A bioassay approach to determine the dioxin-like activity in sediment extracts from the Danube River : Ethoxyresorufin-0-deethylase induction in gill filaments and liver of three-spined sticklebacks (Gasterosteus aculeatus L.) (original) (raw)

Abstract

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 β-naphthoflavone or to the solvent. Measurements of EROD activity in gill filaments and liver microsomes followed the exposure. Concentrationdependent induction of EROD in both gill and liver was found for all sediment extracts. The highest ERODinducing potency was determined for extracts of sediments from the sites "Öpfinger 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.

Figures (10)

Fig. 1. Overview of the sampling sites along the Upper Danube in Germany. a) Sigmaringen, b) Riedlingen, c) Opfinger See, d) Bad Abbach (Keiter et al., 2008, modified).

Fig. 1. Overview of the sampling sites along the Upper Danube in Germany. a) Sigmaringen, b) Riedlingen, c) Opfinger See, d) Bad Abbach (Keiter et al., 2008, modified).

Fig. 2. Mean EROD activity in gill (a) and liver (b) in sticklebacks (n=6) exposed to DMSO (100 ppm), to a process control (100 ul DMSO solution/litre test medium) and to B-NF (1 pV A probability value of p<0.01 is indicated by two asterisks. PC=process control. eet ee Se ss Ts Moraes SSeS Miler Saw ners mene SS te Si Certain chemicals, such a as ; PAHs, PCBs and PCDD/Fs, induce cytochrome P450 1A (CYP1A) by ligand-activation of the aryl hydro- carbon receptor (AhR). There is strong evidence that CYP1A-induction in fish is dose-dependently related to levels of halogenated and non- halogenated aromatic compounds in the organism and the environment (Guiney et al., 1997; Giesy et al., 2002), and several stages of the AhR- mediated pathway have been used or proposed as biomarkers in the assessment of these compounds (Goksgyr and F6rlin, 1992; Bucheli and Fent, 1995; Behnisch et al., 2001; van der Oost et al., 2003). Hence, induction of CYP1A is one of the best studied biomarkers for environ- mental contamination in aquatic ecosystems and it is often quantified using enzyme assays, especially measurement of 7-ethoxyresorufin-O- deethylase (EROD) activity (Whyte et al., 2000; Hollert et al., 2002; Sediment samples for this investigation were from the upper Danube River, Germany. These samples had previously been char- acterized as ecotoxicologically hazardous and as potentially contribut- ing to a decline of fish populations in the upper Danube River (Keiter et al., 2006). A following integrated approach using triad investigations (Chapman, 2000; Chapman and Hollert, 2006) was carried out and focused on chemical analysis of water quality and on monitoring of

Fig. 2. Mean EROD activity in gill (a) and liver (b) in sticklebacks (n=6) exposed to DMSO (100 ppm), to a process control (100 ul DMSO solution/litre test medium) and to B-NF (1 pV A probability value of p<0.01 is indicated by two asterisks. PC=process control. eet ee Se ss Ts Moraes SSeS Miler Saw ners mene SS te Si Certain chemicals, such a as ; PAHs, PCBs and PCDD/Fs, induce cytochrome P450 1A (CYP1A) by ligand-activation of the aryl hydro- carbon receptor (AhR). There is strong evidence that CYP1A-induction in fish is dose-dependently related to levels of halogenated and non- halogenated aromatic compounds in the organism and the environment (Guiney et al., 1997; Giesy et al., 2002), and several stages of the AhR- mediated pathway have been used or proposed as biomarkers in the assessment of these compounds (Goksgyr and F6rlin, 1992; Bucheli and Fent, 1995; Behnisch et al., 2001; van der Oost et al., 2003). Hence, induction of CYP1A is one of the best studied biomarkers for environ- mental contamination in aquatic ecosystems and it is often quantified using enzyme assays, especially measurement of 7-ethoxyresorufin-O- deethylase (EROD) activity (Whyte et al., 2000; Hollert et al., 2002; Sediment samples for this investigation were from the upper Danube River, Germany. These samples had previously been char- acterized as ecotoxicologically hazardous and as potentially contribut- ing to a decline of fish populations in the upper Danube River (Keiter et al., 2006). A following integrated approach using triad investigations (Chapman, 2000; Chapman and Hollert, 2006) was carried out and focused on chemical analysis of water quality and on monitoring of

Fig. 3. EROD activity in gill (a) and liver (b) after exposure to sediment extracts from the sites “Sigmaringen’”, “Riedlingen”, “Opfinger See” and “Bad Abbach”. Sticklebacks (n=5-6) were exposed for 48 h under static conditions to sediment extracts at the sediment equivalent concentrations of 4 mg/I, 0.8 mg/I, 0.16 mg/l or 0.032 mg/I or to a positive control (1 uM B-NF) or a solvent control (100 ppm DMSO). Activities that differ significantly from the solvent control are indicated by one (p<0.05) or by two (p<0.01) asterisks. CALUX®) and RTL-W1. Comparison of the concentrations of persistent (PCDDs, PCDFs and PCBs) and non-persistent (PAHs) organic com- pounds with the total dioxin-like activity measured in vitro led to only partial explanation of the obtained activity (Keiter et al., 2008). different hydrological and biological parameters (Boettcher et al., submitted for publication; Keiter et al., 2008; Seitz et al., 2008). High dioxin-like activity was assessed in vitro for sediments at different sites along the river course using the cell lines GPC.2D.Luc, H4IIE (DR-

Fig. 3. EROD activity in gill (a) and liver (b) after exposure to sediment extracts from the sites “Sigmaringen’”, “Riedlingen”, “Opfinger See” and “Bad Abbach”. Sticklebacks (n=5-6) were exposed for 48 h under static conditions to sediment extracts at the sediment equivalent concentrations of 4 mg/I, 0.8 mg/I, 0.16 mg/l or 0.032 mg/I or to a positive control (1 uM B-NF) or a solvent control (100 ppm DMSO). Activities that differ significantly from the solvent control are indicated by one (p<0.05) or by two (p<0.01) asterisks. CALUX®) and RTL-W1. Comparison of the concentrations of persistent (PCDDs, PCDFs and PCBs) and non-persistent (PAHs) organic com- pounds with the total dioxin-like activity measured in vitro led to only partial explanation of the obtained activity (Keiter et al., 2008). different hydrological and biological parameters (Boettcher et al., submitted for publication; Keiter et al., 2008; Seitz et al., 2008). High dioxin-like activity was assessed in vitro for sediments at different sites along the river course using the cell lines GPC.2D.Luc, H4IIE (DR-

Fig. 4. Lowest observed effect concentrations (LOECs) for induction of gill and liver EROD activities for the four sites investigated. SEQ=Sediment equivalents.

Fig. 4. Lowest observed effect concentrations (LOECs) for induction of gill and liver EROD activities for the four sites investigated. SEQ=Sediment equivalents.

Fig. 5. EROD activity in gill (a) and liver (b) in males and females after exposure to the solvent control (100 ppm DMSO) as well as in gill (c) and liver (d) of males and females after exposure to the positive control (1 tM B-NF). The number of sticklebacks was eight for each sex for the exposure to the solvent and 14-18 for each sex for the positive control. Significant differences between the sexes are indicated by one (p<0.05) or by three (p<0.001) asterisks. The procedure described by Jonsson et al. (2002) for rainbow trout, adapted to sticklebacks by Andersson et al. (2007), was used for determination of the EROD activity in gill filaments. The gill filaments were cut off from the cartilage part of the gill arches and triplicates of about 15 filaments were arranged in a 12-well tissue culture plate. Following steps were done under light-protected conditions. Reaction buffer, consisting of 1 uM 7-ethoxyresorufin and 20 uM dicumarol in HEPES-Cortland buffer, was added in each well. The incubation took place under constant shaking for 10 min at room temperature. Subsequently, the reaction buffer was renewed and incubation proceeded. After 40 and 60 min, 0.2-ml samples were transferred from each well to a Fluoronunc

Fig. 5. EROD activity in gill (a) and liver (b) in males and females after exposure to the solvent control (100 ppm DMSO) as well as in gill (c) and liver (d) of males and females after exposure to the positive control (1 tM B-NF). The number of sticklebacks was eight for each sex for the exposure to the solvent and 14-18 for each sex for the positive control. Significant differences between the sexes are indicated by one (p<0.05) or by three (p<0.001) asterisks. The procedure described by Jonsson et al. (2002) for rainbow trout, adapted to sticklebacks by Andersson et al. (2007), was used for determination of the EROD activity in gill filaments. The gill filaments were cut off from the cartilage part of the gill arches and triplicates of about 15 filaments were arranged in a 12-well tissue culture plate. Following steps were done under light-protected conditions. Reaction buffer, consisting of 1 uM 7-ethoxyresorufin and 20 uM dicumarol in HEPES-Cortland buffer, was added in each well. The incubation took place under constant shaking for 10 min at room temperature. Subsequently, the reaction buffer was renewed and incubation proceeded. After 40 and 60 min, 0.2-ml samples were transferred from each well to a Fluoronunc

Fig. 6. EROD activity in gill (a) and liver (b) after exposure to sediment extracts from the sites “Sigmaringen”, “Riedlingen”, “Opfinger See” and “Bad Abbach”. Data is shown as s{ (male/female) and mixed groups. Within 4 weeks after sampling of the tissue, EROD activity in the liver was measured according to a method described by Kennedy and Jones (1994). The liver was homogenized in ice-cold homogenization buffer (0.15 M KCI, 1 mM EDTA in 0.1 M phosphate buffer; pH 7.4) using a Potter-Elvehjem homogeniser (B. Braun, Melsungen, Germany) with 1200 revolutions per minute. The homogenate was centrifuged at 10000 g (Rotanda 460R; Hettich Zentrifugen, Tuttlingen, Germany) for 15 min (4 °C) and then the supernatant was transferred and centrifuged again (L8-70 ultracentrifuge; Beckman Instruments, Fullerton CA, USA) at 105000 g for 1 h (4 °C). The resulting pellet, containing the microsomes, was resuspended in 1 ml HEPES-Cortland buffer (pH 8.0) and, if necessary, the suspension was diluted in the same buffer. Standard solutions of resorufin (0-500 nM) and bovine serum albumin (0-6 mg/ml) were also made in HEPES-Cortland buffer. For each microsome sample and each concentration of resorufin standard, 45-ul samples were transferred to 4 Fluoronunc 96-well plate (Nunc A/S, Roskilde, Denmark) as triplicates. Subsequently, 160 of a reaction solution (15 uM 7-ethoxyresorufin and 2.1 mM NADPH in HEPES-Cortland buffer) was added to all wells and the fluorescence was immediately measured in 4 Wallac3 plate reader (Wallac Oy, Turku, Finland) (excitation 544 nm, emission 590 nm) and several times within 20 min. To determine the protein concentration, 45-ul aliquots of each microsome suspension and of the protein standard were added to a Fluoronunc 96-well

Fig. 6. EROD activity in gill (a) and liver (b) after exposure to sediment extracts from the sites “Sigmaringen”, “Riedlingen”, “Opfinger See” and “Bad Abbach”. Data is shown as s{ (male/female) and mixed groups. Within 4 weeks after sampling of the tissue, EROD activity in the liver was measured according to a method described by Kennedy and Jones (1994). The liver was homogenized in ice-cold homogenization buffer (0.15 M KCI, 1 mM EDTA in 0.1 M phosphate buffer; pH 7.4) using a Potter-Elvehjem homogeniser (B. Braun, Melsungen, Germany) with 1200 revolutions per minute. The homogenate was centrifuged at 10000 g (Rotanda 460R; Hettich Zentrifugen, Tuttlingen, Germany) for 15 min (4 °C) and then the supernatant was transferred and centrifuged again (L8-70 ultracentrifuge; Beckman Instruments, Fullerton CA, USA) at 105000 g for 1 h (4 °C). The resulting pellet, containing the microsomes, was resuspended in 1 ml HEPES-Cortland buffer (pH 8.0) and, if necessary, the suspension was diluted in the same buffer. Standard solutions of resorufin (0-500 nM) and bovine serum albumin (0-6 mg/ml) were also made in HEPES-Cortland buffer. For each microsome sample and each concentration of resorufin standard, 45-ul samples were transferred to 4 Fluoronunc 96-well plate (Nunc A/S, Roskilde, Denmark) as triplicates. Subsequently, 160 of a reaction solution (15 uM 7-ethoxyresorufin and 2.1 mM NADPH in HEPES-Cortland buffer) was added to all wells and the fluorescence was immediately measured in 4 Wallac3 plate reader (Wallac Oy, Turku, Finland) (excitation 544 nm, emission 590 nm) and several times within 20 min. To determine the protein concentration, 45-ul aliquots of each microsome suspension and of the protein standard were added to a Fluoronunc 96-well

Concentrations of selected PCDD/Fs with affinity for the Ah receptor in sediment extracts from the upper Danube River expressed as pg/g sediment dry weight (Keiter et al., 2008)

Concentrations of selected PCDD/Fs with affinity for the Ah receptor in sediment extracts from the upper Danube River expressed as pg/g sediment dry weight (Keiter et al., 2008)

Concentrations of selected PCBs with affinity for the Ah receptor in sediment extracts from the upper Danube River given in pg/g sediment dry weight (Keiter et al., 2008) plate in triplicates and duplicates respectively. An aliquot of 160 tl fluorescamine in acetonitrile (300 pg/ml) was filled in each well and incubated for 15 min in darkness. The fluorescence was measured by using an excitation wavelength of 355 nm and an emission wavelength of 460 nm. EROD activity was calculated and expressed as picomole resorufin formed per mg protein and minute.

Concentrations of selected PCBs with affinity for the Ah receptor in sediment extracts from the upper Danube River given in pg/g sediment dry weight (Keiter et al., 2008) plate in triplicates and duplicates respectively. An aliquot of 160 tl fluorescamine in acetonitrile (300 pg/ml) was filled in each well and incubated for 15 min in darkness. The fluorescence was measured by using an excitation wavelength of 355 nm and an emission wavelength of 460 nm. EROD activity was calculated and expressed as picomole resorufin formed per mg protein and minute.

Concentrations of 16 PAHs (USEPA 610) in sediments from the upper Danube River in ng/g sediment dry weight (n.d.=not detectable) (Keiter et al., 2008)

Concentrations of 16 PAHs (USEPA 610) in sediments from the upper Danube River in ng/g sediment dry weight (n.d.=not detectable) (Keiter et al., 2008)

Fig. 7. PAH levels (ng/g sediment dry weight) in sediments from the upper Danube River (Keiter et al., 2008) in relation to measured EROD-inducing potency of the sediment extracts. n.d.=not detectable.

Fig. 7. PAH levels (ng/g sediment dry weight) in sediments from the upper Danube River (Keiter et al., 2008) in relation to measured EROD-inducing potency of the sediment extracts. n.d.=not detectable.

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