The role of metallothionein and selenium in metal detoxification in the liver of deep-sea fish from the NW Mediterranean Sea.

Seven deep-sea fish species were sampled in the Blanes Canyon area (NW Mediterranean) at a depth of 1200 m during winter. The concentrations of nine metals were determined in the liver of these species by ICP-MS. Furthermore, the metal detoxification potential was determined for each species by analysing the hepatic metallothionein (MT) content, relations between metals and the molar ratio between MT and/or selected metals. The potential effect of metal content on their physiology was assessed using general stress markers such as the enzyme activities of acetylcholinesterase (AChE) and lactate dehydrogenase (LDH) in muscle. Levels of metals in the seven Mediterranean deep-sea fish species studied were intermediate to equivalent species of fish either from Atlantic waters or hydrothermal vents. The metal detoxification potential varied among species depending on MT, selenium (Se) or zinc (Zn) as reliable mechanisms to handle potential metal toxicity. The role of Se was especially relevant when the liver content of mercury (Hg) was higher. AChE and LDH activities did seem to be affected by metal loads and thus the activities reported would correspond to baseline activities of the selected species.

Muscular cholinesterase and lactate dehydrogenase activities in deep-sea fish from the NW Mediterranean.

Organisms inhabiting submarine canyons can be potentially exposed to higher inputs of anthropogenic chemicals than their counterparts from the adjacent areas. To find out to what extend this observation applies to a NW Mediterranean canyon (i.e. Blanes canyon) off the Catalan coast, four deep-sea fish species were collected from inside the canyon (BC) and the adjacent open slope (OS). The selected species were: Alepocephalus rostratus, Lepidion lepidion, Coelorinchus mediterraneus and Bathypterois mediterraneus. Prior to the choice of an adequate sentinel species, the natural variation of the selected parameters (biomarkers) in relation to factors such as size, sex, sampling depth and seasonality need to be characterised. In this study, the activities of cholinesterases (ChEs) and lactate dehydrogenase (LDH) enzymes were determined in the muscle of the four deep-sea fish. Of all ChEs, acetylcholinesterase (AChE) activity was dominant and selected for further monitoring. Overall, AChE activity exhibited a significant relationship with fish size whereas LDH activity was mostly dependent on the sex and gonadal development status, although in a species-dependent manner. The seasonal variability of LDH activity was more marked than for AChE activity, and inside-outside canyon (BC-OS) differences were not consistent in all contrasted fish species, and in fact they were more dependent on biological traits. Thus, they did not suggest a differential stress condition between sites inside and outside the canyon.

Comparative xenobiotic metabolism capacities and pesticide sensitivity in adults of Solea solea and Solea senegalensis.

The measurement of enzymatic activities involved in xenobiotic biotransformation was carried out in adults of Solea solea and Solea senegalensis. The hepatic enzymes analysed were cytochrome P450 (CYP) related activities using eight fluorometric substrates and carboxylesterases (CbE). The conjugating activities of glutathione S-transferase (GST) and UPD-glucuronosyltransferase (UDPGT) were also assessed. Specific mammalian inhibitors were used as diagnostic tools for related activities of CYP1A (α-naphthoflavone; αNF), CYP2B6 and CYP2C19 (ticlopidine) and CYP3A4 (ketoconazole). The in vitro sensitivity to organophosphorous pesticides (OP) was tested in the S10 homogenate of brain (acetylcholinesterase-AChE) and liver (CbE). Furthermore, the pesticide chlorpyrifos oxon (CLPO) was used to explore the OP sensitivity of CbE of both species in two subcellular fractions (microsomes and cytosol), using two substrates. Overall, only two parameters confirmed species differences: EROD and cytosolic CbE being significantly elevated (p < 0.05) in the common sole, S. solea. A high inhibition of CYP1A related activities using several fluorometric substrates (ER, MR and CEC) after in vitro incubation with αNF confirmed all measure CYP1A1-related activities whereas ketoconazole was more specific for BFCOD (CYP3A4). Pesticide sensitivity was similar for brain AChE but hepatic CbE had a protective role that was species and pesticide dependent.

Biliary PAH and alkylphenol metabolites, biomarker enzyme activities, and gene expression levels in the deep-sea fish Alepocephalus rostratus.

Biliary polycyclic aromatic hydrocarbon (PAH) and alkylphenol (AP) metabolites, hepatic gene expression, and corresponding enzyme activities were determined in the deep-sea fish Alepocephalus rostratus from two sites within the Mediterranean. Biliary metabolites included the hydroxylated PAH metabolites (OH-PAHs) 1-naphthol, 2-naphthol, 9-fluorenol, 9-phenanthrol, and 1-pyrenol and the APs 4-nonylphenol (NP) and 4-tert-octylphenol (OP). Five biomarker genes, namely, cytochrome P450 1A (CYP1A), vitellogenin (Vtg), catalase (CAT), Cu/Zn-superoxide-dismutase (Cu/Zn-SOD), and glutathione reductase (GR), were quantified using qRT-PCR. Moreover, corresponding enzyme activities (ethoxyresorufin-O-deethylase (EROD), CAT, SOD, and GR) were also determined. The ΣOH-PAHs detected ranged from 21.1 to 300.3 ng/g bile and were mainly composed of 1-naphthol. Both NP and OP metabolites were detected in all samples with concentrations ranging from 17.4 to 107.2 ng/g bile and 4.9 to 17.3 ng/g bile, respectively, and levels were significantly higher in samples from the western Mediterranean (WM) compared to those from the Catalan slope (CS). Accordingly, gene expression was significantly induced in male fish from the WM; however, these results were not reflected in enzyme activity levels. In particular, males caught at 2000 m in the WM exhibited 35-times higher Vtg levels compared to those from the CS, suggesting that endocrine-disrupting effects may potentially be occurring in such remote environments as the deep-sea.

Legacy and emergent persistent organic pollutants (POPs) in NW Mediterranean deep-sea organisms.

The levels and profiles of organochlorine (OC) contaminants, including polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethanes (DDTs), hexachlorocyclohexanes (HCHs) and penta- (PeCB) and hexachlorobenzene (HCB), as well as polybrominated diphenyl ethers (PBDEs) were determined in muscle samples of the deep-sea fish Alepocephalus rostratus, Coelorinchus mediterraneus and Lepidion lepidion and the red-shrimp Aristeus antennatus from the NW Mediterranean Sea. Mean PCB and DDT levels ranged from the highest concentrations in the fish A. rostratus (Σ(7)PCBs 6.93±0.71 ng/g w.w. and ΣDDTs 8.43±1.10 ng/g w.w.) to the lowest concentrations in the crustacean A. antennatus (Σ(7)PCBs 1.17±0.24 ng/g w.w. and ΣDDTs 2.53±0.26 ng/g w.w.). The concentrations of ΣHCHs and HCB were more than one order of magnitude lower, ranging from 0.07-0.36 ng/g w.w. and 0.03-0.15 ng/g w.w., respectively, while PeCB was only detected in a few samples. PBDE levels were approximately ten times lower than PCB and DDT concentrations, ranging from 0.47±0.20 ng/g w.w. in A. antennatus to 0.92±0.13 ng/g w.w. in A. rostratus. The high-molecular-weight PCBs 153, 138 and 180 represented 69-79% of Σ(7)PCBs in fish and 60% in the red shrimp. Moreover, in fish, the main DDT compound detected was the metabolite p,p'-DDE (70-80% of ΣDDTs), indicative of old DDT residues. In contrast, o,p'-DDE was the main DDT metabolite (49% of ΣDDTs) in shrimp, while the parent compound p,p'-DDT and its metabolite p,p'-DDE exhibited similar proportions of 16% and 21%, respectively. For PBDEs, the most abundant congeners were BDEs 28, 47, 99, 100 and 154 in fish (>70% Σ(14)PBDEs), while BDEs 153 and 209 were also important in A. antennatus, suggesting different uptake and/or biotransformation rates of PBDEs between fish and crustacea. In this sense, the ratios BDE 99/100, BDE 153/154, and BDE 47/99 were determined as proxies for BDE metabolization capacities and contrasted among species.

New insights into mercury bioaccumulation in deep-sea organisms from the NW Mediterranean and their human health implications.

A number of studies have found high levels of mercury (Hg) in deep-sea organisms throughout the world's oceans, but the underlying causes are not clear as there is no consensus on the origin and cycling of Hg in the ocean. Recent findings suggested that Hg accumulation may increase with increasing forage depth and pointed to the deep-water column as the origin of most Hg in marine biota, especially its organic methylmercury (MeHg) form. In the present study, we determined the total mercury (THg) levels in 12 deep-sea fish species and a decapod crustacean and investigated their relationship with the species' nitrogen stable isotope ratio (δ(15)N) as an indicator of their trophic level, average weight and habitat depth. THg levels ranged from 0.27 to 4.42 µg/g w.w. and exceeded in all, except one species, the recommended 0.5 µg/g w.w. guideline value. While THg levels exhibited a strong relationship with δ(15)N values and to a lesser extent with weight, the habitat depth, characterized as the species' depth of maximum abundance (DMA), had also a significant effect on Hg accumulation. The fish species with a shallower depth range exhibited lower THg values than predicted by their trophic level (δ(15)N) and body mass, while measured THg values were higher than predicted in deeper-dwelling fish. Overall, the present results point out a potential risk for human health from the consumption of deep-sea fish. In particular, for both, the red shrimp Aristeus antennatus, which is one of the most valuable fishing resources of the Mediterranean, as well as the commercially exploited fish Mora moro, THg levels considerably exceeded the recommended 0.5µg/g w.w. limit and should be consumed with caution.

Natural variability of hepatic biomarkers in Mediterranean deep-sea organisms.

Biomarker assays are widely used as proxies for contaminant-induced effects in aquatic organisms. However, in many cases, their intrinsic natural variability due to exogenous and endogenous factors makes the interpretation of biomarker data difficult. In the present study, we investigated the natural fluctuations of six hepatic biomarkers, namely ethoxyresorufin-O-deethylase (EROD) in fish and pentoxyresorufin-O-deethylase (PROD) in crustacea, catalase (CAT), carboxylesterase (CbE), glutathione-S-transferase (GST), total glutathione peroxidase (GPX) and glutathione reductase (GR) in two deep-sea fish species, namely Alepocephalus rostratus and Lepidion lepidion and the decapod crustacean Aristeus antennatus. The NW Mediterranean deep-sea environment is characterized by very stable temperature and salinity conditions, allowing the exclusion of these two factors as potential sources of interference with biomarker activities. Biomarker results exhibited a clear influence of reproductive processes on enzyme activities, in particular in A. rostratus, which presented a pronounced seasonal pattern linked to variations in the gonadosomatic index (GSI). In addition, other factors such as food availability may also have influenced the observed variability, in particular in specimens of L. lepidion, which did not exhibit variations in reproductive activity throughout the sampling period. Depth-related variability did not exhibit a clear trend and fluctuations across sampling depths were not attributable to any specific factor. Body size had also a significant influence on some biomarkers, although allometric scaling of certain enzyme activities appears to be species-specific. The present work has thus shown that despite the lack of fluctuations of abiotic parameters such as temperature and salinity, biomarker activities in deep-sea organisms still exhibit significant variability, mainly as a result of reproductive processes and food availability.

Differences in cytochrome P450 enzyme activities between fish and crustacea: relationship with the bioaccumulation patterns of polychlorobiphenyls (PCBs).

Variations in cytochrome P450 enzyme (CYPs) distribution and function between animal groups could result in differential metabolism and elimination kinetics for certain contaminants. Although a number of studies have suggested that differences in polychlorobiphenyl (PCB) accumulation profiles between crustacea and fish might result from differential CYP patterns, the relationship between PCB bioaccumulation and CYP capacities has not been demonstrated in these organisms. In the present study we investigated the hepatic microsomal catalytic activities in three deep-sea fish species, Alepocephalus rostratus (Alepocephalidae), Coelorinchus mediterraneus (Macrouridae), and Lepidion lepidion (Moridae), and the decapod crustacean Aristeus antennatus (Decapoda), using six fluorescent CYP-mediated substrates, namely ER (7-ethoxyresorufin), PR (7-pentoxyresorufin), BR (7-benzyloxyresorufin), CEC (3-cyano-7-ethoxycoumarin), DBF (dibenzylfluorescein) and BFC (7-benzyloxy-4-trifluoromethylcoumarin). Furthermore, we related the metabolic activities to the accumulation patterns of 41 PCB congeners in the muscle of these organisms. The results indicated a marked difference in the presence and activities of CYP isoforms between fish and the crustacean A. antennatus. Liver microsomes of the three selected fish species were capable of metabolizing all six CYP-mediated substrates and enzymes were identified as primarily belonging to CYP1A and CYP3A subfamilies. In contrast, hepatopancreas microsomes from A. antennatus only showed activity for PR and DBF substrates, generally related to mammalian CYP2-like enzymes. Furthermore, a direct relationship between metabolic activities and PCB accumulation profiles could be established. Results revealed that A. antennatus accumulated significantly higher proportions of PCBs 28, 52, 118, 138, 158 and 169 than fish, which is in accordance with the previously observed lack of CYP1A-like biotransformation capacities. Moreover, A. antennatus exhibited lower levels of PCBs 87, 149, 153, 170, 180, 183, 194 and 206 indicating that this crustacean is able to metabolize congeners considered mammalian CYP2B inducers. Hence, the present findings highlight the role of CYP-mediated metabolism in the congener-specific accumulation of PCBs in aquatic organisms and stress the need to further investigate quantitative and qualitative differences in xenobiotic metabolism among animal groups.

Two novel non-destructive biomarkers to assess PAH-induced oxidative stress and porphyrinogenic effects in crabs.

Two novel, non-destructive assays were developed to evaluate contaminant-induced lipid peroxidation (thiobarbituric acid-reacting substances, TBARS, levels) and haem biosynthesis disruption (porphyrin excretion) in decapod crabs. A laboratory experiment was conducted whereby pie-crust crabs (Cancer novaezelandiae) were fed cockles (Austrovenus stutchburyi) collected from a contaminated and reference site and TBARS levels and porphyrin excretion determined using fluorometric analysis in urine samples. Pyrene metabolite levels were also measured in the same urine samples to assess polycyclic aromatic hydrocarbon (PAH) exposure. Contaminant-exposed crabs exhibited elevated urinary TBARS and porphyrin levels and a strong correlation was found between these two assays and the urinary pyrene metabolite concentrations. However, there was large within-treatment variability, which precluded a clear separation between the control and the impacted group. Nevertheless, consistency in the direction of the response shows that the biomarkers reflect pollutant levels and validates the use of these simple techniques from human medicine for environmental assessments.

Non-destructive assessment of polycyclic aromatic hydrocarbon (PAH) exposure by fluorimetric analysis of crab urine.

The detection of urinary polycyclic aromatic hydrocarbon (PAH) metabolites by fluorescence spectrophotometry is particularly effective as a practical means to assess PAH exposure in decapod crabs. However, the practical application of this technique has thus far only been tested for the European shore crab (Carcinus maenas) and only a few field studies have been conducted in heavily polluted areas. The present study evaluated the adaptability of this method as a rapid, cost-effective and non-destructive biomonitoring tool for the New Zealand crab species, Macrophthalmus hirtipes (stalk-eyed mud crab). A field gradient could be detected among the sites and different input sources of PAH contamination could be discerned through the differentiation of pyrogenic and petrogenic PAH signatures. The present study shows that the fluorescence screening method is sensitive to relatively low levels of PAH contamination and more broadly applicable to smaller crab species than C. maenas, for which the technique was developed.