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Metallothionein in liver of eels Anguilla anguilla from the Thames Estuary: an indicator of environmental quality?

The purpose of this study was to examine the role of the cysteine-rich protein metallothionein (MT) in the detoxification and metabolism of metals in livers of eels Anguilla anguilla from the Thames Estuary, and to assess the value of MT measurements in environmental monitoring. Hepatic MT levels and associated metal concentrations were determined in A. anguilla collected on three occasions during 1998 at sites along the tidal Thames (from freshwater to the sea) and also from a reference site (Tamar Estuary) in southwest England. MT was present at basal levels of 2 mg g(-1) dry weight (dw), predominantly in association with the essential metals Cu and Zn. MT concentrations were variable within each eel 'population' but were generally highest (up to 11 mg g(-1) in individual eels) at the more contaminated upper- and mid-estuarine sites in the Thames (e.g. Brentford and Kew). Lowest MT levels were those in eels from Blythe Sands at the mouth of the estuary. Season, sex, reproductive status and salinity may have influenced MT levels to some extent, although metals were the most significant factor. Hepatic MT concentrations were highly correlated with associated metal burdens (Zn, Cu, Ag and Cd),--notably reflecting Cu and Ag enrichment in the upper- and mid-estuary. With the exception of Zn, metals in the supernatant fraction of eel livers were primarily associated with MT. Proportions of Cu, Ag and Cd bound to MT increased as a function of accumulated metal burdens, with no indication of saturation. Thus, despite causing induction of MT, excess bioavailable Cu, Ag and Cd appear to be successfully detoxified in eels over the range of environmental contamination encountered along the Thames Estuary. Paradoxically, it may be argued from conventional doctrine that the presence of raised levels of MT, at upstream sites, constitutes a response to contamination and, by definition, signals that the fish are affected by metals; however, obvious signs of deleterious effects were not detected. Superficially, from liver MT data, it would seem that eel populations have become biochemically adapted to metal contamination. The approach tested here has proved viable in helping to interpret trends in bioavailability and the toxicological significance of accumulated metal burdens in Thames eels. A. anguilla is a common inhabitant of estuarine and fresh waters throughout Europe, and related species occur world wide. Therefore, determination of hepatic MT (and associated metals) in eels could be useful for monitoring responses to metal exposure and environmental quality on a much broader basis. Ideally these determinations should be performed in tandem with other sublethal-effects measurements to maximise information.

Anguilla↗

Genotoxic and hepatic biotransformation responses induced by the overflow of pulp mill and secondary-treated effluents on Anguilla anguilla L.

Pulp and paper mill effluent compounds pollute the aquatic environment and are responsible for increased biochemical alterations and genotoxicity in aquatic organisms such as fish. Adult eels (Anguilla anguilla L) were exposed during 8, 16, 24, and 72 h to the following conditions: (1) aerated, filtered, and dechlorinated tap water (C); (2) 2.5% (v/v) sewage water previously treated with activated sludge (T); (3) bleached kraft pulp and paper mill effluent collected at the river Vouga, close to an ancient sewage outlet (Portucel), diluted in tap water [25% (E25) and 50% (E50)]; and (4) bleached kraft pulp and paper mill effluent sediment [water-soluble fraction (S)]. Liver biotransformation induced by the above conditions was measured as ethoxyresorufin-O-deethylase (EROD), cytochrome P450 (P450) (Phase I), and glutathione-S-transferase (GST) (Phase II). Genotoxicity was also determined as blood/liver DNA strand breaks and erythrocytic nuclear abnormalities (ENA) induced on European eel (A. anguilla L). Liver EROD activity was significantly increased in eels at 8 and 16 h exposure to E25, as well as at 16, 24, and 72 h exposure to E50. S exposure induced liver EROD activity only at 24h. A significant decrease in liver P450 was observed at 72 h exposure to T, whereas a significant P450 increase at 16 h was followed by a significant decrease at 24h exposure to E25. Another P450 significant increase was noticed at 72 h exposure to S. Liver GST activity (Phase II) demonstrated a significant increase at 72 h exposure to E50 and to S. A significant decrease in blood DNA integrity was observed at 72 h exposure to T and at 24 and 72 h to S. Blood DNA integrity significantly decreased at 16 and 24 h exposure to E25, as well as at 8, 16, and 24 h exposure to E50. Liver DNA integrity significantly decreased at 72 h exposure to T and at 16 h exposure to S. Moreover, liver DNA integrity was significantly decreased at 24h exposure to E25 and E50, and 72 h to E50. A. anguilla L. increased ENA frequency was detected in T at 16, 24, and 72 h, whereas in E25 and S it was observed at 8, 16, and 24 h. Furthermore, E50 ENA frequency increased at 24 h exposure.

Anguilla↗

Anguilla anguilla L. antioxidants responses to in situ bleached kraft pulp mill effluent outlet exposure.

This study assesses the antioxidant enzymes activities viz., catalase, glutathione peroxidase, glutathione S-transferase and nonenzymatic antioxidant molecule such as glutathione in Anguilla anguilla L. gill, kidney and liver in response to 8- and 48-h exposure to bleached kraft pulp mill effluent (BKPME). A. anguilla were caged and plunged at three different sites-50 (Site 1), 100 (Site 2) and 2000 m (Site 3) away from the closed BKPME outlet. A significant gill (8 and 48 h) and kidney (48 h) catalase activity decrease was observed at site 2 exposure whereas liver showed a significant increase in catalase activity after 8 and 48 h to site 1 exposure. Glutathione peroxidase (GPX) activity was significantly decreased in gill after 8-h exposure to site 1 and 48-h exposures to sites 1 and 2, respectively. Concerning gill, kidney and liver glutathione S-transferase (GST) activity, a significant gill GST activity decrease after 8 h at site 2 and 48 h at sites 1 and 2 was observed; in kidney, a significant decrease in its activity was observed after 48 h at sites 1 and 2, respectively, whereas in liver, the decrease was significant only at site 2 after 48-h exposure. The in situ BKPME exposure caused a significant total gill and kidney reduced glutathione (GSH) decrease after 8 h at site 2 exposure and after 48 h at site 1 and 2 exposures, respectively. However, a biphasic response was observed in liver, i.e. initial significant increase after 8 h at site 2 followed by a significant decrease after 48 h to the same site exposure. The enzymatic and nonenzymatic antioxidants pattern in gill and kidney, as observed in this study, was different than liver, demonstrating that the liver was more resistant to oxidative damage than gill and kidney. In addition, A. anguilla gill, kidney and liver antioxidants adaptation potentials may serve as a surrogate biomarker to BKPME exposure.

Adaptation, Physiological↗

Single-dose pharmacokinetics of flumequine in the eel (Anguilla anguilla) after intravascular, oral and bath administration.

Knowledge of the pharmacokinetic properties of drugs to combat bacterial infections in the European eel (Anguilla anguilla) is limited. One antimicrobial agent likely to be effective is flumequine. The aim of this study was to investigate the pharmacokinetic properties of flumequine in European eels in fresh water. Flumequine was administered to eels (Anguilla anguilla) intravenously (i.v.) and orally (p.o.) at a dose of 10 mg/kg body weight, and as a bath treatment at a dose of 10 mg/L water for 2 h. The study was performed in fresh water with a temperature of 23 + 0.3 degrees C, pH 7.15. Identical experimental designs were used. Two additional bath treatments were also performed, one in which the pH in the water was lowered by approximately 1 unit to 6.07 (dose: 10 mg/L) and one at a dose of 40 mg/L for 2 h in a full-scale treatment. Following i.v. administration, the volume of distribution at steady state was 3.4 L/kg. Total body clearance was 0.012 L/h per kg and the elimination half-life (t1/2lambda z) was calculated to be 314 h. Mean residence time was 283 h. Following oral administration, the t1/2lambda z was 208 h. Maximal plasma concentration (Cmax) was 9.3 mg/L, at 7 h after administration (Cmax). The oral bioavailability (F) was calculated to be 85%. Following bath administration in 10 mg/L for 2 h, maximal plasma concentration was 2.1 mg/L, observed immediately after the end of the bath. The 'bioavailability' in eel following a 2-h bath treatment was 19.8%. Reducing the pH in the bath to 6.07 produced a maximal plasma concentration of 5.5 mg/L, observed immediately after the end of the bath. The 'bioavailability' was increased to 41% by the lowering of the pH. A similar effect was observed in a full-scale treatment (1 kg eels/L water). The CO2 produced by the eel lowered the pH and increased 'bioavailability' to 35%.

Administration, Oral↗

Beta3-adrenoceptor in the eel (Anguilla anguilla) heart: negative inotropy and NO-cGMP-dependent mechanism.

Neuroendocrine regulation of cardiac function involves a population of three types of beta-adrenoceptors (ARs). In various mammalian species, beta1- and beta2-AR stimulation produces an increase in contractility; whereas beta3-AR activation mediates negative inotropic effects. At the moment, nothing is known about the physiological role of beta3-AR in fish. Using an isolated working heart preparation, we show that a beta3-AR selective agonist BRL(37344) (0.1-100 nmol l(-1)) elicits a dose-dependent negative inotropism in the freshwater eel Anguilla anguilla. This effect was insensitive to the beta1/beta2-AR inhibitor nadolol (10 mumol l(-1)), but was blocked by the beta3-AR-specific antagonist SR(59230) (10 nmol l(-1)). The analysis of the percentage of stroke work (SW) variations, in terms of EC(50) values, induced by BRL(37344) alone (10 nmol l(-1)), and in presence of SR(59230) (10 nmol l(-1)), indicated a competitive antagonism of SR(59230). In addition to the classic positive inotropism, the non-specific beta agonist isoproterenol (100 nmol l(-1)) induced, in 30% of the preparations, a negative inotropic effect that was abrogated by pre-treatment with SR(59230), pointing to a beta3-mediated pathway. The BRL(37344)-induced negative inotropic effect was abolished by exposure to a G(i/o) proteins inhibitor pertussis toxin (PTx; 0.01 nmol l(-1)), suggesting a G(i/o)-dependent mechanism. Using L-N5(l-imino-ethyl)ornithine (L-NIO; 10 mumol l(-1)), as a nitric oxide (NO) synthase (NOS) blocker and haemoglobin (Hb; 1 mumol l(-1)), as a NO scavenger, we demonstrated that NO signalling is involved in the BRL(37344)-induced response. Pre-treatment with either an inhibitor of soluble guanylate cyclase (GC) 1H-(1,2,4) oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ; 10 mumol l(-1)), or an inhibitor of the cGMP-activated protein kinase (PKG) KT(5823) (100 nmol l(-1)), abolished the beta3-dependent negative inotropism, indicating the cGMP-PKG component as a crucial target of NO signalling. Taken together, our findings provide functional evidence for the presence of beta3-like adrenoceptors in the eel Anguilla anguilla heart identifying, for the first time in a working fish heart, the beta3-AR-dependent negative inotropy discovered in mammals.

Adrenergic beta-3 Receptor Agonists↗

Metabolic effects of kraft mill effluents on the eel Anguilla anguilla L.

Yellow eels (Anguilla anguilla L.) with an average weight of 60 g were used in this experiment. The fish were caught in June/July at the Aveiro Lagoon on the Portuguese West Coast, transported to the Department of Biology, Aveiro University, and kept in aerated aquaria for 1 week before the experiment started. The eels were then exposed for 1 and 3 weeks to 75 and 50% of the kraft pulp mill effluent. The eels exposed to the kraft pulp mill effluent developed an increase in red blood cell number per cubic millimeter and several biochemical changes, such as an increase in plasma lactate and sodium and a decrease in plasma pyruvate and potassium. Histological examination of the experimental eels exposed to the 50% kraft pulp mill effluent revealed deep alteration of the tissue structure, such as disruption of the skin and edematous hypertrophy of covering epithelial cells in secondary gill lamellae. The kidney had damage of the renal tubules. The liver developed necrosis supported by a significant decrease in GOT and GPT activity. The spleen had an increase in blood content as well as in pigment centers. Previous results indicated the kraft pulp mill effluent causes tissue damage and consequent metabolic changes in the eel Anguilla anguilla L.

Alanine Transaminase↗

Tissue distribution and temperature-dependence of Anguilla anguilla L. EROD activity following exposure to model inducers and relationship with plasma cortisol, lactate and glucose levels.

Anguilla anguilla L. ethoxyresorufin-O-deethylase (EROD) elevation by 2.7 microM beta-naphthoflavone (BNF) 3 days water exposure, or 4 mg/kg ip exposure, was studied in four different organs--liver, kidney, gills, and intestine. The results demonstrated a significant increase in liver EROD activity for the two previous conditions, whereas kidney EROD activity only increased during the intraperitoneal exposure. A. anguilla was also exposed during 8, 16, and 24 h to water contaminated with 2.7 microM BNF or benzo[a]pyrene (BaP) at 20 degrees C and 25 degrees C. Both compounds significantly increased liver EROD activity from 8 up to 24 h. There was no significant difference in liver EROD activity elevation by both compounds, either at 20 degrees C or 25 degrees C. Liver EROD activity was demonstrated to be one of the first warning systems concerning the presence of polycyclic aromatic hydrocarbons (PAHs) in water. A. anguilla 3 h exposure to diesel oil water-soluble fraction (DWSF) significantly increased plasma cortisol and significantly decreased plasma lactate. A prolonged exposure beyond 3 h, i.e. 4 h, 2, 3, 4, and 6 days to the previous conditions demonstrated a significant liver EROD activity elevation from Day 2 up to 6, and a significant increase in erythrocytic nuclear abnormalities (ENA) at Day 6.

Animals↗

[Studies on blood morphology in the European eel (Anguilla anguilla). I. Erythrocytes and their developmental stages].

By means of heart tapping we produced a larger loss of blood in the European eel, Anguilla anguilla. This resulted in enriching the peripheric blood with erythroblasts and proerythrocytes. By panoptic staining erythroblasts, proerythrocytes and mature erythrocytes could be prepared and described. By the aid of supravital staining it was possible to detect mitochondria and Substantia granulofilamentosa in all stages of development. Phase contrast observations proved that the erythrocytes of Anguilla anguilla do not possess any locomotion ability. Cytochemical investigations showed that the erythrocytes weakly or very weakly react to acid phosphatases, lipids and ribonucleic acids and positively to unspecific esterases. The artificially produced loss of blood and the following continuous extraction of blood gave indications of how rapidly the erythrocytes form and how much time their maturing takes. 48 hours after tapping erythroblasts appeared in large amounts. The number of proerythrocytes increased 24 hours later. The transformation of proerythrocytes into mature erythrocytes does not take more than 48 hours. The loss of erythrocytes caused by heart tapping is already compensated six days later by new growth. During this investigation division processes in erythrocytes were observed. These are endomitotic or amitotic division or mitotic ones which are very rarely observed.

Acid Phosphatase↗

Effect of deterlon (DBS) on the electrocardiogram of eel Anguilla anguilla (L).

Effect of Deterlon (DBS) on the electrocardiogram of eel Anguilla anguilla (L). Acta Physiol. Pol. 1977, 28 (2): 179--186. The investigations of the effect of a detergent Deterlon-DBS present in industrial and community sewage on ECG changes in the eel Anguilla anguilla (L) showed that it causes disturbances in the impulse-generating centres and in the mechanism of impulse conduction in the heart. It was found that the threshold concentration of Deterlon-DBS in aqueous medium is 3 mg SA/1 for the eel, the lethal concentration lying in the range of 60--100 mg SA/1.

Animals↗

Inhibition of gill Na+,K+-ATPase activity in the eel, Anguilla anguilla, by fenitrothion.

European eels (Anguilla anguilla) were exposed to sublethal fenitrothion concentrations (0.02 and 0.04 mg/liter) in a continuous flow-through system for 4 days. Gill Mg2+- and Na+,K+-ATPase activities were evaluated after 2, 8, 12, 24, 32, 48, 56, 72, and 96 h of pesticide exposure. Results indicated that ATPase activity in gill tissue decreased as concentration of fenitrothion increased. Pesticide induced significant inhibitory effects on the Na+, K+-ATPase activity of A. anguilla, ranging from >56% inhibition at a sublethal concentration of 0.02 ppm to >73% inhibition at a sublethal concentration of 0.04 ppm. Eels were exposed to both fenitrothion concentrations for 96 h and then allowed a period of recovery in pesticide-free water. Samples were removed at 8, 12, 24, 48, 72, 96, 144, and 192 h and eel gill ATPase activity was evaluated. Following 1 week of recovery, the Na+,K+-ATPase activity for those animals previously exposed to 0.04 ppm fenitrothion was still different from that of the controls.

Anguilla↗

Response and recovery of brain acetylcholinesterase activity in the European eel, Anguilla anguilla, exposed to fenitrothion.

European eel (Anguilla anguilla) were exposed to sublethal fenitrothion concentrations in a continuous flow-through system for 4 days. Brain acetylcholinesterase (AChE) activity was evaluated after 2, 8, 12, 24, 32, 48, 56, 72, and 96 h pesticide exposure. Results indicated that AChE activity in eel brains decreased as the concentration of fenitrothion increased. The pesticide induced significant inhibitory effects on the AchE activity of A. anguilla, ranging from > 40% inhibition at a sublethal concentration of 0.02 ppm to > 60% inhibition at a sublethal concentration of 0.04 ppm. Eel were exposed to both fenitrothion concentrations for 96 h and then allowed a period of recovery in pesticide-free water. Samples were removed at 8, 12, 24, 48, 72, 96, 144, and 192 h and eel brain AChE activity was evaluated. Following 1 week of recovery, the AChE activity of those animals previously exposed to 0.02 and 0.04 ppm fenitrothion was still different from that of the controls. So, the AChE activity of eel brains at the end of the recovery phase remained significantly depressed.

Acetylcholinesterase↗

Induction of liver EROD and erythrocytic nuclear abnormalities by cyclophosphamide and PAHs in Anguilla anguilla L.

The induction of liver ethoxyresorufin O-deethylase (EROD) activity and erythrocytic nuclear abnormalities (NA) after treatment with two different polycyclic aromatic hydrocarbons (PAHs) and cyclophosphamide (CP) was investigated in the eel Anguilla anguilla L. EROD activity significantly increases either 3 days after one single i.p. injection or 6 days after two i.p. injections (on days 0 and 3) of 4 mg/kg beta-naphthoflavone (BNF). EROD activity was determined after different lengths of exposure to 4 mg/kg BNF (i.p. injection). The results indicated significant increases from 8-h to 12-day exposure, with a maximum increase at 4 days and a decline between 4 and 12 days. The induction of liver EROD activity and erythrocytic NA was studied, 3 days after one 14.7-mumol/kg treatment (i.p. injection) with BNF, benzo[a]pyrene (BaP), and CP, in three three different groups of eels. The EROD activity significantly increases after BNF and BaP treatment, whereas the erythrocytic NA frequency was kept constant; however, CP induces a significant increase in the erythrocytic NA frequency, but it does not induce a significant increase in liver EROD activity. The sensitivity of A. anguilla liver EROD assay was assessed 3 days after one i.p. injection of BNF (0.0, 0.03, 0.06, 0.125, 0.25, 0.5, 1.0, 2.0, and 4.0 mg/kg). This species exhibits a dose response to BNF at the concentration range 0-4 mg/kg. The NOEL is between 0.125 and 0.25 mg/kg. The eels 3-day exposure to water containing BNF (0.0, 0.1, 0.3, 0.9, and 2.7 microM) induced a significant increase in liver EROD activity, specifically in concentrations of 0.9 and 2.7 microM. The NOEC for the eels external exposure to BNF is between 0.3 and 0.9 microM.

Anguilla↗

Anguilla anguilla L. biochemical and genotoxic responses to benzo[a]pyrene.

Eels (Anguilla anguilla L.) were exposed for 2, 4, 6, 8, 16, 24, 48, 72, 144, and 216 h to 0 (control), 0.3, 0.9, and 2.7 microM benzo[a]pyrene (BaP). The biotransformation induced by BaP was measured as liver ethoxyresorufin O-deethylase (EROD) activity and cytochrome P450 content, and compared with the genotoxic effects, such as erythrocytic nuclear abnormalities (ENA), and blood and liver DNA strand breaks. The liver exhibited a highly significant EROD activity increase from 2 up to 216 h exposure to 0.9 and 2.7 microM BaP, whereas 0.3 microM BaP exposure induced a significant liver EROD increase from 2 up to 144 h. Liver cytochrome P-450 content was significantly increased at 8 h to 2.7 microM BaP exposure. Liver DNA integrity was decreased at 16 h, from 8 up to 144 h and 8 up to 72 h exposure to 0.3, 0.9, and 2.7 microM BaP, respectively. A significant decrease in blood DNA integrity was observed at 48, 72, 144 h, from 8 up to 72, and from 6 up to 72 h exposure to 0.3, 0.9, and 2.7 microM BaP, respectively. The A. anguilla L. genotoxic response to BaP, measured as ENA induction, was significantly increased at 144 h exposure to 0.3 microM BaP. The intermediate BaP concentration tested (0.9 microM) induced a significant three fold ENA increase at 48 and 72 h exposure compared to their controls. The highest BaP concentration (2.7 microM) induced a significant increase in ENA frequency at 72, 144 and 216 h exposure.

Anguilla↗

Bioaccumulation and the effects of organochlorine pesticides, PAH and heavy metals in the Eel (Anguilla anguilla) at the Camargue Nature Reserve, France.

Pesticides (organochlorines-OC), polycyclic aromatic hydrocarbons (PAH) and heavy metals are toxic to fish and may be taken in through gills, skin and contaminated foods. Here we measure concentrations of OC, PAH and heavy metals, and their effects in the eel Anguilla anguilla from three locations in the Camargue Reserve in southern France. The Camargue Biosphere Reserve is the largest coastal wetland in Western Europe, and A. anguilla is a common predator at the top of the food chain. Livers and spleens were analyzed for histopathological, chemical and organo-somatic (HSI and SSI) effects. Gill, liver and spleen samples were collected for histopathological studies. Livers and muscles were sampled for metabolic parameters and persistent organic pollutant analysis. Total lipids were estimated by spectrophotometry and lipid-free residues were used in protein and glycogen analysis. OC pesticides were extracted from lipids of muscles and livers, analyzed by gas chromatography, and PAH from bile were analyzed by fixed wavelength fluorescence spectrofluorimetry. Heavy metals were measured by inductively coupled plasma with optical or with mass spectrometers. High concentrations of contaminants were found in eel tissues. La Capelière had the greatest OC and PAH concentrations; unexpected lesions in gills, livers and spleens were more common at the other sites. Liver and spleen tumors and lipidosis in livers were associated with chronic, and gill lesions with acute exposure. High pesticide and PAH concentrations and lesions in eels from the Camargue reserve demonstrate the contamination of the area. A more complete study in the Camargue reserve is necessary to better understand the impact on wildlife and humans. Also, this study suggests that eel biology must be better understood before continued use of this species as a biomonitor of polluted areas.

Analysis of Variance↗

Adenosine/nitric oxide crosstalk in the branchial circulation of Squalus acanthias and Anguilla anguilla.

The potent vasomodulator adenosine (AD), thanks to the interaction with by A(1) and A(2) receptors, dilates systemic, coronary and cerebral vasculatures but exert a constrictor action in several vessels of respiratory organs. Recent investigations suggest that nitric oxide (NO) contributes to AD effects. In fish, both NO and AD induce atypical effects compared to mammals. Since there is very little information on the role of NO and its involvement in mediating the actions of AD in fish, we have analysed this question in the branchial vasculature of the elasmobranch Squalus acanthias and the teleost Anguilla anguilla using an isolated perfused head and a branchial basket preparation, respectively. In both dogfish and eel, AD dose-response curves showed a biphasic effect: vasoconstriction (pico to nanomolar range) and vasodilation (micromolar range). Both effects were abolished by the classic xanthine inhibitor theophylline (Theo) and also by specific antagonists of A(1) and A(2) receptor subtypes. To analyse the involvement of the NO/cGMP system in the AD responses, we tested a NOS inhibitor, l-NIO, and a specific soluble guanylate cyclase (sGC) blocker, ODQ. In both dogfish and eel preparations l-NIO abrogated all vasomotor effects of AD, whereas ODQ blocked the AD-mediated vasoconstriction without affecting the vasorelaxant response. This indicates that only AD-induced vasoconstriction is mediated by a NO-cGMP-dependent mechanism. By using the NO donor SIN-1, we showed a dose-dependent vasoconstrictory effect which was completely blocked by ODQ. These results provide compelling evidence that the vasoactive role of AD in the branchial circulation of S. acanthias and A. anguilla involves a NO signalling.

Adenosine↗

Naphthalene and beta-naphthoflavone effects on Anguilla anguilla L. hepatic metabolism and erythrocytic nuclear abnormalities.

The effects of a polycyclic aromatic hydrocarbon (PAH) such as naphthalene (NAP)--an environmental contaminant--and beta-naphthoflavone (BNF)--a model substance (PAH-like compound)--were investigated in European eel (Anguilla anguilla L.) over 3-, 6-, and 9-day exposure (0.1-2.7 microM). Both xenobiotics revealed to be strong biotransformation (phase I) inducers. After 3-day exposure, liver ethoxyresorufin O-deethylase (EROD) activity was significantly increased by all NAP and BNF tested concentrations. At 6 and 9 days, liver EROD activity was significantly induced mainly by the highest NAP and BNF concentrations. Liver cytochrome P450 content was significantly induced after 3-day exposure to 0.9 and 2.7 microM BNF and 9-day exposure to 0.1, 0.3 and 0.9 microM NAP. Liver alanine transaminase (ALT) activity was measured as an indicator of hepatic health condition, revealing a significant decrease after 6-day exposure to 0.9 microM BNF. Genotoxicity measured as erythrocytic nuclear abnormalities (ENA) was detected in all BNF treated fish on day 6, whereas on day 9, ENA frequencies returned to control levels, significantly decreasing at 0.9 microM BNF exposure. Immature erythrocytes (IE) frequency demonstrated a decreasing tendency along the BNF experiment and concomitantly with the above ENA response. The present experimental results elect EROD activity in A. anguilla as a useful short- to medium-term biomarker of exposure to both PAH and PAH-like compounds. However, some problems can emerge in the presence of high xenobiotic concentrations. Concerning genotoxicity, it is hypothesized that ENA response depends on different factors such as the exhaustion of the detoxification process, the balance erythropoiesis/erythrocytic catabolism and the DNA repairing capacity.

Alanine Transaminase↗

Induction of EROD activity in European eel (Anguilla anguilla) experimentally exposed to benzo[a]pyrene and beta-naphthoflavone.

The induction of liver ethoxyresorufin O-deethylase (EROD) activity was investigated in the European eel, Anguilla anguilla, collected from a Mediterranean brackish environment and experimentally exposed to benzo[a]pyrene (B[a]P) and beta-naphthoflavone (BNF). Eels were injected intraperitoneally at increasing doses (0.1, 1, 10, and 50 mg/kg wet body weight) using corn oil as a carrier and sacrificed after 7 days. The main objectives of the present study are: (1). to assess of the sensitivity of EROD induction as a biomarker to polycyclic aromatic hydrocarbon (PAH) exposure; (2). to determine an EROD dose-response relationship of the contaminants used; and (3). to compare the efficiency of B[a]P and BNF as inducers of EROD activity. Results showed that both chemicals resulted in a dose-dependent EROD induction, but increases were not linear. EROD activity seemed to reach a plateau at the exposure of 10 mg/kg in both treatment groups; B[a]P was a more potent inducer than BNF was at the higher doses (10 and 50 mg/kg), while the opposite result was observed at the lower ones (0.1 and 1 mg/kg). The greatest induction occurred in eels treated with 10 mg/kg B[a]P, in which a 261-fold increase in EROD activity was observed. Results showed that EROD activity in A. anguilla is significantly induced by B[a]P and BNF exposure, responding to a wide range of concentrations of these contaminants. We infer that this tool may be suited as a diagnostic biomarker for biomonitoring PAHs pollution in Mediterranean brackish environments and further field research is suggested.

Anguilla↗

In vivo inhibition of AChE activity in the European eel Anguilla anguilla exposed to technical grade fenitrothion.

European eel (Anguilla anguilla) were exposed to sublethal fenitrothion concentrations in a continuous flow-through system for 4 days. Plasma acetylcholinesterase (AChE) activity was evaluated after 2, 8, 12, 24, 32, 48, 56, 72 and 96 h pesticide exposure. AChE activity in the plasma of the eel decreased as concentration of fenitrothion increased. Pesticide induced significant inhibitory effects on the AChE activity of A. anguilla ranging from 51% inhibition at sublethal concentration of 0.02 ppm to 57% inhibition at sublethal concentration of 0.04 ppm. Eel were exposed to both fenitrothion concentrations for 96 h and then allowed a period of recovery in pesticide-free water. Following 1 week of recovery, the AChE activity for those animals previously exposed to fenitrothion was still different from the controls. Animals transferred to clean water showed plasma AChE activities reduced in a 34 and 51% when previously exposed to 0.02 and 0.04 ppm pesticide, respectively. This is probably due because regeneration of the enzyme is mainly by the novo synthesis after exposure to organophosphates and levels need a large extent to recover.

Acetylcholinesterase↗