Validation of Bioassays for Assessing the Contamination of Marine Environments

Various biomarkers were used to determine the exposure of fish (Arius felis and Micropogon undulatus) from Galveston Bay (GB), Texas, USA to organic contaminants. Sediment levels of polynuclear aromatic hydrocarbons (PAHs) in GB ranged from 81 to >1000 ng/g and polychlorinated biphenyls (PCBs) were <20 ng/g at all stations. No significant differences in hepatic concentrations of contaminants and ethoxyresorufin O–deethylase (EROD) activity, CYPIA mRNA levels, and DNA adducts were found in A. felis from GB. However, significant differences in biliary concentrations of naphthalene, phenanthrene, and benzo[a]pyrene metabolites were observed. Induced EROD activities and elevated levels of biliary PAH metabolites were measured in M. undulatus from the two most contaminated sites in GB. Induction toxic equivalents (I-TEQs), derived from dosing rat hepatoma H4IIE cells with hepatic extracts of A. felis, were correlated with tissue levels of ≥4–ring PAHs.     

Effect of Benzo(a)pyrene on Hepatic Biotransformation Activities: Time Course of Induction in Aquaculture European Sea Bass (Dicentrarchus labrax)

The responses of phase I and phase II enzymes to benzo(a)pyrene (BaP) exposure were characterized in liver of fish farm European sea bass (Dicentrarchus labrax). Treatment of sea bass with BaP by intraperitoneal injection produced a rapid putative MFO induction. Highest ethoxyresorufin-O-deethylase (EROD), ethoxycoumarin-O-deethylase (ECOD) and benzo(a)pyrene monooxygenase (BaPMO) activities were observed in microsomes 14–24 h after BaP injection. EROD was induced 14.5 fold, ECOD 3.9 and BaPMO 13 fold. Spectrophotometrically measured microsomal cytochrome P-450 and NADPH-cytochrome c reductase activity were not induced by BaP injection, whereas a low increase in cytochrome b₅ level was observed (induction factor 1.6 fold). Induction of phase II enzyme activities was lower than that of phase I enzymes: microsomal uridine diphospho-glucuronyl transferase (UDPGT) and cytosolic glutathion-S-transferase (GST) were induced 3.9 and 1.5 fold, respectively. The results indicate that sea bass is a sensitive species with respect to exposure to polycyclic aromatic hydrocarbons, exhibiting early marked responses of phase I and phase II biotransformation enzymes. The species offers potential as a tool for both laboratory studies and pollution monitoring. In the latter context it could be employed in the fish farms that occur alongside coastal regions.     

HYDROCARBON POLLUTION ALONG MOROCCAN COASTS AND BPH ACTIVITY IN THE MUSSEL PERNA PERNA

Specimens of the mussel Perna perna were collected along Moroccan coasts to determine the concentrations of polycyclic aromatic hydrocarbons (PAHs) bioaccumulated in the tissues, and to measure benzo[a]pyrene hydroxylase (BPH) activity. Chemical analysis of PAHs show that the Mediterranean (Nador, Martil, Tanger) and central Atlantic coasts (from Rabat to Jorf Lihoudi) are those most contaminated (351 245 ng.g^{? 1} dry weight in Tanger). The mussel contaminants were of mixed origin for most of the locations with non negligible inputs of petrogenic origin in many of them. Baseline levels of PAHs were between 6 and 55 ng.g^{? 1} dry weight. BPH activity showed significant correlation (r _s = 0.64, P < 0.05) with total PAH concentrations at the six most contaminated stations. The baseline level of BPH activity can be identified as in the range 0.1 to 13 pmol.min^{? 1}.mg prot^{? 1} along the Mediterranean and Atlantic coasts.     

Subterminal hydroxylation of lauric acid by microsomes from a marine fish

Microsomes from the liver of sea bass (Dicentrarchus labrax) were shown to hydroxylate lauric acid at subterminal positions. The cytochrome P-450 system converted lauric acid to several mono-hydroxylated metabolites including ω-1 hydroxylaurate, which was the major metabolite (44% of total products). In addition, ω-2, ω-3, ω-4 and a small amount (2.3%) of ω hydroxylaurates were found. Reaction products were identified using thin-layer chromatography (TLC) and gas chromatography/mass spectrometry (GC/MS). Oxidation reactions were dependent upon O₂ and NADPH, and did not occur with boiled microsomes or in the presence of a mixture of CO/O₂. Hydroxylation proceeded linearly up to 20 min at 28°C for protein concentrations below 380 μg. Treatment of fish with benzo(α)pyrene (BP) (20 mg/kg) drastically increased xenobiotic metabolism (ECOD, EROD and BPMO activities), but no difference in laurate hydroxylase activity was observed between untreated and treated fish. Starvation strongly enhanced laurate hydroxylase activity, and resumption of feeding reduced by half this increase of activity. In all of the experiments we did not observe any modification of the regioselectivity of lauric acid hydroxylation by this microsomal in-chain hydroxylating system. We suggest that cytochrome P-450 enzymes involved in lauric acid and xenobiotics metabolism are regulated independently.     

THE TOXICITY OF BENZO[a]PYRENE ON SOLE (SOLEA SOLEA) HEPATOCYTES: ASSESSMENT OF GENOTOXIC AND ENZYMATIC EFFECTS

The benzo[a]pyrene is a polycyclic aromatic hydrocarbon known to be genotoxic, mutagenic and carcinogenic in higher vertebrates. The aim of this study was to evaluate in vitro the enzymatic and genotoxic effects of BaP in a benthic fish species, Solea solea. Sole hepatocytes were exposed to BaP in order to measure the modulation of ethoxyresorufin-o-deethylase (EROD) activity and the DNA strand breaks induced by BaP metabolism. Exposures were performed in both culture flasks and microplate wells in order to check for the possible miniaturization of the exposure system. Moreover, sole liver microsomes were exposed to BaP in the presence of standard DNA in order to assess the potential formation of DNA adducts in sole. The results demonstrated the ability of sole hepatic enzymes to metabolize BaP into reactive species responsible for bulky DNA adducts and DNA strand breakage, whatever the tested exposure concentration and the mode of exposure.     

Relative Genotoxicities of PAH of Molecular Weight 252 AMU in Coal Tar-Contaminated Sediment

Bioassay-directed chemical fractionation methodology was used to calculate relative mutagenic potencies of polycyclic aromatic hydrocarbons (PAH) of molecular weight 252 amu in coal tarcontaminated sediment from Sydney Harbour, Nova Scotia. A normal phase HPLC technique was used to separate organic solvent extracts into fractions containing isomeric PAH of a single benzologue class. Bioassays with Salmonella typhimurium strain YG1025 with the addition of oxidative metabolism (S9) showed that approximately 50% of the mutagenic activity observed in the sediment extract was associated with PAH of molecular weight 252 amu. Further separation of the 252 PAH fraction using reversed phase HPLC yielded subfractions containing individual compounds; bioassay dose-response curves for these subfractions showed that benzo[a]pyrene was responsible for approximately 75% of the activity of the 252 PAH fraction.     

Effect of the degree of hydrogenation of dietary fish oil on the trans fatty acid content and enzymatic activity of rat hepatic microsomes

The degree of fat hydrogenation and the trans fatty acid content of the diet affect the fatty acid composition of membranes, and the amount and the activity of some membrane enzymes. We describe the effects of four isocaloric diets containing either sunflower oil (SO, 0% trans), fish oil (FO, 0.5% trans), partially hydrogenated fish oil (PHFO, 30% trans), or highly hydrogenated fish oil (HHFO, 3.6% trans) as fat sources on the lipid composition and the trans fatty acid content of rat hepatic microsomes. We also describe the effect of these diets on the cytochrome P-450 content and on the aminopyrine N-demethylase, aniline hydroxylase, and UDP-glucuronyl transferase microsomal activities. Cytochrome P-450 content was dependent on the degree of unsaturation of the diet, being higher for the FO-containing diet and lower for the HHFO diet. Aminopyrine N-demethylase activity also correlated with the degree of unsaturation of the diet as did the cytochrome P-450 content did (FO>SO>PHFO>HHFO). Aniline hydroxylase activity appeared to be independent of the degree of unsaturation of the dietary fat, but correlated with the trans fatty acid content of the diet, which was also reflected in the trans content of the microsomal membranes. UDP-glucuronyl transferase activity was higher for the FO-containing diet than for the SO diet, showing intermediate values after the PHFO and HHFO diets.     

Effect of freezing on quality of sea bass and gilthead sea bream

Freezing is an efficient method of fish preservation. The aim of the present work was to examine the impact of freezing in fatty acid composition and in the in vitro inhibitory activity of sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) fillet lipids against platelet activating factor (PAF). The Bligh and Dyer extraction method and the counter‐current distribution method were used to obtain total, polar and neutral lipids. The fatty acid analysis conducted using the internal standard method and the biological assay on washed rabbit platelets took place calculating the in vitro inhibitory activity of fish lipids against 2.5 × 10^?11 M of PAF. No statistical changes (p<0.05) occurred in fatty acid content of fresh and thawed gilthead sea bream, while fatty acid amount in thawed sea bass was significantly higher (p<0.05) compared to fresh fish. Total lipids of both thawed fish species exhibited stronger anti‐thrombotic activity compared to fresh fish. Freezing preserved fish quality and reinforced the anti‐thrombotic properties of fish oils, since even after 6 months of freezing, fish oils preserve their nutritional value in terms of protecting against cardiovascular diseases. Practical applications: Fish fillets contain high amount of unsaturated lipids that may easily undergo lipid oxidation. Freezing and frozen storage prevent such oxidative changes so fish quality is retained. Fatty acids and PAF‐antagonists in fish are of major importance since they contribute to the nutritional value of fish. The practical application of this work lies on the evaluation of the nutritional value of fish in terms of cardio protection by examining the impact of freezing on the levels of fatty acids and PAF‐antagonists in aquacultured fish fillets.     

Evaluation of PAH Metabolites in Bile of Common Carp,Cyprinus carpio L. with Fixed Wavelength Fluorescence in a Field and Laboratory Study

The seasonal and inter-gender variability of polycyclic aromatic hydrocarbon (PAH) metabolites in the bile of common carp (Cyprinus carpio) was investigated with a field study in Karakaya Dam Lake (KDL) and a laboratory-conducted study with the fish samples of KDL and Sultansuyu Dam Lake (Turkey). The KDL has been polluted by urban, industrial, and agricultural activities for many years. A seasonal trend was seen in PAH metabolite levels with higher levels detected in May 2005 samples in KDL. Their levels in the bile of fish were not found to be related to sex which showed that gender is not a confounding factor on PAH uptake. The results showed significant decreases in bile metabolite levels in the KDL fish by approximately 75% versus increases in the SDL fish after acclimation at laboratory conditions. These results supported that PAH metabolites in bile were not stable and short-term differences in living conditions may lead to great differences in their levels. Moreover, these rapid changes in bile metabolites with acclimation may also indicate that dam lakes were not badly polluted with PAHs and the feeding status of fish should be taken into consideration when using bile metabolites as biomarkers.     

Organ Specific Distribution of PAHs in a Carnivorous Fish Species Following Chronic Exposure to Used Synthetic-Based Drilling Mud

The rapid growth of offshore oil and gas exploration on the Western Indian Continental shelf has generated the need for both general and region-specific scientific information on the environmental consequences of drilling activities. Much of the toxicity related to drilling mud discharges has been attributed to Polycyclic Aromatic Hydrocarbons (PAH) content. This study addressed concerns related to the potential for contamination and subsequent bioaccumulation of PAHs in Oreochromis Mossambicus from the disposal of used synthetic-based drilling mud (SBM). The LC₅₀, 96 h for solid phase and suspended particulate liquid phase, was 37,550 mg/L and 40,390 mg/L, respectively, which was within the acceptable limits as per the Ministry of Environment and Forests (MoEF) Notification, New Delhi, dated August 30, 2005. After chronic exposure (for 55 days) to used SBM, higher mean values of PAHs accumulation were obtained from the Tilapias exposed to higher SBM concentration. The highest level of accumulation was noted for Naphthalenes and the lowest for Benzo (a) pyrenes. Liver was the primary organ for PAH accumulation, while least was observed in muscle. From the observations made on PAH levels in control as well as exposed fish, we found SBM concentration dependant bioaccumulation, suggesting the potential high risk of PAH toxicity to the fish inhabiting in the vicinity of disposal site.     

Stability of Polycyclic Aromatic Hydrocarbons in Frozen Mussel Tissue

The stability of polycyclic aromatic hydrocarbons (PAHs) in frozen mussel tissue (stored at -80 °C and -120 °C was assessed by analyzing samples of SRM 1974 and SRM 1974a, Organics in Mussel Tissue (Mytilus edulis), since the initial certification analyses. Results of analyses of SRM 1974 and SRM 1974a after 10 years and 6 years of storage, respectively, indicate that the PAH concentrations have not changed. Comparison of results from the analyses of frozen versus freeze-dried mussel tissue samples indicate significant losses of the more volatile PAH such as naphthalene and methylnaphthalenes during the freeze-drying process.     

Long-term feeding of dietary oils alters lipid metabolism, lipid peroxidation, and antioxidant enzyme activities in a teleost (Anabas testudineus bloch)

Anabas testudineus (climbing perch), average body weight 21±1 g, were maintained in culture tanks and fed a 35% protein feed plus an additional supplementation of three dietary oils (20% each of coconut oil, palm oil, or cod liver oil). Body weight gain was similar among all groups. However, several hepatic lipogenic enzymes such as malic enzyme (ME), NADP-isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and β-hydroxy-β-methyl glutaryl CoA reductase (HMG CoA reductase) were assayed, and they responded differently. Hepatic ME and G6PDH activities showed a significant decrease in the coconut oil and palm oil groups, but there was no significant change in ICDH activity. The 6PGDH activities were reduced, whereas HMG CoA reductase activity was increased in the palm oil-treated group. Cholesterol synthesis in the liver and muscle increased in the palm oil-treated group, but liver phospholipids did not show any significant change in fish supplemented with oils rich in saturated fatty acids. Triacyl-glycerol and free fatty acid concentrations were high in the coconut oil- and palm oil-supplemented groups. Lipid peroxidation products such as thiobarbituric acid-reactive substances and conjugated dienes decreased in the same two groups. Antioxidant potential was high in all groups as evidenced by increased activity of superoxide dismutase, glutathione peroxidase, and glutathione content. The results of this study indicate that in fish, dietary lipids depress hepatic lipogenic activity as well as lipid peroxidation products by maintaining high levels of antioxidant enzymes.     

The Cyanobacterial “Nutraceutical” Phycocyanobilin Inhibits Cysteine Protease Legumain

The blue biliprotein phycocyanin, produced by photo-autotrophic cyanobacteria including spirulina (Arthrospira) and marketed as a natural food supplement or “nutraceutical,” is reported to have anti-inflammatory, antioxidant, immunomodulatory, and anticancer activity. These diverse biological activities have been specifically attributed to the phycocyanin chromophore, phycocyanobilin (PCB). However, the mechanism of action of PCB and the molecular targets responsible for the beneficial properties of PCB are not well understood. We have developed a procedure to rapidly cleave the PCB pigment from phycocyanin by ethanolysis and then characterized it as an electrophilic natural product that interacts covalently with thiol nucleophiles but lacks any appreciable cytotoxicity or antibacterial activity against common pathogens and gut microbes. We then designed alkyne-bearing PCB probes for use in chemical proteomics target deconvolution studies. Target identification and validation revealed the cysteine protease legumain (also known as asparaginyl endopeptidase, AEP) to be a target of PCB. Inhibition of this target may account for PCB's diverse reported biological activities.     

Sphingomyelin metabolism is linked to salt transport in the gills of euryhaline fish

Byin vivo andin vitro studies ofl-(3-³H)serine and [9,10(n)-³H]palmitic acid incorporation into phospholipids, we show a change in the renewal of the ceramide moiety of sphingomyelin in the gills of euryhaline fish (sea bass and eels) when the animals were subjected to abrupt alterations in environmental salinity.In vivo, decrease of the salinity from sea water (salinity 3.7%) to diluted sea water (salinity 1%) induced an increase of label incorporation into gill sphingomyelin. The same was true when gills from sea water-adapted sea bass or sea water-adapted eels were incubated in diluted sea water. A decrease in free ceramides synthesis was also observed in the gills of sea water-adapted sea bass when the salinity of the incubation medium was reduced. Direct inhibition of Na⁺/K⁺-ATPase activity with ouabain decreased the sphingomyelin synthesis in the gills of sea bass duringin vitro incubation in diluted sea water, whereas treatment with furosemide stimulated sphingomyelin synthesis in the same gills incubated in sea water. These findings indicate that changes in Na⁺ fluxes modify the sphingomyelin turnover and control the production of free ceramides and sphingosine in gill cells of euryhaline fish. In view of the well-known effects of these sphingomyelin degradation products on isolated tumor cell differentiation, we suggest that they play a very important role in modulating chloride cell distribution and metabolism of fish gills during abrupt changes in environmental salinity.     

Alteration of kairomone-induced antipredator response of the freshwater amphipod Gammarus roeseli by sediment type

The importance of sediment type and of kairomones released by juvenile burbot (Lota lota) on behavioral responses of the freshwater amphipod Gammarus roeseli was studied in laboratory experiments. In the absence of kairomones, G. roeseli preferred coarse substrates; the highest preference was shown for the most coarse sediment. In the presence of kairomones, the amphipods preferred the less-coarse substrate pebbles over gravel. This difference is an effective antipredator response; G. roeseli is able to hide efficiently in the interstices of the pebbles. Juvenile burbot have a low foraging efficiency on pebbles and preferentially forage in the interstices of coarser sediments. G. roeseli responded differently to kairmones in different sediment types. The amphipods decreased their activity only in pebbles. In gravel, the rate of exploration behavior increased, and on a bare surface, the area containing fish kairomones was avoided.     

Optimization of activated carbon‐based decontamination of fish oil by response surface methodology

The effect of activated carbon (AC) adsorption on the reduction of persistent organic pollutants (POP) in fish oil was studied based on response surface methodology at a 5‐g/kg AC inclusion level. Pretreatment of the oil by alkali refining and bleaching increased the POP levels. The tested process variables (contact time and temperature) affected the AC adsorption rate and significant first‐ and second‐order response models could be established. Polychlorinated dibenzo‐p‐dioxins and dibenzofurans (PCDD/F) showed a very rapid adsorption behavior and the concentration and toxic equivalent (TEQ) level could be reduced by 99%. Adsorption of dioxin‐like polychlorinated biphenyls (DL‐PCB) was less effective and depended on ortho substitution, i.e. non‐ortho PCB were adsorbed more effectively than mono‐ortho PCB with a maximum of 87 and 21% reduction, respectively, corresponding to a DL‐PCB‐TEQ reduction of 73%. A common optimum for both PCDD/F and DL‐PCB adsorption could not be identified. AC treatment had no effect on the level of polybrominated diphenyl ether flame retardants. The differences in adsorption patterns may be explained based on molecular conformation. No change in oil quality could be observed based on oxidation parameters. Compliance with present PCDD/F and DL‐PCB legislation levels in fish oil can be achieved based on AC adsorption.     

Eicosapentaenoic acid,but not docosahexaenoic acid,increases mitochondrial fatty acid oxidation and upregulates 2,4-dienoyl-CoA reductase gene expression in rats

The aim of the present study was to investigate whether eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) was responsible for the triglyceride-lowering effect of fish oil. In rats fed a single dose of EPA as ethyl ester (EPA-EE), the plasma concentration of triglycerides was decreased at 8 h after acute administration. This was accompanied by an increased hepatic fatty acid oxidation and mitochondrial 2,4-dienoyl-CoA reductase activity. The steady-state level of 2,4-dienoyl-CoA reductase mRNA increased in parallel with the enzyme activity. An increased hepatic long-chain acyl-CoA content, but a reduced amount of hepatic malonyl-CoA, was obtained at 8 h after acute EPA-EE treatment. On EPA-EE supplementation, both EPA (20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3) increased in the liver, whereas the hepatic DHA (22:6n-3) concentration was unchanged. On DHA-EE supplementation retroconversion to EPA occurred. No statistically significant differences were found, however, for mitochondrial enzyme activities, malonyl-CoA, long-chain acyl-CoA, plasma lipid levels, and the amount of cellular fatty acids between DHA-EE treated rats and their controls at any time point studied. In cultured rat hepatocytes, the oxidation of [1-¹⁴C]palmitic acid was reduced by DHA, whereas it was stimulated by EPA. In thein vivo studies, the activities of phosphatidate phosphohydrolase and acetyl-CoA carboxylase were unaffected after acute EPA-EE and DHA-EE administration, but the fatty acyl-CoA oxidase, the rate-limiting enzyme in peroxisomal fatty acid oxidation, was increased after feeding these n-3 fatty acids. The hypocholesterolemic properties of EPA-EE may be due to decreased 3-hydroxy-3-methylglutaryl-CoA reductase activity. Furthermore, replacement of the ordinary fatty acids, i.e., the monoenes (16:1n-7, 18:1n-7, and 18:1n-9) with EPA and some conversion to DPA concomitant with increased fatty acid oxidation is probably the mechanism leading to changed fatty acid composition. In contrast, DHA does not stimulate fatty acid oxidation and, consequently, no such displacement mechanism operates. In conclusion, we have obtained evidence that EPA, and not DHA, is the fatty acid primarily responsible for the triglyceride-lowering effect of fish oil in rats.     

Water Sorption and Glass Transition Temperature of Spray-Dried Mussel Meat Protein Hydrolysate

The water sorption behavior at 25°C and glass transition temperature (T _g ) of mussel meat protein hydrolysate powder without and with maltodextrin 10 DE or gum arabic at 15 and 30% (w/w) were studied in this work. The sorption isotherms were determined by the gravimetric method, and the glass transition temperature was obtained by differential scanning calorimetry (DSC) after powder conditioning at various water activities. Sorption isotherms data were well fitted by a modified Brunauer-Emmett-Teller (BET) model. Powder without additives showed the highest water adsorption followed by those produced with 15 and 30% of carrier agent, respectively. The Gordon-Taylor model was able to predict the effect of water on the glass transition temperature. At 25°C, the critical water content that ensured the glassy state of the mussel hydrolysate powder during storage increased from 0.05 to 0.12 g water/g product and the critical water activity increased from 0.24 to 0.60 when the concentration of carrier agents was increased to 30%.     

Marine mussel adhesion: biochemistry,mechanisms, and biomimetics

Common blue mussel (Mytilus edulis) is a sessile organism that has unique ability to attach to a wide array of organic and inorganic marine surfaces using its holdfast structures. Strong adhesion to surfaces is essential for mussel survival, movement, and self-defense. Mussel proteins from byssal thread are structural components connecting soft mussel tissues to marine surfaces via an adhesive plaque in the distal end, while adhesive proteins from byssal plaque are responsible for mussel adhesion. Adhesive proteins are small molecules containing a high proportion of post-translationally modified amino acids such as 3,4-dihydroxyphenylalanine (DOPA). High DOPA content, small molecular size, protein flexibility, the presence of metal ions, and a high oxidation state enable strong mussel adhesion to surfaces. Mussel adhesion mechanisms depend on the composition and interactions of mussel proteins, as well as their interactions with the environment. Difficulties in the extraction of mussel adhesion proteins hamper mechanism studies and their practical applications. Development of recombinant mussel proteins and biomimetics will advance our understanding of adhesion mechanisms. In this paper, recent advances in the characterization of mussel adhesive proteins (MAPs), mussel adhesion mechanisms, application of MAPs, and the development of biomimetic biopolymers are reviewed.     

Fatty acid composition of wild and cultivated gilthead seabream (Sparus aurata) and sea bass (Dicentrarchus labrax)

Total lipid content and fatty acid composition were determined for wild and cultivated gilthead seabream and sea bass. Fatty acid analyses were carried out by gas chromatography‐ mass spectrometry. Respective total lipid content of flesh in cultivated gilthead seabream and sea bass were 1.7‐5.0‐times those of wild samples. Palmitic acid (C16:0) and oleic acid (C18:1n‐9) were the major fatty acids respectively among the saturated fatty acids and monounsaturated fatty acids of each fish species. It is noteworthy that both linoleic acid (C18:2n‐6) and arachidonic acid (C20:4n‐6) were predominant in total n‐6 polyunsaturated fatty acids in the respective cultivated and wild types. Eicosapentaenoic acid (C20:5n‐3) and docosahexaenoic acid (C22:6n‐3) amounts were significantly higher in flesh of cultivated fish than in wild fish.