Liver Ethoxyresorufin O-deethylase Activity Research Articles

Selection of biochemical and physiological parameters in the croaker Micropogonias furnieri as biomarkers of chemical contamination in estuaries using a generalized additive model (GAM)

Biochemical and physiological parameters in the croaker Micropogonias furnieri were evaluated as biomarkers of chemical contaminants in estuaries. Juvenile croakers (10–20 cm total body length) were collected in summer and winter (2011 and 2012), in two sites at the Lagoa dos Patos estuary (southern Brazil). Fish were also collected in summer (2011 and 2012) in one site at the Barra do Chui estuary (southern Brazil). Tissue (gills, muscle or liver) samples were dissected and analyzed for contaminants [metals, polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs)] and selected biochemical and physiological parameters [metallothionein-like proteins (MTLP), acetylcholinesterase (AChE) activity, ethoxyresorufin-O-deethylase (EROD) activity, and lipid peroxidation (LPO)]. Additionally, water samples were collected for water chemical analyses (salinity, pH, temperature, dissolved oxygen, total alkalinity, and sulfate concentration). Data obtained were integrated and analyzed, using Principal Component Analysis (PCA) and Generalized Additive Model (GAM) approaches. Results showed that changes in concentrations of chemical contaminants and responses of biochemical and physiological parameters did not show any pattern according to the site, season and year of fish collection. However, they were influenced by fish body length and water temperature and salinity. Liver LPO and EROD activity were not responsive to PAHs. However, liver LPO was responsive to HCB, p,p-DDD, p,p-DDT and endosulfan sulfate. In turn, gill MTLP concentration, muscle AChE activity and liver EROD activity were responsive to non-essential metals (Pb, Cd and Ag). Considering that the ecotoxicological modeling approach adopted (GAM) accounted for biological, spatial and temporal variability of data associated with fish body size and site, season and year of fish collection, gill MTLP concentration, muscle AChE activity, as well as liver LPO and EROD activity can be considered as reliable biomarkers of fish exposure to chemical contaminants in estuaries.

Biomarkers in fish as a measure of the state of marine environment of Kuwait.

The health of a marine ecosystem can effectively be monitored by studying the levels of biomarkers in a representative species. A change in background level of a biomarker indicates exposure to a specific type of pollutants. It also identifies bioavailability and the organism response to the causative agent among the compounds present in the surrounding water body. Yellowfin seabream (Acanthopagrus latus), a local variety of fish, was examined for parent PAHs in the liver, its metabolites in bile by the GC-MS method as exposure biomarkers and cytochrome P4501A1 by assay of ethoxyresorufin-o-deethylase (EROD) in the liver as an effect biomarker. A comparison was made between fish collected in 2015 with the fish collected in 2005-2006 and stored at - 80°C in the fish bank. The objective was to examine the extent of changes in the environmental quality of the Kuwait marine area and the status of fish health concerning oil-related pollutants since Arabian Gulf is surrounded by oil-producing countries. Interestingly, insignificant differences between the liver PAH content and EROD activity were observed in fish over the sampling periods. The fish efficiently metabolized PAHs and excreted hydroxy-metabolites in bile. The study suggested that environmental quality of the Kuwait marine area was not deteriorated to any serious extent in the last decade and biomarkers can be used effectively in assessing the thrust of sub-optimal levels of various contaminants present in the marine area on the resident biota.

Effects of water accommodated fractions of crude oils and diesel on a suite of biomarkers in Atlantic cod (Gadus morhua)

The aim of this study was to characterize concentration- and time-dependent responses in juvenile Atlantic cod (Gadus morhua) following exposure for one and three weeks to the water-soluble fraction (WAF) of three weathered oils: Arabian Light crude oil (ALC), North Sea crude oil (NSC) and ship-diesel. The sum of polycyclic aromatic hydrocarbons (PAH) in water was highest after one week of exposure and within environmentally relevant concentrations. PAH metabolites in bile confirmed exposure to and uptake of PAHs. Hepatic cytochrome P450 1A (CYP1A) gene expression (mRNA quantification) increased dramatically following exposure to all three oil types (fold-change up to 165) and there was a time lag between gene and protein expression. Hepatic CYP1A protein concentration and ethoxyresorufin-O-deethylase (EROD) activity were more variable among individuals and treatments than gene expression. EROD activity in liver and gills increased in fish exposed to WAF from the two crude oils, but not in fish exposed to WAF from diesel. Exposure to diesel appeared to induce oxidative stress to a greater extent than exposure to crude oils. Other biomarkers (glutathione S-transferases, acetylcholine esterase, vitellogenin) did not appear to respond to the exposure and hence did not discriminate among oils. Biomarker responses in cod after exposure to weathered crude oils and diesel suggested that the CYP1A system and oxidative stress markers have the highest potential for discriminating among different oil types and to monitor the environmental consequences of spills.

EROD activity in liver and gills of polar cod ( Boreogadus saida) exposed to waterborne and dietary crude oil

Polar cod Boreogadus saida an indicator species for biomonitoring in the Arctic was exposed to crude oil in waterborne and dietary experiments. Ethoxyresorufin O-deethylase (EROD) activity was measured in liver and gills of polar cod at weeks 0, 2 and 4 of exposure and following 2 weeks of depuration. EROD increased significantly and dose-dependently in both tissues through both exposure routes. Levels were very low in gills compared to liver reflecting the tissue-specific metabolism capacities and tissue-specific response kinetics were also observed. Furthermore, a significant increase of gill EROD was shown in dietary exposed fish, demonstrating a substantial transport of PAHs via the systemic circulation. To conclude, this study gave some preliminary information on the EROD response in terms of levels, dose dependency and timing, in gills of PAH exposed polar cod.

Biochemical biomarkers of exposure to deltamethrin in freshwater fish, Ancistrus multispinis

This study aimed to determine the effect of sublethal doses of deltamethrin, using biochemical biomarkers as activities of cholinesterase (ChE), ethoxyresorufin-O-deethylase (EROD) and the Na+K+- ATPase and levels of total cytochrome P450 (CYP450). Fishes received sublethal doses of deltamethrin and were sacrificed after 96 h of exposure. Samples of gills, heart, brain, liver and muscle were collected for enzymatic analyses. Deltamethrin inhibited the activity of the gills and heart Na+ K+-ATPase, induced the liver total CYP450, as well as the liver EROD activity. The activity of the ChE was not inhibited by deltamethrin. Deltamethrin altered the hepatic metabolism and the normal ionic flux in Ancistrus multispinis.

The Dose–Response Relationships for EROD and GST Induced by Polyaromatic Hydrocarbons in Carassius auratus

Freshwater fish Carassius auratus were chosen as experimental animals, the hepatic biochemical responses to medium-term exposure of five PAHs were measured as ethoxyresorufin O-deethylase (EROD) activity (phase I) and glutathione S-transferase (GST) activity (phase II) to assess sub-lethal effects. The fold increases of EROD and GST activity were calculated and both increased in the order Fluoranthene < Fluorene < Benzo(b)fluoranthene < Benzo(g,h,i)perylene < Indeno(1,2,3-cd)-pyrene. The clear dose-response relationships were found for liver EROD and GST activity induced by PAHs. The enzyme EROD and GST in Carassius auratus were confirmed as useful biomarkers of exposure to both PAH and PAH-like compounds.

Biochemical Markers for Assessing Aquatic Contamination.

Biochemical markers, specifically enzymes of the first phase of xenobiotic transformation - cytochrome P450 and ethoxyresorufin-O-deethylase (EROD) - were used to determine the quantities of persistent organic pollutants (POPs) in fish muscle (PCB, HCB, HCH, OCS, DDT). Eight rivers were monitored (Orlice, Chrudimka, Cidlina, Jizera, Vltava, Ohře and Bílina; and the River Blanice was used as a control). The indicator species selected was the chub (Leuciscus cephalus L.). There were no significant differences in cytochrome P450 content between the locations monitored. The highest concentration of cytochrome P450 in fish liver was in the Vltava (0.241 nmol mg-1 protein), and the lowest was in the Orlice (0.120 nmol mg-1 protein). Analysis of EROD activity showed a significant difference between the Blanice and the Vltava (P< 0.05), and also between the Orlice and the Vltava (P< 0.01), the Orlice and the Bílina (P< 0.01), and the Orlice and the Ohře (P< 0.05). The highest EROD activity in fish liver was in the Vltava (576.4 pmol min-1 mg-1 protein), and the lowest was in the Orlice (63.05 pmol min-1 mg-1 protein). In individual locations, results of chemical monitoring and values of biochemical markers were compared. A significant correlation (P< 0.05) was found between biochemical markers and OCS, and PCB. Among the tributaries studied those that contaminated the Elbe most were the Vltava and the Bílina. These tributaries should not be considered the main sources of industrial contamination of the River Elbe, because the most important contamination sources were along the river Elbe itself.

Using Semipermeable Membrane Devices, Bioassays, and Chemical Analysis for Evaluation of Bioavailable Polycyclic Aromatic Hydrocarbons in Water

Meiliang Bay is a sublake of Taihu Lake and has been polluted by domestic and industrial effluents. As part of a comprehensive risk assessment project in this region, semipermeable membrane devices (SPMDs) were applied to evaluate the levels and potential toxic potency of polycyclic aromatic hydrocarbons (PAHs) in lakewater, in combination with chemical analysis and in vitro bioassay using H4IIE rat hepatoma cells. In addition, induction of hepatic ethoxyresorufin-O-deethylase (EROD) activity, in crucian carp (Carassius auratus), caged in the vicinity of SPMD sampling sites was also used as a biomarker of exposure to PAHs and related chemicals. The caged crucian carp accumulated similar PAH profile patterns (p < 0.001) but at lower levels compared with SPMDs on a wet-weight basis. Total concentrations of PAHs in crucian carp muscle tissues ranged from 35.6 to 69.1 ng/g after the 32-day exposure, whereas total PAHs in SPMDs ranged from 716.9 to 1007.8 ng/g. Dialysates from SPMDs exposed to Meiliang Bay water caused marked EROD activity in H4IIE cells. Toxic potency of dialysates expressed as bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQ) ranged from 3.8 to 6.2 pg TCDD-EQ/g SPMD for 32-day exposure samples. A linear correlation of total amount of PAHs and in vitro TCDD-EQ of SPMD dialysates yielded an R (2) of 0.82. Empirical evidence suggests that aryl hydrocarbon receptor-active PAHs can account for about 42-56% of the potency observed. Significant induction of liver EROD activity was also observed in crucian carp caged alongside SPMDs and there was a good correlation between the results of EROD assays in vivo and in vitro (R (2) = 0.62, p = 0.02). It is, therefore, suggested that the SPMD technique in connection with chemical analysis and specific in vitro bioassays might be a valuable tool to assess the levels and effects of bioavailable hydrophobic pollutants in water.

Cytochrome P4501A, genotoxic and stress responses in golden grey mullet ( Liza aurata) following short-term exposure to phenanthrene

This study represents a first approach to short-term effects of phenanthrene (Phe) in fish. The teleost Liza aurata was exposed to 0.1–2.7 μM Phe during 16 h. CYP1A induction was assessed as liver ethoxyresorufin- O-deethylase (EROD) activity. Genotoxicity was evaluated in gill and liver as DNA integrity (by alkaline unwinding), whereas in blood the erythrocytic nuclear abnormalities (ENA) frequency was determined. Stress responses were determined as cortisol, glucose and lactate plasma levels. Liver EROD activity was significantly increased by Phe 0.3–2.7 μM. Phe genotoxicity in gill was not found, whereas liver DNA integrity significantly decreased after exposure to Phe 0.1 and 0.9 μM demonstrating its genotoxicity which did not correlate with liver CYP1A induction. Phe genotoxicity in blood was demonstrated by a significant ENA increase from 0.1 up to 2.7 μM. In terms of stress responses, plasma cortisol was significantly increased by Phe 0.3–2.7 μM, though plasma glucose was only significantly increased by Phe 0.9 and 2.7 μM. The Phe observed effects on L. aurata detected at different levels demonstrate a physiological unbalance and a probable ecological risk to ichthyofauna.

Oxidative stress, liver biotransformation and genotoxic effects induced by copper in Anguilla anguilla L. – the influence of pre-exposure to β-naphthoflavone

Fish are exposed in the aquatic ecosystems to different classes of pollutants. Polycyclic aromatic hydrocarbons (PAHs) and heavy metals represent two important classes of aquatic contaminants. Thus, one lot of European eels ( Anguilla anguilla L.) was pre-exposed during 24 h to 2.7 μM β-naphthoflavone (BNF; a PAH-like compound), and subsequently exposed during 24 h to 0, 1 and 2.5 μM copper (Cu). Additionally, another lot not pre-exposed to BNF was exposed to the same Cu concentrations. BNF pre-exposure promoted a significant increase in liver ethoxyresorufin O-deethylase (EROD) activity, but did not change the other responses investigated in eels. On the other hand, both Cu concentrations did not modify the liver EROD activity either in eels pre-exposed to BNF or not. Liver total cytochrome P450 was increased in eels exposed to Cu 2.5 μM, being significantly only in eels not pre-exposed to BNF. Free sulfhydryl group content was decreased by 1 and 2.5 μM in eels pre-exposed to BNF or not pre-exposed, being significant at 2.5 μM Cu in eels not pre-exposed compared to its control. Liver total glutathione (TG), reduced glutathione (GSH) and GSH/oxidized glutathione (GSSG) levels were slightly decreased by 1 and 2.5 μM Cu in eels pre-exposed to BNF, whereas a slight tendency to increase was observed in eels not pre-exposed. Thus, liver TG and GSH significantly decreased in 2.5 μM Cu BNF pre-exposed eels compared to eels not pre-exposed to BNF. Liver glutathione reductase and catalase activities were significantly inhibited by 1 and 2.5 μM Cu in eels pre-exposed to BNF, concomitantly with a slight liver glutathione peroxidase tendency to decrease. Lipid peroxidation was significantly increased by 1 μM Cu in eels either pre-exposed or not pre-exposed to BNF. Liver H 2O 2 was significantly increased by 1 μM Cu in eels pre-exposed to BNF. Liver DNA integrity was significantly decreased by 1 and 2.5 μM Cu in eels pre-exposed to BNF. The oxidative stress and genotoxic effects induced by Cu in eels pre-exposed to BNF revealed that the metal effects are potentiated by previous exposure to BNF.

Ethoxyresorufin- O-deethylase (EROD) activity and bile metabolites as contamination indicators in Baltic Sea perch: Determination by HPLC

Exposure of Baltic Sea perch ( Perca fluviatilis) to organic contaminants was investigated by means of liver enzyme activity, measured as ethoxyresorufin- O-deethylase (EROD) activity, and PAH metabolites excreted to bile. Female perch used in the study were caught in coastal waters of the western Gulf of Finland. Reaction conditions were optimised for determination of EROD activity in liver. A high-performance liquid chromatography (HPLC) method with fluorescence detection was developed to separate the interfering fluorescent signal from the signal due to EROD activity. EROD activity in perch varied within a concentration range of 0.30–14 pmol min −1 mg −1 protein. Recent PAH exposure was indicated in enhanced levels of the bile metabolite 1-hydroxypyrene, which varied between 213 and 1149 μg kg −1. No correlation was indicated between hepatic EROD activity and concentration of 1-hydroxypyrene.

Biotransformation, stress and genotoxic effects of 17β-estradiol in juvenile sea bass (Dicentrarchus labrax L.)

The effects of 17β-estradiol (E2) on fish became a matter of concern, since significant levels of this hormone were detected in the aquatic environment released mainly by domestic sewage treatment plants. In this perspective, the current study was focused on E2 effects upon biotransformation, stress and genotoxic responses of juvenile Dicentrarchus labrax L. (sea bass). Fish were exposed to E2 during 10 days in two different ways: water diluted (200 ng/L or 2000 ng/L) and i.p. injected (0.5 mg/kg or 5 mg/kg). A battery of biological responses was evaluated: liver ethoxyresorufin-O-deethylase (EROD) and alanine transaminase (ALT) activities, liver somatic index (LSI), plasma cortisol, glucose and lactate concentrations, as well as erythrocytic nuclear abnormalities (ENA).All the exposure conditions induced endocrine disruption, measured as plasma cortisol decrease, and genotoxicity, measured as ENA increase. Thus, no differences were detected either between different exposure routes or tested concentrations. Concerning liver EROD and ALT activities, as well as plasma glucose and lactate concentrations no differences were found between treated and control groups. LSI was the only parameter to respond differently in the two exposure routes, as only E2 water diluted induced a significant increase in this hepatic indicator.

Sparus aurata L. liver EROD and GST activities, plasma cortisol, lactate, glucose and erythrocytic nuclear anomalies following short-term exposure either to 17β-estradiol (E 2) or E 2 combined with 4-nonylphenol

Immature Sparus aurata L. (gilthead seabream) were exposed to 17β-estradiol (E 2) 4000 ng/l and to the same E 2 concentration mixed with 50,000 ng/l 4-nonylphenol (E 2+NP) during 4, 8, 12 and 16 h. E 2 availability and E 2 plasma level variations were assessed. Liver biotransformation capacity was measured as ethoxyresorufin- O-deethylase (EROD) and glutathione- S-transferase (GST) activities. Plasma cortisol, lactate and glucose were also determined. Genotoxicity was assessed through erythrocytic nuclear anomalies (ENA) frequency. Liver EROD activity significantly decreased during the whole experiment for both treatments, with the exception of 16 h exposure to E 2. Liver GST activity was significantly increased after 8 and 12 h of exposure either to E 2 or E 2+NP. An endocrine disruption expressed as plasma cortisol decrease was observed after 16 h exposure under both tested conditions, concomitantly with a plasma lactate increase. No genotoxic responses, measured as ENA frequency, were detected. Analyzing the E 2 water concentration in aquaria without fish it was demonstrated an intense and fast E 2 loss, considerably reducing its availability to fish. In the presence of fish, E 2 water levels were drastically reduced after 4 h exposure, being this reduction more pronounced in E 2 aquarium when compared to E 2+NP aquarium. In addition, it was demonstrated a rapid E 2 uptake from the water since the highest E 2 plasma concentrations were observed after 4 h exposure, followed by a continuous decrease, which became more pronounced between 8 and 12 h of exposure. Furthermore, during the first 8 h exposure to E 2 and E 2+NP, seabream plasma E 2 concentrations were higher than the initial water exposure concentration. Comparing the E 2 plasma levels in both seabream-exposed groups, it was clear that its concentration is always higher in E 2+NP-treated fish. Despite the previous results, no significant differences were found in the measured responses between E 2 and E 2+NP.

Juvenile sea bass biotransformation, genotoxic and endocrine responses to β-naphthoflavone, 4-nonylphenol and 17β-estradiol individual and combined exposures

Juvenile sea bass, Dicentrarchus labrax L., were exposed during 2, 4, 8, and 24 h to 0.9 μM β-naphthoflavone (BNF), 131 nM 17β-estradiol (E 2), 4.05 μM 4-nonylphenol (NP), as well as to BNF combined either to E 2 or NP (maintaining the previous concentrations). Liver cytochrome P450 content (P450), ethoxyresorufin- O-deethylase (EROD), and glutathione S-transferase (GST) activities were measured in order to evaluate biotransformation responses. Genotoxicity was assessed as erythrocytic nuclear abnormalities (ENA) frequency. The effects on endocrine function were evaluated as plasma cortisol and glucose. Cortisol was not affected by xeno/estrogens tested, either in single exposure or mixed with BNF. Nevertheless, the intermediary metabolism was affected since glucose concentration increased after 4 h exposure to E 2, and after all BNF + NP exposure lengths. Moreover, a synergism between BNF and NP was thoroughly demonstrated, whereas a sporadic antagonistic interaction was found at 4 h BNF + E 2 exposure. Liver EROD and GST activities were not significantly altered by single E 2 or NP exposure. However, both compounds were able to induce EROD activity in the presence of BNF. NP single exposure was able to significantly increase liver P450 content, while its mixture with BNF displayed an antagonistic interference. Considering the xeno/estrogens single exposures, only NP induced an ENA increase; however, both mixtures (BNF + E 2 and BNF + NP) displayed genotoxic effects. Fish responses to mixtures of xenobiotics are complex and the type of interaction (synergism/potentiation or antagonism) in a particular mixture can vary with the evaluated biological response.

Glutathione protects heavy metal-induced inhibition of hepatic microsomal ethoxyresorufin O-deethylase activity in Dicentrarchus labrax L.

The in vitro effects of chromium (Cr(VI)), copper (Cu 2+), iron (Fe 2+), mercury (Hg 2+), and zinc (Zn 2+) were assessed on liver microsomal ethoxyresorufin O-deethylase (EROD) activity from a sea bass ( Dicentrarchus labrax L.) preexposed under laboratory conditions to 2.7 μM β-naphthoflavone. The reduced glutathione (GSH) protection potential against heavy metal effects was also studied. The heavy metal concentration ranges used for this study were as follows: 10 pM–5 mM Cr(VI), 10 pM–100 μM Cu 2+, 10 pM–1 mM Fe 2+, 10 pM–10 μM Hg 2+, and 10 pM–100 μM Zn 2+. Liver microsomal EROD activity was significantly inhibited after in vitro exposure to Cr(VI) (500 μM), Cu 2+ (1 μM), Fe 2+ (100 μM), Hg 2+ (100 pM), and Zn 2+ (10 μM). Heavy metals inhibitory effect on liver EROD activity was ordered as follows: Hg 2+>Cu 2+>Zn 2+>Fe 2+>Cr(VI). Protective effects against Hg 2+ (1 and 10 μM), Cu 2+ (1, 10, and 100 μM), and Zn 2+ (10, 50, and 100 μM) were observed in the presence of 0.5 mM GSH by a decrease in liver microsomal EROD activity inhibition. However, 0.5 mM GSH did not protect liver microsomal EROD activity from Cr(VI), and Fe 2+-induced inhibition. The effect of metal mixtures (Cu 2++Zn 2+, Zn 2++Fe 2+, Zn 2++Cr(VI), and Cr(VI)+Fe 2+) (100 μM) on liver microsomal EROD activity was also assessed, revealing a synergistic interaction.

Anguilla anguilla L. genotoxic and liver biotransformation responses to abietic acid exposure

Adult eels ( Anguilla anguilla L.) were exposed for 8, 16, 24, and 72 h to 0, 0.1, 0.3, 0.9, and 2.7 μM abietic acid (AA). Genotoxicity was measured as erythrocytic nuclear abnormalities (ENA), as well as DNA strand breaks in blood and liver. Liver cytochrome P450 (P450) content, liver ethoxyresorufin O-deethylase (EROD), and glutathione S-transferase (GST) activities were determined as biotransformation biomarkers. Liver alanine transaminase (ALT) activity was also measured as an indication of tissue damage. Low AA concentrations, such as 0.1 and 0.3 μM, result in a delayed induction of A. anguilla L. liver EROD activity, whereas the higher AA concentration (2.7 μM AA) also has a delayed effect probably as a consequence of liver tissue high inhibitory concentration. The current eel liver GST activity results demonstrate that only low AA concentrations promote liver increases in GST, whereas high AA concentrations, such as 0.9 and 2.7 μM, do not alter it. The results concerning eel liver ALT activity indicate that significant liver damage is induced by high AA concentrations, such as 2.7 and 0.9 μM. The eel ENA result analysis reveals that AA is a weak ENA inducer in A. anguilla L. Blood DNA integrity results suggest that low AA concentrations promote late decreases in blood DNA integrity; nevertheless, high AA concentrations are early blood genotoxic inducers compared with low AA doses. According to the present research results with respect to eel liver DNA damage, all of the AA exposure concentrations decreased liver DNA integrity.

Enzymatic and nonenzymatic antioxidants as an adaptation to phagocyte-induced damage in Anguilla anguilla L. following in situ harbor water exposure

Anguilla anguilla L. were caged for 8 and 48 h in harbor water of Aveiro Lagoon, Portugal. Respiratory burst activity (RBA) of peritoneal, head kidney, and gill phagocytes was measured. Lipid peroxidation (LPO) was estimated in gill, kidney, and liver. Liver ethoxyresorufin- O-deethylase (EROD) activity, cytochrome P450 (Cyt P450) content, and bile metabolites were assayed. Various antioxidant enzymes, viz., glutathione peroxidase, catalase, and glutathione S-transferase and nonenzymatic antioxidant, viz., total reduced glutathione were also studied. Harbor water xenobiotics induced a significant RBA increase in gill after 8 h; whereas in peritoneum and head kidney it increased after 48 h exposure. These responses were adversely associated with tissue-specific peroxidative damage since significant LPO increase was observed in gill (8 and 48 h), kidney (48 h), and liver (48 h). The tissue most affected was gill. Moreover, liver EROD activity, Cyt P450 content and bile metabolites remain unaltered after 8 h; in contrast, 48 h exposure showed significant EROD activity decrease and pyrene-type bile metabolites increase. Decreased EROD activity may be associated with concomitant liver damage, as increased LPO was observed after 48 h. Furthermore, the tissue-specific damage corresponded to the differences in the antioxidant potentials of the tissues, since the initial exposure period caused a significant increase in liver antioxidant activities, whereas gill and kidney showed a significant decrease, demonstrating that liver is highly adaptive to oxidative damage. However, at 48 h exposure gill, kidney, and liver showed a suppressive antioxidant effect, probably due to PAHs, since a significant induction at PAH-type bile metabolites has been seen. Our results demonstrate that phagocyte activation and associated peroxidative damage are concomitantly corroborated with enzymatic and nonenzymatic antioxidant activity differences. In addition, hepatic antioxidant induction after short-term exposure may serve as a potent biomarker for water pollutants in fish.

Dicentrarchus labrax biotransformation and genotoxicity responses after exposure to a secondary treated industrial/urban effluent

The present research work was designed to study Dicentrarchus labrax (sea bass) biotransformation and genotoxicity responses to the soluble fraction of a secondary treated industrial/urban effluent (SF-STIUE) discharged through a submarine pipe outlet into the Aveiro coastal area. Sea bass was exposed for 4, 8, 16, 24, 48, and 96 h to 0%, 0.1%, and 1% SF-STIUE and the following biological responses were measured: (1) liver cytochrome P450 (P450) content and ethoxyresorufin- O-deethylase (EROD) activity, as phase I biotransformation parameters; (2) liver gluthathione S-transferase (GST) activity as a phase II conjugation enzyme; (3) biliary and liver cytosol naphthalene (Naph)- and benzo( a)pyrene (B( a)P)-type metabolites, by fixed wavelength fluorescence detection (FF); (4) liver DNA strand breaks, erythrocytic micronuclei (EMN), and erythrocytic nuclear abnormalities (ENA) as genotoxicity parameters. Both SF-STIUE dilutions (0.1% and 1%) failed to significantly increase liver EROD activity, despite a significant increase of liver P450 at 16 and 48 h exposure to 0.1%. Liver GST activity increased significantly at 4 h of sea bass exposure to 1% SF-STIUE, being inhibited at 96 h of exposure to this SF-STIUE dilution. Naph- and B( a)P-type metabolite contents were not significantly increased in bile. However, Naph-type metabolite contents increased significantly in liver cytosol at 4 h exposure to 1% SF-STIUE, and at 24 h exposure to 0.1% and 1% SF-STIUE. Furthermore, B( a)P-type metabolites increased significantly in liver cytosol at 4 h exposure to 1% SF-STIUE, and 16 h exposure to 0.1% and 1% SF-STIUE. EMN and ENA frequencies increased significantly at 4, 8, 16, 24, 48, and 96 h exposure to 0.1% and 1% SF-STIUE. Liver DNA integrity decreased significantly at 96 h of sea bass exposure to 1% SF-STIUE. The STIUE discharged into Aveiro coastal area is of great ecotoxicological concern due to its genotoxic potential.

Genotoxicity biomarkers’ association with B( a)P biotransformation in Dicentrarchus labrax L.

Dicentrarchus labrax (sea bass) were exposed during 0, 4, 8, 16, 24, 48, and 96 h to 0 and 0.1 μM benzo( a)pyrene (B( a)P), an environmental pollutant, and the following biomarkers were measured: (1) liver cytochrome P450 (P450) content and ethoxyresorufin- O-deethylase (EROD) activity as phase I biotransformation parameters, (2) liver gluthathione- S-transferase (GST) activity as a phase II biotransformation conjugation enzyme, (3) biliary and liver cytosolic B( a)P-type metabolites by fixed wavelength fluorescence detection (FF), and (4) erythrocytic micronuclei (EMN) and erythrocytic nuclear abnormalities (ENA) as genotoxicity biomarkers. Liver EROD activity (4 h), P450 content (24 h), GST activity (4, 8, and 96 h), bile (4–96 h), and liver cytosolic (4–24 h) B( a)P-type metabolites increased significantly in sea bass exposed to B( a)P as well as EMN (8–96 h) and ENA (4–96 h) frequencies. B( a)P genotoxicity is associated with increase in B( a)P-type metabolites in liver cytosol due to an impaired phase II conjugation. This increase seems to be responsible for the decrease in liver EROD and GST activities.

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% ( E 25 ) and 50% ( E 50 )]; 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 E 25 , as well as at 16, 24, and 72 h exposure to E 50 . S exposure induced liver EROD activity only at 24 h. 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 24 h exposure to E 25 . 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 E 50 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 E 25 , as well as at 8, 16, and 24 h exposure to E 50 . 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 24 h exposure to E 25 and E 50 , and 72 h to E 50 . A. anguilla L. increased ENA frequency was detected in T at 16, 24, and 72 h, whereas in E 25 and S it was observed at 8, 16, and 24 h. Furthermore, E 50 ENA frequency increased at 24 h exposure.