Arch. Environ. Contam. Toxicol. 51, 232–236 (2006) DOI: 10.1007/s00244-005-0064-1
Polycyclic Aromatic Hydrocarbon Metabolites and 7-Ethoxyresorufin O-Deethylase Activity in Caged European Eels H. Fenet, E. Gomez, D. Rosain, C. Casellas UMR Hydrosciences Montpellier, Dpartement Sciences de lÕEnvironnement et Sant Publique, UFR des Sciences Pharmaceutiques et Biologiques, Universit Montpellier I, Av. C. Flahault, 34060 Montpellier, France
Received: 21 March 2005 /Accepted: 24 December 2005
Abstract. This study investigated the contribution of two biomarkers, bile polycyclic aromatic hydrocarbon (PAH) metabolites and 7-ethoxyresorufin O-deethylase (EROD), activity in the assessment of PAH contaminated sites. European eels (Anguilla anguilla) were caged in a freshwater stream upstream and downstream from local industrial effluent outlets. Bile PAH metabolites were recorded as fluorescent aromatic compounds by synchronous fluorescence spectroscopy and as a marker for total PAH metabolism: 1-hydroxypyrene (1-OH Pyr) was isolated by high-pressure liquid chromatography and quantified. After 14 and 28 days of caging, EROD activity, bile fluorescence (synchronous fluorometric measurement), and 1-OH Pyr concentrations in bile were higher at the downstream site than at the upstream site. This increase was similar after 2 and 4 weeks of caging. During a reversibility study, EROD activity, bile fluorescence, and 1-OH Pyr concentrations decreased, and this trend was similar for the three markers. These results suggest that PAHs could be the main factor responsible for EROD induction in eels caged at the downstream site.
Polycyclic aromatic hydrocarbons (PAHs) are known to be environmental aquatic pollutants (Van der Oost et al. 2003). Mutagenic and carcinogenic effects have been observed in fish and other vertebrates (Stein et al. 1990; Myers et al. 2003), so assessment of their potential impacts within aquatic ecosystems is of considerable environmental interest. In rivers, PAHs tend to accumulate in sediment (Notar et al. 2001), and therefore sediment PAH concentrations can be measured to determine contamination levels at sites of concern. However, this measurement provides neither information on the fraction of PAHs that are bioavailable for the biota nor any insight into their potential biological impacts.
Correspondence to: H. Fenet; email:
[email protected]
Fish absorb PAHs through the gills or by way of ingestion of contaminated food or suspended particles. PAHs are rapidly metabolized in fish (Livingstone 1998), so measurement of parent PAHs in tissues does not provide an adequate assessment of exposure. PAHs are transformed by phase I enzymes of the mixed-function oxygenase system into more lipophilic products through insertion of an oxygen atom; most PAH metabolites are combined with endogenous molecules to form conjugates by phase II enzymes (Vermeulen et al. 1992). The liver is the major organ involved in PAH metabolism, and the metabolites produced are secreted into the bile and stored in the gallbladder. Induction of cytochrome P4501A1 in fish liver is recognized as being an excellent biomarker for evaluating PAH exposure (Fenet et al. 1998; Beyer et al. 1996; Narbonne et al. 1991). However, this induction could be caused by other organic contaminants such as planar congeners of polychlorinated biphenyls, dioxins and furans (Hewitt et al. 1998; JedamskiGrymlas et al. 1995; Monod et al. 1988), so it does not enable discrimination between these classes of compounds. Measuring PAH metabolites in bile is thus useful to gain further insight into the nature of exposure. PAH metabolites in bile can be determined by synchronous fluorometric measurement (SFS). SFS was introduced by Ariese et al. (1993) and has been successfully used in many PAH monitoring studies (Barra et al. 2001; Lyons et al. 1999). This technique is rapid and cost-effective and can be implemented to directly assess bile without any preliminary hydrolyses or extraction steps. Alternatively, after deconjugation, the different phase I metabolites could be separated and identified by high-performance liquid chromatography (HPLC) with fixed fluorescence detection (FFD) (Ruddock et al. 2003). This study investigated the contribution of two biomarkers, bile PAH metabolites and EROD activity, for the assessment of PAH-contaminated sites. European eels (Anguilla anguilla) were caged in a freshwater stream upstream and downstream from local industrial effluent outlets. PAH exposure was evaluated through measurement of bile aromatic metabolites using SFS and HPLC-FFD. The relations between these two measurements were evaluated. The recovery of bile fluorescence was also studied by transferring fish pre-exposed to pollutants at the downstream site to the upstream site.
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Materials and Methods Field Experiments European eels (A. anguilla) of approximately similar weight (approximately 25 g) were fished from a low-contamination brackish lagoon located in southern France. The eels were acclimatized for 1 week before the experiments and maintained unfed in tanks with aerated filtered freshwater. At the end of the acclimation period (day 0), eels were killed to evaluate EROD activity and bile fluorescence. Eels were caged at two sites in a freshwater stream located in southern France (4311¢N, 256¢E). The upstream (U) site was situated in an agricultural area. The downstream (D) site was located 6 km from U site, 3 km from local industrial effluent outlets, and near a discharge outlet from a wastewater treatment plant (50,000 inhabitants). The downstream site was also located under a highway bridge. Thirty-liter cages were set in the stream near the riverbanks in the water column (50 cm depth). The eels were not fed throughout this period. Ten to twelve eels were placed in each cage. The field study was performed in April, and water temperature was 15C € 2C. Two cages were immersed at the upstream site, and three cages were immersed at the downstream site. One cage from each site was removed after 14 days, and another cage from each site was removed after 28 days (U site = 28 days, and D site = 28 days), and the eels were subsequently killed. On day 14, one cage from the downstream site was transferred to the upstream site, where it was maintained until day 28. The eels were killed 14 days after transfer. The livers were removed, and bile was collected from the gallbladder. Excised livers were frozen in liquid nitrogen before analyses. Bile samples were stored at )20C for further processing. Because bile volume was limited, some pooling was necessary for the fluorescence measurements.
EROD Activity Analysis Liver samples were thawed at 4C and homogenized in Hepes buffer (pH 7.4) using a potter Elvehjem. The homogenate was centrifuged at 9500 g for 20 minutes at 4C. The supernatant was transferred and centrifuged at 105,000g for 60 minutes. The supernatant was removed, and EROD activity was determined using the methodology derived from Burke and Mayer (1974) as described by Fenet et al. (1996). EROD activity was normalized to the protein content and expressed as pmol resorufin min)1 mg protein)1.
SFS Analysis Because the total bile sample was too small for analysis, pooling was necessary. Gallbladder bile samples were diluted 1:1500 in 48% ethanol before determination of bile fluorescence by SFS. The SFS was performed on a Shimadzu luminescence spectrometer (RF 5301, Shimadzu Europe GmbH). Spectra were recorded in 1-cm quartz cuvettes with spectral split widths set at 5 nm for both excitation and emission. Fluorescence signals were measured with a fixed wavelength difference of 41 nm. The peak area was obtained by integrating emission fluorescence from 260 to 450 nm wavelength, and results were expressed in unit of fluorescence.
PAH Metabolite Analysis When there was not enough material to perform PAH metabolites analysis, some samples used for SFS analysis were pooled again.
There were always at least three pooled samples analyzed. Bile metabolites were prepared for HPLC-FFD analyses. Bile samples were slowly thawed. Bile, 50 ll (weighed), was mixed with 2000 U b-glucuronidase, 50 U sulfatase (Sigma), and 500 ll 0.4 M acetate buffer (pH 5) in an Eppendorf tube. The mixture was incubated in a water bath at 40C for 2 hours. Ethyl acetate, 500 ll, was added and mixed, and the sample was centrifuged for 10 minutes at 3500g in an Eppendorf centrifuge; this procedure was carried out in triplicate. The ethyl acetate extract was evaporated under nitrogen to 50 ll and 150 ll ethanol-water (80:20)-5% ascorbic acid was added. The extract was weighed. The analysis was done on a Spectra system P1000 XR equipped with a Shimadzu RF-530 fluorescence detector and a 25-cm length 3-mm i.d. Nucleosil MN-100-5 C18 column, a Lichrocart Lichrospher PAH guard column (Merck), and a 20-ll rheodyne loop. A water-acetonitrile gradient was used (5% to 100% acetonitrile in 40 minutes). The wavelength pair was excitation/emission 256/380 nm. Quantification was performed by external calibration for 1-OH Pyr.
Statistical Analysis Results were expressed as mean € SD. Significant differences among values were analyzed by Student t test. Significance was set at p < 0.05.
Results and Discussion The EROD activity results are displayed in Figure 1. Eels from the control group (day 0) showed significantly lower EROD activity than eels exposed in the field upstream or downstream from the effluent outlets. As expected, EROD activity was significantly higher in downstream eels than in upstream eels. This induction of EROD activity (threefold) was similar after 2 and 4 weeks of caging. Transferring the eels from the downstream to the upstream site resulted in a significant decrease in EROD activity, which was still significantly higher than noted at the upstream site. Several laboratory studies have highlighted that in European eels intermittently exposed to PAHs, the induction of EROD activity was maximum within 3 or 4 days and that the subsequent decrease to control levels was achieved within 3 weeks (Fenet et al. 1996; Gorbi and Regoli 2004). The field results showed the same decrease in EROD activity after changing the exposure level. To determine the extent to which PAHs contributed to the observed EROD induction, bile aromatic metabolites were assessed by SFS (Fig. 2) and by measurement of individual 1-OH Pyr metabolite (Fig. 3). Caging at the upstream site did not have a significant impact on the level of bile metabolites (SFS and 1-OH Pyr) compared with the control group (day 0). Exposure at the downstream site resulted in a significant (threefold to fourfold) increase in bile fluorescence and 1-OH Pyr concentration. This increase was similar after 2 and 4 weeks of caging. During the reversibility study, the bile fluorescence and 1-OH Pyr concentration decreased, but bile fluorescence was still significantly higher than that noted in eels maintained solely at the upstream site. The difference observed between SFS and HPLC measurements could be due to SFS analysis, which is not specific to 1-OH Pyr and takes into account other PAH metabolites. However, a linear relationship between 1-OH Pyr concentration and SFS measurement was observed in this study (y = 0.637x + 7.022,
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Fig. 1. EROD activity (mean € SD) in European eel at day 0 and after 2 and 4 weeks of caging at D and U sites. *EROD activity was significantly different from that at U site (p < 0.05). The number in the histogram corresponds to the number of liver samples
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r2 = 0.896; Fig. 4), which provides evidence that fluorescence in bile was representative of PAH ingestion and metabolization. 1-OH Pyr is the main metabolite of pyrene, a widespread and common PAH generated by many pyrolytic and petrogenic industrial processes. Moreover, the bioavailability of pyrene is relatively high for aquatic organisms (Ariese et al. 1993), and it is often considered to be one of the most abundant compounds in fish bile (Krahn et al. 1987). This metabolite is thus regarded as being the best general indicator of PAH exposure in fish (Van der Oost et al. 2003). More specifically, for European eel, Ruddock et al. (2003) showed that 1-OH Pyr was the most frequently observed metabolite in industrialized United Kingdom estuaries. When bile metabolite concentrations are used as biomarkers of exposure, confounding factors must be taken into account. Individual PAH metabolite concentrations in bile vary between
Fig. 2. Bile fluorescence measured by SFS (mean € SD) in bile of European eels at day 0 and after 2 and 4 weeks of caging at D and U sites. *EROD activity was significantly different from that at U site (p < 0.05). The number in the histogram corresponds to the number of pooled bile samples
fish species (Porte and Escartin 2000) and are affected by factors such as dietary status (Collier and Varanasi 1991; Brumley et al. 1998). However, these sources of variation are minimized by caging the fish. During our caging studies, we were not able to control the feeding status of the fish. We hypothesized that the impact of feeding status was similar between the two sites. However, in fish that are not fed, metabolites accumulate in the bile and become further concentrated because of water resorption across the gallbladder wall. The bile metabolite concentrations measured by SFS or by 1-OH Pyr suggest that PAHs could be the main factor responsible for EROD induction by eels caged at the downstream site. PAH metabolite patterns measured by SFS or by 1-OH Pyr concentration were similar to EROD activity patterns, although no differences were detected between the laboratory control eels and those caged at the upstream site. A
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Fig. 3. 1-OH Pyr concentrations (mean € SD) in bile of European eel at day 0 and after 2 and 4 weeks of caging at D and U sites. *1-OH Pyr concentrations were significantly different from those at U site (p < 0.05). The number in the histogram corresponds to the number of pooled bile samples
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Fig. 4. Relation between 1-OH Pyr concentrations and peak area SFS (mean € SD) in bile of European eel at day 0 and after caging experiments. The line represents a linear regression curve
y = 21.279x - 5.2237 R2 = 0.9368
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linear relationship between EROD activity and 1-OH Pyr concentration or SFS (Fig. 5) was observed in this study (r2 = 0.974 and r2 = 0.937, respectively). A positive relationship between the MFO system and the presence of PAH metabolites in bile has been reported in a number of studies in which fish were exposed to PAHs in marine and estuarine
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Fig. 5. Relation between EROD activity and peak area SFS in bile of European eel at day 0 and after caging experiments. The line represents a linear regression curve
environments with species such as European eel (Ruddock et al. 2003) and Atlantic cod (Beyer et al. 1996). Studies have been conducted on rainbow trout in freshwater (Barra et al. 2001). The results of the current study indicate that caged European eels are effective for evaluating PAH contamination in freshwater streams.
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