Age-dependent in situ hepatic and gill CYP1A activity in the see-through medaka (Oryzias latipes)

We used a recently introduced strain of medaka, the see-through medaka, whose internal organs can be seen through the skin, to develop an in situ toxicity assay of ethoxyresorufin-O-deethylase (EROD) activity that detected fluorescence from resorufin, a metabolite of ethoxyresorufin and thus an indicator of CYP1A activity. EROD activity in the liver and gills of 2-week post-hatch see-through medaka exposed simultaneously to various concentrations of 3-methylcholanthrene and 200 microg/L ethoxyresorufin for 24 h was proportional to the 3-methylcholanthrene dose. Activities in the liver and gills peaked at 40 microg/L of 3-methylcholanthrene and then decreased at higher doses, possibly because of 3-methylcholanthrene toxicity. At 1-week post-hatch stage, however, constant high EROD activity was observed in controls and at all 3-methylcholanthrene doses. Four-week post-hatch see-through medaka exhibited less EROD activity than 2-week post-hatch see-through medaka, and activity in the liver peaked at 100 microg/L of 3-methylcholanthrene. Adult see-through medaka were not suitable for fluorescence detection owing to their thick skin, muscle and/or tissue. In tests of oxidative activity response to ethoxyresorufin, 1-day and 1-week post-hatch see-through medaka exhibited high intrinsic EROD activity in the liver, gills, and other organs in the absence of 3-methylcholanthrene. This intrinsic activity declined with growth and explained the high constant EROD activity at 1-week post-hatch stage.     

Determination of cytochrome P450 1A activities in mammalian liver microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) in liver microsomes from human, monkey, rat and mouse by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than previous methods using a spectrofluorimeter and fluorescence plate reader. The detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol/assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver microsomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in liver microsomes from all animal species exhibited a monophasic kinetic pattern, whereas the pattern of EROD activities in rat and mouse liver microsomes was biphasic. In addition, the method could determine the non-inducible and 3-methylcholanthrene-inducible activities of EROD and MROD in rat and mouse liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with cytochrome CYP1A isoforms in mammals.     

A new technique for assaying cytochrome P450 enzyme activity in a single cell

A new microspectrofluorometric technique for measuring the ethoxyresorufin-O-deethylase (EROD) activity of cytochrome P450 (CYP)1A1 in single living cells is described. The system, which uses a perfusion chamber and an HPLC pump, allowed cells to be stained, fixed, blocked, and washed by injecting each treatment solution into the on-line carrier stream of buffer from the sampling block of the HPLC pump. After addition of the substrate 7-ethoxyresorufin, the fluorescence intensity of the metabolite resorufin was measured in individual cells. Fluorescence intensity steeply increased to a unique peak for each cell and then decreased to the basal level. Furthermore, CYP1A1 in each cell was stained with its antibody and quantified using the fluorescence intensity of an FITC-conjugated secondary antibody. EROD activity was normalized using the FITC fluorescence. The results show that the initial slopes and peak values of resorufin production by the cells were dependent on the CYP1A1 level. Treatment of hepatocytes with two nonspecific P450 inhibitors, cimetidine and SKF-525A, suppressed EROD activity.     

Characterization of CYP1A enzyme in Adélie penguin liver

As CYP1A enzymes are induced by certain contaminants, their induction pattern has been used as a biomarker for exposure of certain pollutants. Ethoxyresorufin O-deethylase (EROD) activities are widely used in environmental assessments of polychlorinated biphenyls in many wildlife species. The EROD activity, a typical probe for CYP1A enzyme was studied in liver microsomes prepared from Adélie penguins (Pygoscelis adeliae) (n=10). Penguin liver microsomes (0.5 mg/mL) were incubated with the substrate ethoxyresorufin and NADPH at 37 degrees C for 10 min, and the reaction was terminated by addition of methanol. The formation of the metabolite resorufin was assayed by an HPLC method. EROD activity was present in all liver samples studied. Penguin liver microsomal fraction exhibits typical Michaelis-Menten kinetics in the O-deethylation of ethoxyresorufin. The data were best described by a biphasic kinetic model, which could be interpreted in terms of two populations of CYP enzyme. Mean (+/-S.D.) K(m) values for high- and low-affinity components of EROD were 51+/-109 (range: 0.16 to 358) and 872+/-703 (range: 303 to 2450) nM, respectively. The corresponding mean V(max) values for the high- and low-affinity enzyme activities were 1.8+/-1.4 (range: 0.21 to 5.1) and 9.6+/-3.7 (range: 6.0 to 18.3) pmol/min/mg. The EROD activity in penguin liver microsomes was inhibited by CYP1A inhibitors (phenacetin, 7-ethoxycoumarin and proportional variant-naphthoflavone), whereas other CYP inhibitors for CYP2C9 (tolbutamide), 2C19 (mephenytoin), 2D6 (debrisoquin) and 2E1 (diethyldithiocarbamate) had no effect. These results suggest that CYP1A-like enzymes are present in penguin livers. The activity of this enzyme may be a useful biomarker for assessing the environmental impact of pollutants on Antarctic wildlife.     

Synthesis,antifungal and antioxidant screening of some novel benzimidazole derivatives

Some novel benzimidazole derivatives were synthesized and their in vitro effects on rat liver microsomal NADPH-dependent lipid peroxidation (LP) level, ethoxyresorufin O-deethylase (EROD) and antifungal activities were determined. A significant decrease in male rat liver microsomal LP level was noted by compounds 4c (52%), 4e (58%) and 4h (43%) at 10^{? 3} M concentration. Compounds 4c (100.0%), 4h (100.0%), 5c (98.0%) and 5h (100.0%) inhibited the microsomal ethoxyresorufin O-deethylase (EROD) enzyme activity better than that of the specific inhibitor caffeine (85%). Among these compounds, only compounds 4b and 4h exhibited moderate activity against C.albicans whereas the others had weak effects.     

Noncompetitive mixed-type inhibition of rainbow trout CYP1A catalytic activity by clotrimazole

Over the past two decades a number of antifungal imidazole derivatives have been approved for use in agricultural. The purpose of this study was to characterize the interaction of a model antifungal imidazole compound with a cytochrome P450 isozyme in a species of fish. Clotrimazole inhibited rainbow trout (Oncorhyncus mykiss) hepatic CYP1A-catalyzed ethoxyresorufin O-deethylase (EROD) activity in vivo and in vitro. Although clotrimazole inhibited EROD activity in vivo, it did not effect CYP1A mRNA levels. Addition of clotrimazole to microsomes produced a type II binding spectrum and clotrimazole was determined to be a noncompetitive mixed-type inhibitor of EROD activity with an IC₅₀ of 190 nM. Since antifungal imidazole compounds may be co-applied with other pesticides, inhibition of cytochrome P450 activity by antifungal imidazole compounds may lead to unexpected toxicological interactions.     

O-Dealkylation of coumarin and resorufin ethers by unicellular green algae: kinetic properties of Chlorella fusca and Chlorella sorokiniana

O-Dealkylations of resorufin and coumarin ethers, mediated by microsomal cytochrome P450 mono-oxygenases from animals, plants and microorganisms, are shown here to be performed also by intact cells of the unicellular green algaeChlorella fusca andChlorella sorokiniana. The activity of theO-dealkylation of these ethers was up to tenfold higher withChlorella sorokiniana. Both algae dealkylated methyl-, ethyl-, and pentylethers of resorufin and coumarin. Dealkylation in vivo indicated efficient absorption of methoxy- and ethoxyresorufin, confirmed by the respective absorption kinetics. Piperonylbutoxide and 1-aminobenzotriazole, known inhibitors of plant and mammalian cytochrome P450s, significantly inhibited theO-dealkylase activity of both algal strains. The use of synchronized cultures of both algae revealed that efficiency ofO-dealkylation depends on the stage of the cell cycle: during the growth phase, theO-dealkylase activities increased more than proportional, and the distinct drop in activity during the last hours of the light period indicated the appearance of an endogenous substrate.     

Stock‐specific variation of trophic position,diet and environmental stress markers in Atlantic salmon Salmo salar during feeding migrations in the Baltic Sea

This study investigated stock‐specific variation in selected ecophysiological variables during the feeding migrations of Atlantic salmon Salmo salar in the Baltic Sea. Oxidative stress biomarkers and EROD (ethoxyresorufin‐O‐deethylase, Cyp1A enzyme) activity were used as indicators of possible environmental stress and stable isotopes as determinants of diet and trophic position. Latvian S. salar stocks Daugava and Gauja had distinct stable‐isotope signatures compared to the other stocks, indicating differences in migration patterns, residency or arrival times, or dietary specialization among stocks. Salmo salar originating from Daugava and Gauja also had lower catalase enzyme activity than the other stocks. Post‐smolts originating from rivers of the Gulf of Finland had elevated EROD activities compared to fish of the same age from Bothnian Bay rivers, which could indicate exposure to organochlorine pollutants. No other stock‐specific differences in oxidative stress biomarkers were found. The study demonstrates how genetic, oxidative stress biomarker, EROD and stable‐isotope data may be combined to study trophic position, prey prevalence and environmental stress of mixed S. salar stocks foraging in the sea.     

Synthesis, antifungal and antioxidant screening of some novel benzimidazole derivatives

Some novel benzimidazole derivatives were synthesized and their in vitro effects on rat liver microsomal NADPH-dependent lipid peroxidation (LP) level, ethoxyresorufin O-deethylase (EROD) and antifungal activities were determined. A significant decrease in male rat liver microsomal LP level was noted by compounds 4c (52%), 4e (58%) and 4h (43%) at 10(-3) M concentration. Compounds 4c (100.0%), 4h (100.0%), 5c (98.0%) and 5h (100.0%) inhibited the microsomal ethoxyresorufin O-deethylase (EROD) enzyme activity better than that of the specific inhibitor caffeine (85%). Among these compounds, only compounds 4b and 4h exhibited moderate activity against C. albicans whereas the others had weak effects.     

Evaluation of chromosomal damage by flow cytometry in turbot (Scophthalmus maximus L.) exposed to fuel oil

Flatfishes, turbots (Scophthalmus maximus), were injected intraperitoneally with two doses of fuel oil number 2. Biliary metabolites were evaluated by fixed fluorescence to verify the efficiency of intoxication. Ethoxyresorufin-O-deethylase (EROD) activity was compared with chromosomal damage measured by flow cytometry. The analysis of biliary metabolites showed a good dose–response relation and constitutes a clear reference for the subsequent measurements. Comparing flow cytometry and EROD results, a shorter delay of response for EROD activity was obtained, but chromosomal damage was significant only after 1 week. The persistence of the EROD response was shorter, while the genotoxic signal still persisted after 1 month. The measurement of chromosomal damage allowed a good differentiation between the two tested doses. In the case of EROD activity, the results were less clear. The results suggest that within a few weeks after exposure to fuel oil number 2, the measurements of chromosomal damage by flow cytometry can be used to detect a dose-dependant genotoxic response in fish.     

Fast determination of low-level cytochrome P-450 1A1 activity by high-performance liquid chromatography with fluorescence or visible absorbance detection

A method to determine the activity of the cytochrome P-450 1A1 enzyme, by measuring 7-ethoxyresorufin-O-deethylase (EROD) activity using high-performance liquid chromatography (HPLC) with fluorescence or with visible absorbance detection of resorufin, is described. The lowest quantifiable activity (0.2 pmol/mg min) is obtained by incubation of 0.3 mg of human duodenal microsomal proteins using HPLC fluorescence detection. Using HPLC with visible absorbance detection, sensitivity was ten times lower. However, the equipment for this last method is available in most laboratories. The use of both HPLC assays allows determination of the low EROD activity level in samples of small size, such as two or three human duodenal biopsies obtained by routine endoscopy. These methods will be a useful tool to study the role of drug intestinal metabolism by cytochrome P-450 1A1.     

Cytochrome P450 1A-dependent enzyme activities in the liver of dab (Limanda limanda): kinetics, seasonal changes and detection limits

Concentrations of total cytochrome P450 and cytochrome P450 1A (CYP 1A) and activities of ethoxycoumarin O-deethylase (ECOD), ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase (PROD) were measured in the liver of prespawning, spawning and postspawning dab (Limanda limanda) from the German Bight. Between all P450-dependent parameters measured significant correlations were found. Generally, during prespawning and spawning season higher values were measured in the liver of males compared to females, but the ratio between sexes changed during spawning time, when concentrations and activities in the liver of males decreased and increased in the liver of females. The activity and the signal-to-noise ratio decrease in the order EROD, ECOD and PROD. This decrease is accompanied by an increase in K_m. The findings indicate that the different activities can be attributed to the strongly overlapping substrate specificity and the different enzyme affinities of one enzyme, CYP 1A, towards the three substrates. A biphasic kinetic of ECOD indicates that in addition to CYP 1A a second isozyme catalyses the O-deethylation of ethoxycoumarin in the liver of dab. Interestingly, the ratio between EROD activity and CYP 1A concentration varied seasonally but did not differ significantly between sexes.     

Induction of liver cytochrome P-450 (CYP) 3A in male and female rats by a steroidal androgen receptor antagonist,Zanoterone

The cytochrome P-450 (CYP) mediated hydroxylation of testosterone to 6β-, 7α-, and 16α-hydroxytestosterone (6β-, 7α-, and 16α-OHT) and the de-alkylation of ethoxycoumarin to 7-hydroxycoumarin (ECOD) and ethoxyresorufin to resorufin (EROD) were used to probe changes in CYP monooxygenase activities in liver microsomes form rats treated with the androgen receptor antagonist, zanoterone (Z). Phenobarbital (PB) and β-naphthoflavone (β-NF) were used as comparators. There were sex-related differences in the constitutive CYP activities and in the responses of CYP activities to Z. The greatest effect of Z administration was on 6β-OHT activity: It was increased up to 5.2-fold in males and 13.9-fold in females (Z high dose). The effect was larger than that produced by PB or β-NF (⊆threefold increases). Z (high dose), PB, and β-NF increased ECOD to a similar extent, e.g., about 1.3-fold in males and 1.2–2.9-fold in females. β-NF increased EROD (11.2-fold males, 6.2-fold females) more than PB (3.4- to 4.6-fold) or Z (1.3- to 1.7-fold). Since hydroxylation of testosterone at the 6β position in rats and humans is catalyzed primarily by CYP isoforms from the 3A subfamily, the increase in 6β-OHT suggests that Z induced CYP 3A activity. Theses findings were confirmed with Western immunoblots with probes for rat CYP 1A1, 2B1/2, 2E1, 3A, and 4A. Z produced a three-to fourfold increase in the 3A isoform for both male and female rats. Results from this study suggest that in a clinical setting Z therapy has the potential to induce CYPs of the 3A subfamily and in so doing alter the metabolism and clearance of drugs that are substrates for the 3A subfamily. © 1997 John Wiley & Sons, Inc.     

Alteration of liver microsomal monooxygenases and substrate competition with aniline hydroxylase from rats chronically fed low-fat and high-fat-containing alcohol diets

Male Sprague-Dawley rats fed ethanol (EtOH) 36% of total calories for four weeks in a liquid diet containing either 34% (HF) or 12% (LF) of calories as fat were studied with respect to induction of microsomal monooxygenases (MFO) and substrate competition with EtOH-inducible aniline hydroxylase. The specific activity and turnover of aniline hydroxylase were induced to similar extents by HF-EtOH and LF-EtOH diets. Whereas, both LF-EtOH and HF-EtOH caused a decrease in the turnover of arylhydrocarbon (benzo[a]pyrene) hydroxylase (AHH) and aldrin epoxidase compared to pair-fed (PF) controls, LF-EtOH but not HF-EtOH increased the turnover of ethoxycoumarin and ethoxyresorufin O-deethylase (ECOD and EROD). The increase in ECOD and EROD and the decrease in AHH by EtOH is contrary to the parallel induction of these activities by J-methylcholanthrene (3-MC) and Aroclor 1254 (Aroclor). Benzo(a)pyrene (BaP) stimulated aniline hydroxylase in the HF-EtOH and PF systems, whereas with LF diet, stimulation was seen only in the EtOH group. Ethoxycoumarin (EC) inhibited aniline hydroxylase by microsomes from EtOH- and pyrazole-treated rats, whereas it stimulated aniline hydroxylase by control microsomes, suggesting that the EC effects were associated with EtOH-inducible cytochrome P-450. Ethoxyresorufin (ER) inhibited aniline hydroxylase in EtOH and PF groups, thus the differential effects of EC were not nonspecific O-deethylase effects. The effects of EtOH feeding on ECOD, EROD, and AHH (ie, substrates for 3-MC-inducible cytochrome P-450) displayed a greater differential between the experimental and control group with the LF- than with the HF-containing diet. The findings suggest that the alteration of certain MFO activities by chronic EtOH ingestion can be modified by the content of dietary fat. Moreover, the competition dynamics of MFO substrates toward EtOH-inducible aniline hydroxylase are altered by EtOH feeding and, in turn, modified by dietary fat.     

Investigation of EROD, CYP1A immunopositive proteins and SOD in haemocytes of Chamelea gallina and their role in response to B[a]P

CYP1A sub-family represents the main form of cytochrome P450 involved in benzo[a]pyrene (B[a]P) detoxification, but there are no clear evidences about its presence in invertebrates. 7-Ethoxy resorufin O-deethylase (EROD) activity is strictly related to CYP1A presence, at the same time P450-dependent oxidative metabolism leads to reactive oxygen species (ROS) production, thought to be an important mechanism of pollutant-mediated toxicity in aquatic organisms. Superoxide dismutases (SODs), EROD and CYP1A activities and/or expressions were detected in haemocytes of pooled clams (Chamelea gallina) and cell-free haemolymph after 24 h, 7 and 12 days of exposure to 0.5 mg/L of B[a]P. After 24 h, B[a]P content was maximum in whole tissues. A 61 kDa band was recognized in haemocytes and cell-free haemolymph by polyclonal anti-fish CYP1A, while 53.5 and 63.8 kDa CYP1A immunopositive proteins were discriminate without differences of expression. Differently, EROD, MnSOD activity/expression and ECSOD expression decreased in haemocytes and haemolymph. C. gallina immune system presents an interesting response dose/time exposure of B[a]P and the 7 days condition highlights the major effects of xenobiotic action. The identification of basal EROD levels supports the possible presence of the CYP1A, never identified in C. gallina and more specifically never isolated in immune cells, as confirmed by CYP1A-immunopositive proteins identification.     

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

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

Assessment of some innate immune responses in dab (Limanda limanda L.) from the North Sea as part of an integrated biological effects monitoring

The marine flatfish dab (Limanda limanda), which lives in direct contact with contaminated sediments, is frequently used as a sentinel species in international monitoring programmes on the biological effects of contaminants. In this study, immune responses were recorded as indicators of sublethal chronic effects of contaminants, in addition to measurement of the induction of mono-oxygenase ethoxyresorufin O-deethylase (EROD) in liver cells, the inhibition of acetylcholin esterase (AChE) in muscle and a quantification of grossly visible diseases and parasites. In total, 336 dab were analysed from five sampling areas in the North Sea, including the German Bight, the Dogger Bank, the Firth of Forth, and two locations close to oil and gas platforms (Ekofisk and Danfield). When considering plasma lysozyme levels, pinocytosis and respiratory burst activity of head kidney leucocytes, a clear gradient could be observed with decreased levels in individuals collected from the Firth of Forth and locations near the oil or gas platforms compared with dab from the Dogger Bank or the German Bight. Individuals with induced EROD activity displayed reduced lysozyme and respiratory burst activities. Lysozyme levels were also reduced in dab with lymphocystis or with nematodes. The data obtained indicate that the assessment of innate immune parameters in a monitoring programme provides supplementary information about immunomodulatory effects associated with the exposure of fish to contaminants. In particular, concentrations of plasma lysozyme, which can be analysed in an easy and inexpensive assay, are considered to be an appropriate parameter for use in a battery of other bioindicators. Communicated by H. von Westernhagen and A. Diamant     

The effects of diet composition and ration on biotransformation enzymes and stress parameters in rainbow trout, Oncorhynchus mykiss

Juvenile rainbow trout (Oncorhynchus mykiss) were fed one of three isoenergetic diets varying in protein (35-55%) and lipid content (8-18%), at full satiation levels or half rations for 6 weeks in order to investigate the effects of diet on baseline stress parameters and biotransformation enzyme activity. Growth was greatest in fish fed to satiation on a low protein and high lipid diet. Stress parameters, including plasma lactate, glucose and cortisol concentrations were not significantly affected by dietary treatment or ration. Basal biotransformation enzymes, including glutathione S-transferase (GST) and ethoxyresorufin O-deethylase (EROD) activity, were also unaffected by dietary treatment. Fish exposed to the biotransformation enzyme inducer beta-naphthoflavone did not exhibit an alteration in stress indicators or GST activity; however, EROD activity was increased (87- to 210-fold) in fish receiving all diets and rations. The results of the present study indicate that, unlike mammals, fish may be more recalcitrant to different levels of ingestion of isoenergetic diets varying in protein and lipid concentration with respect to stress responses and the maintenance of basal titers of biotransformation enzymes and their induction.     

Use of Biochemical Endpoints to Determine Relationships between Contaminants and Impaired Fish Health in a Freshwater Stream

Bayou Bartholomew is a low gradient stream predominantly influenced by agricultural inputs and stormwater discharge from the urban areas of Pine Bluff, Arkansas. Preliminary studies indicated induction of hepatic cytochrome P450 1A protein (CYP1A) and CYP1A-catalyzed enzyme activity (ethoxyresorufin O-deethylase-EROD) in several fish species collected in the waterway, which meanders through residential areas of Pine Bluff, and forested regions in the outskirts of the city. Largemouth bass (Micropterus salmoides) showed CYP1A repression and a lack of correlation between CYP1A and EROD activity. In addition, hepatic heme oxygenase activity was elevated in several species and demonstrated an inverse relationship with hepatic CYP1A. Hepatic metallothionein was unchanged in any species collected from these locations. Four years after this evaluation, sediments were collected and analyzed for agents that could be mechanistically consistent with the biochemical responses observed. Utilizing seven criteria for causality, these data indicated that PCBs appear to be at least one group of agents responsible for the biological effects observed in fish species from the upper Bayou Bartholomew. While this supports causality under the criteria, further studies in this waterway would strengthen the relationship between this class of compound and the effects observed in resident fish species.     

Biomarkers in Zebra mussel for monitoring and quality assessment of Lake Maggiore (Italy)

Three different biomarkers (acetylcholinesterase (AChE), ethoxy resorufin-O-deethylase (EROD) and DNA strand breaks) were measured in Zebra mussel (Dreissena polymorpha) specimens collected in April 2005 at six different sampling sites on Lake Maggiore, the second largest Italian lake in terms of depth and volume, in order to assess the spatial variation of exposure to man-made contaminants. Mussels maintained at fixed laboratory conditions were used as controls to eliminate potential interference due to environmental factors. Biomarker data were also supported by the analysis of several chemicals (six dichlorodiphenyltrichloroethane (DDTs), 23 polychlorinated biphenyls (PCBs), 14 polybrominated diphenyl ethers (PBDEs), 11 polycyclic aromatic hydrocarbons (PAHs) and hexachlorobenzene (HCB)) measured in the mussel soft tissues by gas chromatographic analyses. We found a negative correlation between temperature and AChE activity, while any measured environmental or physiological factor seemed to influence EROD activity and DNA strand breaks. A positive relationship was found between EROD activity and all of the measured chemicals, except for PAHs, which correlated with the amount of DNA strand breaks. Significant differences were noted for all biomarkers, both among sampling stations and between control and experimental data, even if the general level of variability was low. The biomarkers showed a distinct pattern of spatial variation, but the evaluation of DNA strand breaks was the strongest discriminating power between sites. In addition, the comparison between AChE and EROD activity measured in 2005 was compared with results obtained in a previous study carried out over the same sampling period in 2003. Results indicated a strong influence of temperature on AChE activity and probable interference of substrate inhibition of EROD activity, pointing out the need to take care in the interpretation of data comparisons. The results obtained with two different metrics used for the measure of DNA strand breaks is also discussed, as well as the relationship between EROD activity data and potential genotoxicity.