) were exposed to HS of different origins to evaluate effects on the 7-ethoxyresorufin O-deethylase (EROD) activity catalyzed by cytochrome P4501A (CYP1A) in the liver and the gill. To that end, three-spined sticklebacks were exposed for 48 h to different concentrations of synthetic humic acid (AHA), Nordic reservoir natural organic matter (N.R.-NOM) and water from six lakes with different concentrations of HS. EROD activity was significantly induced (3-6-fold) in the gills of fish exposed to water from all lakes except the lake with the lowest concentration of HS. All tested concentrations of AHA and N.R.-NOM significantly induced gill EROD activity and the induction was dose-dependent. AHA, but neither N.R.-NOM nor lake water, induced EROD activity in the liver. In addition, fish were exposed to the potent CYP1A inducers benzo(a)pyrene (BaP) and PCB126 in combination with AHA. Presence of AHA had no significant effect on EROD induction by BaP or PCB126. The components in HS responsible for EROD induction remain to be identified. Our finding that HS of both natural and synthetic origin induce EROD activity in the gill is of significance for the interpretation of biomonitoring data on EROD activity as well as for the choice of suitable reference waters.

CYP1A inhibition in fish gill filaments: a novel assay applied on pharmaceuticals and other chemicals.

The gill filament 7-ethoxyresorufin O-deethylase (EROD) assay was originally developed as a biomarker for cytochrome P4501A (CYP1A) induction by Ah-receptor agonists in water. In this study, the assay was adapted to measure inhibition of CYP1A activity in fish gill filaments ex vivo. The experiments were carried out using gill arch filaments from beta-naphthoflavone (betaNF)-exposed three-spined stickleback (Gasterosteus aculeatus). Candidate CYP1A inhibitors were added to the assay buffer. Nine selected pharmaceuticals and five known or suspected CYP1A-modulating chemicals were examined with regard to their ability to reduce EROD activity in gill filaments. Ellipticine, a well characterized CYP1A inhibitor, was the most effective inhibitor of the compounds tested. At a concentration in the assay buffer of 1 microM the antifungal azoles ketoconazole, miconazole and bitertanol, and the plant flavonoid acacetin reduced gill EROD activity by more than 50%, implying IC50 values below 1 microM. These compounds have previously been shown to inhibit EROD activity in liver microsomes from fish and mammals at similar concentrations. The proton pump inhibitor omeprazole reduced the gill EROD activity by 39% at 10 microM. It is concluded that the modified gill filament EROD assay is useful to screen for waterborne pollutants that inhibit catalytic CYP1A activity in fish gills.

EROD activity in gill filaments of anadromous and marine fish as a biomarker of dioxin-like pollutants.

The applicability of a gill filament-based ethoxyresorufin O-deethylase (EROD) assay, originally developed in rainbow trout, was examined in Atlantic salmon (Salmo salar), Arctic charr (Salvelinus alpinus), Atlantic cod (Gadus morhua), saithe (Pollachius virens) and spotted wolffish (Anarhichas minor). All species but spotted wolffish showed strong EROD induction in tip pieces of gill filaments following 48 h of exposure to waterborne beta-naphthoflavone. Atlantic salmon parr, smolts held in freshwater and smolts transferred to seawater showed EROD induction of similar magnitude. Arctic charr, differing 11-fold in body weight, showed similar EROD activities as expressed per gill filament tip. Laboratory exposure of saithe to water and sediments collected at polluted sites, resulted in strong EROD induction. In conclusion, the gill filament assay seems useful for monitoring exposure to aryl hydrocarbon receptor agonists in various species. Furthermore, smoltification status, water salinity and body size proved to have minor influence on gill filament EROD activity. However, the results in spotted wolffish show that some species may be less suitable for monitoring using the gill assay. Assessment of gill filament EROD activity in fish exposed to polluted water and sediments in the laboratory proved to be an easy and cost-effective way to survey pollution with dioxin-like chemicals.