Characterization and in vitro sensitivity of cholinesterases of gilthead seabream (Sparus aurata) to organophosphate pesticides.

The characterization of cholinesterase activity in brain and muscle of gilthead seabream was carried out using four specific substrates and three selective inhibitors. In addition, K m and V max were calculated from the Michaelis-Menten equation for ASCh and BSCh substrates. Finally, the in vitro sensitivity of brain and muscle cholinesterases to three organophosphates (OPs) was also investigated by estimating inhibition kinetics. The results indicate that AChE is the enzyme present in the brain, whereas in muscle, a typical AChE form is present along with an atypical form of BChE. Very low ChE activity was found in plasma with all substrates used. The inhibitory potency of the studied OPs on brain and muscle AChEs based on bimolecular inhibition constants (k i ) was: omethoate < dichlorvos < azinphosmethyl-oxon. Furthermore, muscle BChE was found to be several orders of magnitude (from 2 to 4) more sensitive than brain and muscle AChE inhibition by dichlorvos and omethoate.

The Bromodomain testis-specific gene (Brdt) characterization and expression in gilthead seabream, Sparus aurata, and European seabass, Dicentrarchus labrax.

Multiple genes and transcription factors are involved in regulation and control of the complex process of sex determination and differentiation of fish species. Also more, several hormonal factors and some environmental conditions can also be adequate spawning strategies and stimuli for inducing reproduction of fish species. Brdt gene belongs to the bromodomain-extraterminal domain (BET) family of transcriptional coregulators. In mammals, Brdt gene is almost exclusively expressed in testis. Furthermore, Brdt protein is involved in elongating spermatids, and is required for proper spermatogenesis and male fertility. However, from our understanding of fish species, the role of this gene as key, during gametogenesis, still remains unknown. In this study, two Brdt mRNA transcripts were isolated from two teleostean fish species, gilthead seabream and European seabass. In both species the shorter form lacked a functional C-terminal domain, which may involve a different function as transcriptional regulator. The pattern of Brdt expression showed that the highest levels occurred in the gonads. Significantly lower levels of expression were detected in brain, pituitary and different organ systems (heart, kidney, gills, among other somatic tissues) from both studied species. In situ hybridization approach evidenced that Brdt mRNA expression was restricted to specific cell-types of the germ line, during both oogenesis and spermatogenesis processes.

Busulfan administration produces sublethal effects on somatic tissues and inhibits gametogenesis in Senegalese sole juveniles.

Busulfan, a cytotoxic alkylating agent used for treatment of chronic myeloid leukemia has effects in mammalian germ cells. In fish species, the use of this compound is of special interest in intra and interspecies germ cell transplants. To determine the effects of busulfan in fish a previous range finding experiment was designed. Survival and growth rate of 150-days after hatching (150DAH) Senegalese sole (Solea senegalensis) juveniles was determined. In a second experiment, the effects of a sublethal busulfan dose in fish germ cell depletion and in somatic tissues were analysed. Sublethal effects of several busulfan treatments (B10-10 days after injection, B20-20 days after injection, B20÷-20 days after injection with double injection) were determined in somatic and gonadal tissues. Alterations were registered through histopathological techniques, TUNEL (cell apoptosis) and quantified at molecular level (Q-PCR analyses) using the vasa mRNAs (Ssvasa1-2 and Ssvasa3-4 mRNAs) as molecular markers for germinal cells in Senegalese sole juveniles. Several sublethal effects were observed with 40 mg kg⁻¹ busulfan, a non-lethal dose, such as: pyknosis in liver, increase of melanomacrophage centres and blood stagnation in spleen and interruption of gonadal development. Females were more affected by busulfan treatments than males in terms of germ cell disruption, since a significant decrease in the expression of both Ssvasa1-2 and Ssvasa3-4 markers was found in the gonad of treated females rather than males. At 10 days post-treatment (B10), females already presented a decrease in germ cell proliferation, as confirmed by Q-PCR. Ssvasa expression proved to be a reliable tool for the direct evaluation of the effects of busulfan on Senegalese sole gonadal development, proving that busulfan can be a suitable treatment for causing transient sterility in recipient gonads for germ cell transplantation.

Effect of two sulfur-containing amino acids, taurine and hypotaurine in European sea bass (Dicentrarchus labrax) sperm cryopreservation.

In the present work, taurine and hypotaurine were evaluated as potential additives to improve European sea bass (Dicentrarchus labrax) sperm quality after cryopreservation. For cryopreservation, three different extenders were used: control extender (NAM), supplemented with 1mM taurine or supplemented with 1mM hypotaurine, all of them containing 10% Me2SO as cryoprotectant. To evaluate sperm quality of fresh and thawed sperm, motility (CASA: computer assisted sperm analysis), viability (SYBR Green/propidium iodide), lipid peroxidation (malondialdehyde level), protein oxidation (carbonyl content), glutathione peroxidase, glutathione reductase and superoxide dismutase activities and DNA fragmentation (comet assay) were quantified. The result demonstrated that 1 mM hypotaurine supplemented extender increased total motility (30.1 ± 3.2%), and that 1 mM taurine extender produced higher velocity (18.1 ± 2.6 µm/s) and linearity (46.0 ± 4.8%) than the control extender (21.8 ± 3.2%, 15.5 ± 1.3 µm/s, 41.8 ± 2.4%, respectively). Cell viability, lipid peroxidation and protein oxidation were not statistically different between treatments. Similar results were obtained for glutathione peroxidase and superoxide dismutase activities. Only glutathione reductase showed differential activity before and after freezing, increasing its activity in thawed sperm. Regarding the comet assay results, taurine and hypotaurine significantly reduced DNA fragmentation (52.8 ± 0.9% and 51.8 ± 0.9%, respectively) in comparison to the control (55.7 ± 0.8%). In conclusion, for European sea bass sperm cryopreservation, extenders supplemented with 1 mM taurine and 1 mM hypotaurine improved some parameters of sperm quality after thawing, resulting in better motility and lower DNA damage than the control, two very important factors related to fertilization success.

Transmission of lymphocystis disease virus to cultured gilthead seabream, Sparus aurata L., larvae.

The transmission of lymphocystis disease virus (LCDV) to gilthead seabream, Sparus aurata L., larvae was investigated using fertilized eggs from a farm with previous reports of lymphocystis disease. LCDV genome was detected by PCR-hybridization in blood samples from 17.5% of the asymptomatic gilthead seabream broodstock analysed. Using the same methodology, eggs spawned from these animals were LCDV positive, as well as larvae hatched from them. The presence of infective viral particles was confirmed by cytopathic effects development on SAF-1 cells. Whole-mount in situ hybridization (ISH) and immunohistochemistry (IHC) showed the presence of LCDV in the epidermis of larvae hatched from LCDV-positive eggs. When fertilized eggs were disinfected with iodine, no viral DNA was detected either in eggs (analysed by PCR-hybridization) or in larvae (PCR-hybridization and ISH). These results suggest the vertical transmission of LCDV, the virus being transmitted on the egg surface. Larvae hatched from disinfected eggs remain LCDV negative during the endotrophic phase, as showed by PCR-hybridization, ISH and IHC. After feeding on LCDV-positive rotifers, viral antigens were observed in the digestive tract, which suggests that viral entry could be achieved via the alimentary canal, and that rotifers can act as a vector in LCDV transmission to gilthead seabream larvae.

Antiviral activity of casein and αs2 casein hydrolysates against the infectious haematopoietic necrosis virus, a rhabdovirus from salmonid fish.

Salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), are responsible for serious losses in the rainbow trout and salmon-farming industries, and they have been the subject of intense research in the field of aquaculture. Thus, the aim of this work is to study the antiviral effect of milk-derived proteins as bovine caseins or casein-derived peptides at different stages during the course of IHNV infection. The results indicate that the 3-h fraction of casein and α(S2) -casein hydrolysates reduced the yield of infectious IHNV in a dose-dependent manner and impaired the production of IHNV-specific antigens. Hydrolysates of total casein and α(S2) -casein target the initial and later stages of viral infection, as demonstrated by the reduction in the infective titre observed throughout multiple stages and cycles. In vivo, more than 50% protection was observed in the casein-treated fish, and the kidney sections exhibited none of the histopathological characteristics of IHNV infection. The active fractions from casein were identified, as well as one of the individual IHNV-inhibiting peptides. Further studies will be required to determine which other peptides possess this activity. These findings provide a basis for future investigations on the efficacy of these compounds in treating other viral diseases in farmed fish and to elucidate the underlying molecular mechanisms of action. However, the present results provide convincing evidence in support of a role for several milk casein fractions as suitable candidates to prevent and treat some fish viral infections.

Sea bass sperm freezability is influenced by motility variables and membrane lipid composition but not by membrane integrity and lipid peroxidation.

Cryopreserved sperm quality depends on the characteristics of fresh sperm. Thus, it is necessary to establish a group of variables to predict the cryopreservation potential of the fresh samples with the aim of optimizing resources. Motility, viability, lipid peroxidation and lipid profile of European sea bass (Dicentrarchus labrax) sperm were determined before and after cryopreservation to establish which variables more accurately predict the sperm cryopreservation potential in this species. Cryopreservation compromised sperm quality, expressed as a reduction of motility (46.5 ± 2.0% to 35.3 ± 2.5%; P<0.01) and viability (91.3 ± 0.7% to 69.9 ± 1.6%; P<0.01), and produced an increase in lipid peroxidation (2.4 ± 0.4 to 4.0 ± 0.4 µmoles MDA/mill spz; P<0.01). Also, significant changes were observed in the lipid composition before and after freezing, resulting in a reduction in the cholesterol/phospholipids ratio (1.4 ± 0.1 to 1.1 ± 0.0; P<0.01), phosphatidylcholine (47.7 ± 0.8% to 44.2 ± 0.8%; P<0.01) and oleic acid (8.7 ± 0.2% to 8.3 ± 0.2%; P<0.05) in cryopreserved sperm, as well as an increase in lysophosphatidylcholine (4.4 ± 0.3% to 4.8 ± 0.3%; P<0.01) and C24:1n9 fatty acid (0.5 ± 0.1% to 0.6 ± 0.1%; P<0.05). Motility, velocity, cholesterol/phospholipids ratio, monounsaturated fatty acids and the n3/n6 ratio were positively correlated (P<0.05) before and after freezing, whereas, viability and lipid peroxidation were not correlated. Motility and the cholesterol/phospholipids (CHO/PL) ratio were negatively correlated (P<0.05) with each other and the CHO/PL ratio was positively correlated (P<0.05) with lipid peroxidation. Therefore, the results demonstrated that motility and plasma membrane lipid composition (CHO/PL) were the most desirable variables determined in fresh samples to predict cryo-resistance in European sea bass sperm, taking into account the effect of both on cryopreserved sperm quality.

Incorporation of ascorbic acid and α-tocopherol to the extender media to enhance antioxidant system of cryopreserved sea bass sperm.

Despite the overwhelming application of sperm cryopreservation in aquaculture and broodstock management, its detrimental effects on sperm quality must be taken into account. Imbalance of reactive oxygen species is considered one of the main triggers of cell damage after cryopreservation, because the spermatozoa antioxidant system is decimated during this process, mainly because the natural antioxidants present in seminal plasma diminish when sperm is diluted in extenders. It has been demonstrated that the addition of antioxidants to the extender improves the quality of thawed sperm. Thus, the aim of the present work was to evaluate the status of the antioxidant system in cryopreserved sea bass sperm, and the possibility of enhancing this system to reduce oxidation of the membrane compounds by extender supplementation with vitamins. To do this, sperm from European sea bass (Dicentrarchus labrax) was cryopreserved using an extender control (NAM), supplemented with 0.1 mm α-tocopherol or 0.1 mm ascorbic acid. Sperm motility (computer assisted sperm analysis (CASA) parameters), viability (SYBR Green/propidium iodide (PI)), lipid peroxidation (malondialdehyde (MDA) levels) and protein oxidation (DNPH levels) were analyzed, as well as the status of the sperm antioxidant system by determining glutathione peroxidase, glutathione reductase and superoxide dismutase (GPX, GSR and SOD) activity. The results demonstrated that extenders containing vitamins significantly increased sperm motility. Total motility, velocity and linearity increased from 31.2 ± 3.0 µm/sec, 18.3 ± 1.7 µm/sec and 46.9 ± 2.0% in extender containing 0.1 mm α-tocopherol or 30.6 ± 3.9 µm/sec, 19.5 ± 1.6 µm/sec and 47.9 ± 2.2% in extender containing 1 mm ascorbic acid respect to the extender control (20.7 ± 3.3 µm/sec, 13.8 ± 1.7 µm/sec and 37.3 ± 4.1%). However, viability and levels of lipid peroxidation and protein oxidation were not affected by the presence of these antioxidants, suggesting that membrane impairment could be more associated to osmotic shock or membrane destabilization than oxidative damage. The increased activity of both GPX and GSR after cryopreservation showed that the antioxidant system of sea bass sperm must play an important role in preventing oxidation of the membrane compounds. In conclusion, the addition of α-tocopherol and ascorbic acid to the extender media, together with the antioxidant system of the spermatozoa improved sea bass sperm motility, which is one of the impairment parameters most affected by cryopreservation.

The influence of certain aminoacids and vitamins on post-thaw fish sperm motility, viability and DNA fragmentation.

During cryopreservation, dilution in the extender media reduces the seminal plasma constituents being cells more vulnerable to oxidative stress. Vitamins (C and E) and the amino acids taurine and hypotaurine are powerful antioxidants naturally present in seminal plasma. Whether their effect may improve sperm quality and reduce sperm DNA damage after cryopreservation in fish sperm still remains unclear. Thus, the aim of the present work was to analyse the effect of extender supplementation with several antioxidant components on post-thawed sperm motility, viability and DNA integrity of two commercial species, gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax). Sperm collected from ten to twelve individuals was cryopreserved in ten different extenders containing: taurine and hypotaurine (1 and 10mM), ascorbic acid (1 and 10mM), α-tocoferol (0.1 and 0.5 mM) or 1 ml/l of a commercial cell antioxidant supplement. Cell viability, motility and DNA fragmentation were determined in post-thawed samples. Addition of antioxidants (vitamins and amino acids) to D. labrax and S. aurata extenders did not significantly increase the parameters of motility (TM, PM, VCL, VSL and Lin) or viability, although 1mM taurine slightly increased the percentage of motile cells (TM) in S. aurata. DNA fragmentation (DNA in tail and Olive tail moment) in D. labrax sperm was higher in treatments containing vitamins than amino acids or control. However in S. aurata sperm, antioxidants especially taurine and hypotaurine, significantly reduced both DNA fragmentation parameters, protecting DNA against strand breaks. These results suggest a species-specific effect depending on the type of antioxidants used.

Lectin-binding pattern of Senegalese sole Solea senegalensis (Kaup) testis.

The localization and characterization of oligosaccharide sequences in the testis of Senegalese sole Solea senegalensis was investigated using 12 lectins in combination with KOH saponification and sialidase digestion (K-s). The interstitial compartment contained all the sugar residues investigated, those bearing oligosaccharides terminating with sialic acid (Neu5Ac) alpha2,3Galbeta1,4GlcNAc, Neu5AcGalNAcalpha1,3(LFucbeta1,2) Galbeta1,3/4GlcNAcbeta1 and GalNAcalpha1,3(LFuc1,2) Galbeta1,3/4GlcNAcbeta1 being more abundant in the medullar region than in the cortex. The melano-macrophage centres found in the interstitial compartment displayed glycans terminating with Galbeta1,3GalNAc. The basal lamina separating the germinal and interstitial compartments exhibited glycans with terminal/internal mannose, internal betaGlcNAc, and terminal Neu5Acalpha2,6Gal/GalNAc, and Neu5AcGalbeta1,3GalNAc, Galbeta1,3GalNAc (PNA), Galbeta1,4GlcNAc, GalNAc, alphaGal, and alphaL-Fuc. In the germinal compartment, the Sertoli cells expressed only glycans terminating with Neu5Acalpha2,3Galbeta1,4GlcNAc in the apical and supra-nuclear lateral surface of the spermatonial cysts located in the distal part of the seminiferous lobules. Primary spermatocytes exhibited oligosaccharides terminating with Galbeta1,3GalNAc and alphaGalNAc in the cytoplasm and nucleus, respectively. The spermatids contained highly mannosylated glycans terminating with GalNac, alphaGal, and alphaL-Fuc. The head of spermatozoa expressed a more complex glycosylation pattern characterized by the additional presence of oligosaccharides terminating with Neu5Acalpha2,3Galbeta1,4GlcNAc, Neu5AcGalbeta1,3GalNAc, Neu5AcGalNAcalpha1,3(LFuca1,2)Galbeta1,3/4GlcNAcbeta1, GalNAcalpha1,3(LFucalpha1,2)Galbeta1,3/4GlcNAcbeta1. The comparison with previous lectin histochemical studies carried out in other fish species reveals a specific glycosylation pattern of Senegalese sole testicular structures and spermatozoa head.

Brain CYP1A in seabream, Sparus aurata exposed to benzo(a)pyrene.

This study compares basal and induced expression of cytochrome P4501A-CYP1A in the brain of gilthead seabream, Sparus aurata. Larval or adult seabream were exposed to benzo(a)pyrene -B(a)P- and the CYP1A response was assessed by analyzing CYP1A mRNA (RT-PCR), CYP1A protein (expression levels: ELISA, western blotting; cellular localization: immunohistochemistry), and CYP1A catalytic activity (7-ethoxyresorufin-O-deethylase-EROD). In the brain of adult S. aurata, CYP1A immunostaining was generally detected in the vasculature. It was present in the neuronal fibers and glial cells of the olfactory bulbs and the ventral telencephalon. ELISA and RT-PCR analyses confirmed CYP1A expression in the brains of non-exposed seabream. B(a)P exposure led to increased CYP1A staining mainly in neuronal fibers and glial cells of the olfactory bulbs, but also in the vascular endothelia. EROD activity, however, could not be detected in the brain of adult seabream, neither in control nor in exposed fish. In the developing brain of S. aurata larvae, immunohistochemical staining detected CYP1A protein exclusively in endothelia of the olfactory placode and in retina. Staining intensity of CYP1A slightly increases with larval development, especially in vascular brain endothelia. Exposing the larvae to 0.3 or 0.5 microg B(a)P/L from hatching until 15 days post hatching (dph) did not result in enhanced CYP1A immunostaining in the brain. In samples of whole seabream larvae, both from controls and BaP treatments, neither CYP1A mRNA, protein nor catalytic activity were detectable. The results demonstrate that CYP1A is expressed already and inducible in the larval brain, but that the regional and cellular expression differs partly between larval and adult brain. This may have implications for the toxicity of CYP1A-inducing xenobiotics on early and mature life stages of seabream.

Comparative gene expression of gonadotropins (FSH and LH) and peptide levels of gonadotropin-releasing hormones (GnRHs) in the pituitary of wild and cultured Senegalese sole (Solea senegalensis) broodstocks.

The Senegalese sole (Solea senegalensis) is a valuable flatfish for aquaculture, but it presents important reproductive problems in captivity. Spawning is achieved by wild-caught breeders but cultured broodstocks fail to spawn spontaneously and, when they do, eggs are unfertilized. To gain knowledge on the physiological basis underlying this reproductive dysfunction, this study aimed at analyzing comparative hormone levels between wild and cultured broodstocks at the spawning season. The Senegalese sole gonadotropin (GTH) subunits, FSHbeta, LHbeta and GPalpha, were cloned and qualitative (in situ hybridization) and quantitative (real-time PCR) assays developed to analyze pituitary GTH gene expression. In females, FSHbeta and GPalpha mRNA levels were higher in wild than in cultured broodstocks, whereas in males all three subunits were highest in cultured. By ELISA, three GnRH forms were detected in the pituitary, displaying a relative abundance of GnRH2>GnRH1>GnRH3. All GnRHs were slightly more abundant in wild than cultured females, whereas no differences were observed in males. Plasma levels of vitellogenin and sex steroids were also analyzed. Results showed endocrine differences between wild and cultured broodstocks at the spawning period, which could be related to the endocrine failure of the reproductive axis in cultured breeders.

Oxidative stress and histopathology damage related to the metabolism of dodecylbenzene sulfonate in Senegalese sole.

Surfactants such as linear alkylbenzene sulfonates (LAS) are widely utilised in the formulation of detergents in commercial products. After use, they pass through waste water treatment plants (WWTP) and are then discharged to aquatic ecosystems, causing risk to aquatic life. The exposure of marine animals to these compounds enhances the production of reactive oxygen species (ROS) with subsequent damage to macromolecules, and produces histological alterations. A flow-through experiment with Senegalese sole (Solea senegalensis) has been devised with the object of correlating the metabolism of LAS including sulfophenylcarboxylic acids (SPCs) by fish with their antioxidant defence system (generation of oxyradicals) and histopathological damage. The generation of intermediate degradation products (SPCs) by the organism, the histopathological responses, the antioxidant enzymes (catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and glutathione S-transferase (GST)), as well as other kinds of enzyme such as acid and alkaline phosphatases (AcP, ALP), were measured. SPCs from 5ØC(6) to 11ØC(12) were identified and quantified in fish and water; their concentrations differed depending on the sampling moment. In general, the responses found in the enzymes were slight: a decrease in the enzymatic activity in gills and activation in the digestive tract. The evidence of histopathological damage identified was also small; the organism's defensive mechanism against pollutants should enable it to recover easily. A direct relationship was established between biotransformation and the generation of SPCs and ROS. In conclusion, the correct functioning of the antioxidant defence system with absence of large variations, the short-term histopathological damage, and the evidence of SPCs indicate an adequate metabolism of 2-phenyl-C(12)-linear alkylbenzene sulfonates (2ØC(12)LAS) by this specie and non-toxic effects at environmentally realistic levels.

Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt-head seabream, Sparus aurata L.

Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques have been used for the detection of lymphocystis disease virus (LCDV) in formalin-fixed, paraffin-embedded tissues from gilt-head seabream, Sparus aurata L. Diseased and recovered fish from the same population were analysed. IHC was performed with a polyclonal antibody against a 60-kDa viral protein. A specific digoxigenin-labelled probe, obtained by PCR amplification of a 270-bp fragment of the gene coding the LCDV major capsid protein, was used for ISH. LCDV was detected in skin dermis and gill lamellae, as well as in several internal organs such as the intestine, liver, spleen and kidney using both techniques. Fibroblasts, hepatocytes and macrophages seem to be target cells for virus replication. The presence of lymphocystis cells in the dermis of the skin and caudal fin, and necrotic changes in the epithelium of proximal renal tubules were the only histological alterations observed in fish showing signs of the disease.

Nutritional cellular biomarkers in early life stages of fish.

Histological (tissular and cellular) indices have a tradition of determining the nutritional condition of fish both in the laboratory and in the wild. The assessment of condition by means of microscopical methods is probably the mos accurate indicator of nutritional status during the early life stages of fish. This success is partly attributable large amount of information that can be derived from their study and because they are thought to be the only true starvation indices. The technique usually consists of the examination of cells and organs and the establishment of a grading system based on the presence/absence of standardised biomarkers. Each organ is examined, and the cellular aspect or tissular cohesion is evaluated qualitatively and even quantitatively in order to obtain a measure of the general condition of a larva. The literature indicates that there are certain tissular and cellular responses to food availability and quality, particularly in the digestive and muscular tissues, which are common to most teleost fish larvae. These responses, which are independent of water temperature, can be used for assessing fish larvae nutritional condition. In this regard, the microscopical organization of the liver hepatocytes, the intestinal mucosa, the exocrine pancreas and the muscular fibers, which are generally used as target tissues and organs to assess the nutritional condition of fish larvae, is deeply reviewed. The advantages and disadvantages of the use of different cellular biomarkers of effect are discussed considering different conditions.

Effects of sediment sorbed linear alkylbenzene sulphonate on juveniles of the Senegal sole, Solea senegalensis: toxicity and histological indicators.

Many synthetic organic substances, including surfactants, tend to be sorbed on suspended solids and to accumulate finally on bottom sediments, where benthic communities may be exposed to them. Concentrations of Linear Alkylbenzene Sulphonates (LAS) have been detected in estuarine and coastal sediments, presenting wide concentration ranges depending on the presence of treatment facilities, hydrodynamic conditions, organic matter content etc. Senegal sole, Solea senegalensis, larvae (40 days posthatching; dph) were exposed to increasing concentrations of LAS spiked sediments, comprised between 0.37 and 880.78 mg LAS x kg(-1) during 30 days. The obtained results showed that survival of exposed larvae was not significantly affected at environmentally relevant concentrations, the LC50 value being obtained after 30 days 876.46 mg x kg(-1). However, the histological and histopathological analyses carried out in target organs revealed, that first alterations from the normal pattern were observed at concentrations of 222.66 mg x kg(-1), presenting effects such as blood extravasation and hyperplasy of the lamellar epithelium in gills, increase of inter-myotomal spaces of the skeletal musculature and edematous separation of the skin from epidermis. At the highest exposure concentrations (755.27 and 880.78 mg LAS x kg(-1)), shrinkage of hepatocytes, nuclear pycnosis and blood stagnation are observed in the liver, degeneration of pancreatic cells, reduction of hemocytopoietic tissue in the kidney and vacuolisation of intestinal enterocytes was observed at histological level, as well as severe separation of the epidermis from the underlying tissues. Simultaneously, a significant increase of the wet weight with exposure concentration was observed in the test organisms.

Tissue-specific induction of EROD activity and CYP1A protein in Sparus aurata exposed to B(a)P and TCDD.

The purpose of this study was to compare xenobiotic CYP1A induction in liver, gills, and excretory kidney of gilthead seabream, Sparus aurata. Fishes were exposed via water for 20 days to different concentrations of benzo(a)pyrene (B(a)P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). CYP1A was measured at the enzyme activity level as 7-ethoxyresorufin-O-deethylase (EROD) activity, and at the protein level by means of ELISA. The liver displayed the highest absolute levels of EROD activity, both under non-exposed and exposed conditions. Organ- or inducer-related differences in the time course of CYP1A induction were moderate; however, the magnitude of the induction response varied between the organs and between B(a)P and TCDD. In the case of TCDD, liver, and kidney yielded a comparable induction response, whereas in the case of B(a)P, the kidney showed a substantially higher maximum induction factor than the liver. In the gills, the two xenobiotics resulted in similar maximum induction factors. In B(a)P-exposed seabream, EROD activities and CYP1A protein levels showed a good correlation in all three organs, whereas with TCDD as inducer the correlation was poor, what was mainly due to a decrease of EROD activities at the higher concentrations of TCDD, while CYP1A protein levels showed no concomitant decline. Overall, the study revealed both similarities and differences in the time-, concentration-, and inducer-dependent CYP1A responses of the three target organs, liver, kidney, and gills. Although, the findings of this study principally confirm the notion of the liver as the major metabolic organ in fish, they also provide evidence for substantial metabolic potential in gills and particularly in the kidney.