Amaia Orbea

Antioxidant enzymes and peroxisome proliferation in relation to contaminant body burdens of PAHs and PCBs in bivalve molluscs, crabs and fish from the Urdaibai and Plentzia estuaries (Bay of Biscay)

With the aim of studying levels of antioxidant and peroxisomal enzymes and the structure of peroxisomes in relation to body burdens of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), mussels Mytilus galloprovincialis, oysters Crassostrea sp., crabs Carcinus maenas and mullets Mugil cephalus were sampled in two Basque estuaries (Bay of Biscay): Urdaibai (Laida, Txatxarramendi, Arteaga, and downstream a sewage treatment plant-STP) and Plentzia. In general, animals showed higher concentrations of contaminants in winter than in summer and no relevant differences were detected among locations. Conversely, antioxidant enzyme activities were higher in summer. Enzyme expression was studied in mullets using immunochemical methods. By immunoblotting season-dependent differences were detected for Mn-superoxide dismutase (Mn-SOD). As for the immunohistochemical staining, mullets sampled in summer in Plentzia showed significantly higher optical densities for acyl-CoA oxidase and lower for both Cu,Zn-SOD and Mn-SOD than those collected downstream a STP as well as higher catalase immunostaining than those collected in winter. Peroxisomal volume density (V(vp)) of mussels sampled in Laida and Txatxarramendi did not show seasonal variations, while for oysters collected in Laida and Arteaga V(vp) was higher in summer. Crab and mullet V(vp) were also higher in summer. In conclusion, the estuaries of Urdaibai and Plentzia can be considered as low to moderately polluted areas and levels of PAHs and PCBs do not show marked variations apart from seasonal variations. Animals can be adapted to low pollution conditions and, under these circumstances, seasonal factors might affect biomarker responses to a greater extent than pollution variations.

Immunolocalization of four antioxidant enzymes in digestive glands of mollusks and crustaceans and fish liver

Abstract. The aim of this work was to determine the immunolocalization of the antioxidant enzymes catalase, Cu,Zn-superoxide dismutase (SOD), Mn-SOD, and glutathione peroxidase (GPX) in the bivalve mollusks Mytilus galloprovincialis and Crassostrea sp., the crab Carcinus maenas, and the teleostean fish Mugil cephalus. By immunoblotting, crossreactivity between antibodies and the corresponding proteins in the digestive gland/hepatopancreas of invertebrates and the fish liver was demonstrated. Immunohistochemical studies showed that the stomach epithelium was strongly immunostained for catalase in mollusks. In crabs, ducts showed stronger immunostaining than tubules and in mullet hepatocytes the reaction appeared in discrete granules corresponding to peroxisomes. With regard to Cu,Zn-SOD, the apex of the tubule cells in mussels and crabs was distinctly immunostained, whereas in oysters the reaction was more marked in ducts and in mullet liver a uniform diffuse cytoplasmic staining was found. Mn-SOD was strongly positive in mollusk and crab ducts and in mullet periportal hepatocytes. Finally, GPX was not detected in mussels while in oysters a slight reaction was noted in all cell types. In crabs, connective tissue cells and the apex of duct cells were immunostained, but in mullet liver only erythrocytes appeared reactive. Immunoelectron microscopy revealed that catalase was localized in peroxisomes with a dense labeling in fish and less intense labeling in invertebrates. Cu,Zn-SOD was mainly a cytosolic protein although additional positive subcellular sites (peroxisomes, nuclei) were also observed, while Mn-SOD was restricted to mitochondria. GPX was localized in the cytosol, nucleus, and lysosomes, occurring also in peroxisomes of the fish liver. The results presented here provide a basis for future application of the immunodetection techniques to study the possible differential induction of antioxidant enzymes in aquatic organisms subjected to oxidative stress as a result of exposure to environmental pollutants.

Structural changes in the digestive lysosomal system of sentinel mussels as biomarkers of environmental stress in mussel-watch programmes

Abstract A field study has been carried out to validate the measure of structural changes in the digestive lysosomal system of sentinel mussels as biomarkers of environmental stress. Previous laboratory studies demonstrated that the digestive lysosomal system of molluscs reponds to a variety of pollutants and to different stress situations by exhibiting significant changes in its structure. Mussels were collected monthly over 1 year at the Abra estuary (Bizkaia, Biscay Bay) from six sites with different degrees of pollution. The changes in the structure of the digestive lysosomes were quantified on cryostat sections of the digestive gland by means of automated image analysis. Four stereological parameters were recorded: lysosomal volume density, surface density, surface-to-volume ratio and numerical density. A seasonal pattern in the structure of the digestive lysosomes was evidenced with reduced volume, surface, size and numbers of lysosomes in winter-spring; increased volume, surface, size and numbers in summer and an intermediate situation in autumn. The structure of digestive lysosomes was also dissimilar among sites, the most significant differences being found between Plentzia (nonpolluted site) and Galea (polluted site). The digestive lysosomes of mussels collected from Galea were smaller and more abundant than in Plentzias mussels in most sampling times. The basis of these differences are discussed to conclude that organic chemical pollution might be the cause for these specific changes which are different from the enlargement of digestive lysosomes described as a result of various sources of environmental stress. It is concluded that structural changes in the digestive lysosomes of sentinel mussels are sensitive to pollution-induced environmental stress even in the complex situation of the field where many factors may interact to affect the structure of the digestive lysosomal system.

Chemical, biochemical and cellular responses in the digestive gland of the mussel Mytilus galloprovincialis from the Spanish Mediterranean coast

Mussels (Mytilus galloprovincialis) were sampled in March 1996 from five stations along the Western Mediterranean coast (Barcelona, Ebro Delta, Alboraya, Cullera, Denia) corresponding to urban, industrial and agricultural areas. Different biochemical and cellular markers were determined in the mussels in order to assess the effects and/or exposure to pollutants. The cytochrome P450 system, acetylcholinesterase and metallothioneins were among the biochemical markers selected for the study. Histochemical analysis of ß-glucuronidase and catalase activity were performed as marker enzymes for lysosomes and peroxisomes. Chemical analyses indicated that mussels from Barcelona and Denia as highly exposed to polycyclic aromatic hydrocarbons (PAHs)(1.8-2.7 µg g(-1) w.w. against 0.02-0.10 µg g(-1) w.w.), and polychlorinated biphenyls (PCBs)(132-260 ng g(-1) w.w. against 8-24 ng g(-1) w.w.). This was in agreement with changes in lysosome structure and higher number of peroxisomes in those organisms. High levels of metals (particularly Cr and Cu) were recorded in the digestive gland of Alboraya mussels, which also had elevated metallothionein content (28 nmol g(-1) w.w.) in comparison with the other stations (15-20 nmol g(-1) w.w.). Benzo(a)pyrene hydroxylase (BPH) activity indicated Cullera and Barcelona as possibly polluted sites. The results support the usefulness of the biomarker approach to assess and diagnose environmental pollution. The use of a battery of biomarkers at different levels of biological organization coupled with chemical analysis is highlighted.Mussels (Mytilus galloprovincialis) were sampled in March 1996 from five stations along the Western Mediterranean coast (Barcelona, Ebro Delta, Alboraya, Cullera, Denia) corresponding to urban, industrial and agricultural areas. Different biochemical and cellular markers were determined in the mussels in order to assess the effects and/or exposure to pollutants. The cytochrome P450 system, acetylcholinesterase and metallothioneins were among the biochemical markers selected for the study. Histochemical analysis of ß-glucuronidase and catalase activity were performed as marker enzymes for lysosomes and peroxisomes. Chemical analyses indicated that mussels from Barcelona and Denia as highly exposed to polycyclic aromatic hydrocarbons (PAHs)(1.8-2.7 µg g-1 w.w. against 0.02-0.10 µg g-1 w.w.), and polychlorinated biphenyls (PCBs)(132-260 ng g-1 w.w. against 8-24 ng g-1 w.w.). This was in agreement with changes in lysosome structure and higher number of peroxisomes in those organisms. High levels of metals (particularly Cr and Cu) were recorded in the digestive gland of Alboraya mussels, which also had elevated metallothionein content (28 nmol g-1 w.w.) in comparison with the other stations (15-20 nmol g-1 w.w.). Benzo(a)pyrene hydroxylase (BPH) activity indicated Cullera and Barcelona as possibly polluted sites. The results support the usefulness of the biomarker approach to assess and diagnose environmental pollution. The use of a battery of biomarkers at different levels of biological organization coupled with chemical analysis is highlighted.Mussels (Mytilus galloprovincialis) were sampled in March 1996 from five stations along the Western Mediterranean coast (Barcelona, Ebro Delta, Alboraya, Cullera, Denia) corresponding to urban, industrial and agricultural areas. Different biochemical and cellular markers were determined in the mussels in order to assess the effects and/or exposure to pollutants. The cytochrome P450 system, acetylcholinesterase and metallothioneins were among the biochemical markers selected for the study. Histochemical analysis of s-glucuronidase and catalase activity were performed as marker enzymes for lysosomes and peroxisomes. Chemical analyses indicated that mussels from Barcelona and Denia as highly exposed to polycyclic aromatic hydrocarbons (PAHs)(1.8-2.7 µg g-1 w.w. against 0.02-0.10 µg g-1 w.w.), and polychlorinated biphenyls (PCBs)(132-260 ng g-1 w.w. against 8-24 ng g-1 w.w.). This was in agreement with changes in lysosome structure and higher number of peroxisomes in those organisms. High levels of metals (pa...

Combined use of native and caged mussels to assess biological effects of pollution through the integrative biomarker approach.

Native and caged mussels were used in combination for the monitoring of pollution biological effects through an integrative biomarker approach. Mussels (Mytilus galloprovincialis) were deployed in cages in two well-known model localities with different pollution levels in the Basque coast. After 3 weeks caged and native mussels were collected from each site and a suite of effect and exposure biomarkers (from molecular/cellular to organism level) was applied and chemical contaminants (metals, PAHs, PCBs, phthalates and nonylphenol ethoxylates) were analytically determined. Integrative biomarker indices and pollutant indices of tissues were calculated. Several biomarkers used herein responded similarly in native and caged mussels, whereas others exhibited significant differences. Overall, biomarkers in-a-suite depicted site-specific profiles useful for the diagnostic of mussel health status and therefore for ecosystem health assessment in marine pollution biomonitoring. On the other hand, biomarkers and bioaccumulation exhibited different response times, which was especially evident when comparing biomarker and pollutant indices of tissues. The suite of biomarkers was more sensitive after caging (short-term response), whereas tissue pollutant concentrations were more sensitive in native mussels (long-term response). Thus, the combination of native and caged mussels is highly recommended to monitor biological effects of pollution in mussels through the integrative biomarker approach, especially in chronically polluted sites.

Effects of exposure to prestige-like heavy fuel oil and to perfluorooctane sulfonate on conventional biomarkers and target gene transcription in the thicklip grey mullet Chelon labrosus.

Thicklip grey mullets Chelon labrosus inhabit coastal and estuarine areas where they can be chronically exposed to commonly released pollutants such as polycyclic aromatic hydrocarbons (PAHs) and perfluorinated compounds. These pollutants can also originate from accidental spills, such as the Prestige oil spill in 2002, which resulted in the release of a heavy fuel oil that affected coastal ecosystems in the Bay of Biscay. Peroxisome proliferation (PP), induced biotransformation metabolism, immunosuppression and endocrine disruption are some of the possible biological effects caused by such chemicals. With the aim of studying the effects of organic toxic chemicals on such biological processes at the transcriptional and at the cell/tissue level, juvenile mullets were exposed to the typical mammalian peroxisome proliferator perfluorooctane sulfonate (PFOS), and to fresh (F) and weathered (WF) Prestige-like heavy fuel oil for 2 and 16 days. First, fragments of genes relevant to biotransformation, immune/inflammatory and endocrine disruption processes were cloned using degenerate primers. Fuel oil elicited a significant PP response as proved by the transcriptional upregulation of palmitoyl-CoA oxidase (aox1), peroxisome proliferator activated receptor alpha (pparalpha) and retinoic X receptor, by the AOX1 activity induction and by the increased peroxisomal volume density. PFOS only elicited a significant induction of AOX1 activity at day 2 and of PPARalpha mRNA expression at day 16. All treatments significantly increased catalase mRNA expression at day 16 in liver and at day 2 in gill. Cyp1a transcription (liver and gill) and EROD activity were induced in fuel oil treated organisms. In the case of phase II metabolism only hepatic glutathione S-transferase mRNA was overexpressed in mullets exposed to WF for 16 days. Functionally, this response was reflected in a significant accumulation of bile PAH metabolites. WF treated fish accumulated mainly high molecular weight metabolites while F exposure resulted in accumulation of mainly low molecular ones. Fuel oil significantly regulated immune response related complement component C3 and hepcidin transcription followed by a significant regulation of inflammatory response related apolipoprotein-A1 and fatty acid binding protein mRNAs at day 16. These responses were accompanied by a significant hepatic inflammatory response with lymphocyte accumulations (IRLA) and accumulation of melanomacrophage centers (MMC). PFOS did not elicit any transcriptional response in the studied biotransformation and immune related genes, although histologically significant effects were recorded in IRLA and MMC. A significant reduction of lysosomal membrane stability was observed in all exposed animals. No endocrine disruption effects were observed in liver while brain aromatase mRNA was overexpressed after all treatments at day 2 and estrogen receptor alpha was downregulated under WF exposure at day 16. These results show new molecular and cellular biomarkers of exposure to organic chemicals and demonstrate that in mullets PP could be regulated through molecular mechanisms similar to those in rodents, although the typical mammalian peroxisome proliferator PFOS and heavy fuel oil follow divergent mechanisms of action.

Marine ecosystem health status assessment through integrative biomarker indices: a comparative study after the Prestige oil spill “Mussel Watch”

Five integrative biomarker indices are compared: Bioeffects Assessment Index (BAI), Health Status Index (HSI), integrated biological response (IBR), ecosystem health condition chart (EHCC) and Integrative Biomarker Index (IBI). They were calculated on the basis of selected biomarker data collected in the framework of the Prestige oil spill (POS) Mussel Watch monitoring (2003–2006) carried out in Galicia and the Bay of Biscay. According to the BAI, the health status of mussels was severely affected by POS and signals of recovery were evidenced in Galicia after April-04 and in Biscay Bay after April-05. The HSI (computed by an expert system) revealed high levels of environmental stress in 2003 and a recovery trend from April-04 to April-05. In July-05, the health status of mussels worsened but in October-05 and April-06 healthy condition was again recorded in almost all localities. IBR/n and IBI indicated that mussel health was severely affected in 2003 and improved from 2004 onwards. EHCC reflected a deleterious environmental condition in 2003 and a recovery trend after April-04, although a healthy ecosystem condition was not achieved in April-06 yet. Whereas BAI and HSI provide a basic indication of the ecosystem health status, star plots accompanying IBR/n and IBI provide complementary information concerning the mechanisms of biological response to environmental insult. Overall, although the integrative indices based on biomarkers show different sensitivity, resolution and informative output, all of them provide coherent information, useful to simplify the interpretation of biological effects of pollution in marine pollution monitoring. Each others’ advantages, disadvantages and applicability for ecosystem health assessment are discussed.

Interactive effects of benzo(a)pyrene and cadmium and effects of di(2-ethylhexyl) phthalate on antioxidant and peroxisomal enzymes and peroxisomal volume density in the digestive gland of mussel Mytilus galloprovincialis Lmk

Exposure of marine animals to certain organic and metal pollutants is thought to enhance reactive oxygen species (ROS) production with concomitant alterations of antioxidant defence mechanisms. Some of these organic pollutants cause peroxisome proliferation, a process resulting also in possible enhanced production of ROS. The aim of this study was to investigate the effects of two organic xenobiotics, benzo(a)pyrene (B(a)P) and di(2-ethylhexyl)phthalate (DEHP), as well as the effects of cadmium (Cd), on antioxidant and peroxisomal enzymes and on peroxisomal volume density in the digestive gland of mussel, Mytilus galloprovincialis Lmk., experimentally exposed for 21 days. Special attention was paid to the interactive effects of organic and metal compounds by exposing one group of mussels to a mixture of B(a)P and Cd. Exposure of mussels to Cd caused a decrease in superoxide dismutase (SOD) activity, in Mn-SOD protein levels and in volume density of peroxisomes. B(a)P exposure significantly increased catalase and glutathione peroxidase (GPX) and inhibited Mn-SOD after 21 days of exposure. B(a)P also caused a slight increase in acyl-CoA oxidase (AOX) activity and peroxisomal volume density after 21 days of exposure. Cd tended to inhibit changes provoked by B(a)P, indicating that responses to organic xenobiotics can be modulated by concomitant exposure to metal contaminants. Exposure to DEHP increased catalase and AOX and inhibited SOD activity and Mn-SOD protein levels. In conclusion, peroxisome proliferation, measured as an increase of the peroxisomal enzymes catalase and AOX (up to 1.53-fold for AOX), is a specific response to organic contaminants such as B(a)P and DEHP, whereas Cd does not cause peroxisome proliferation. Thus, peroxisome proliferation may be a specific biomarker of organic pollutants in mussels. Both organic and metal pollutants inhibited SOD activity and protein levels (up to 0.21-fold for Mn-SOD protein levels), the latter offering potential as general marker of pollution.

Peroxisome Proliferation in the Digestive Epithelium of Mussels Exposed to the Water Accommodated Fraction of Three Oils

Abstract Recent studies show that the peroxisomal marker enzyme catalase is induced in the digestive epithelium of mussels exposed to petroleum-derived hydrocarbons. The aim of the present study was to investigate whether this effect is accompanied by a proliferative response of the peroxisomes. Mussels were treated for 21, 49 and 91 days with three different doses of the water accommodated fraction (WAF) of two different crude oils (Ural and Maya types) and of one commercial lubricant oil. The volume density, surface density, surface-to-volume ratio and numerical density of peroxisomes were quantified by stereology on cryostat sections of digestive glands stained for the histochemical demonstration of catalase activity. The results show that after 21 days, the three WAFs act as typical peroxisome proliferators, inducing a significant increase in the volume, surface and numerical densities of peroxisomes in the digestive epithelium. The response is diminished or completely abolished after 49 and 91 days of exposure to the WAFs. The ability to induce the proliferative response differs depending on the WAF type, the lubricant oil showing the maximal induction ability. These results suggest that peroxisome proliferation could be used as a diagnostic biomarker for both exposure and effects of petroleum hydrocarbons on marine biota.

Assessing the effects of treated and untreated urban discharges to estuarine and coastal waters applying selected biomarkers on caged mussels

To assess effects of urban discharges, biomarkers were measured in caged mussels in northern Iberian Peninsula. Lysosomal membrane stability and histopathology of gonad and digestive gland were analysed as general effect biomarkers. Exposure to specific pollutants was evaluated by autometallographical detection of metals, peroxisomal acyl-CoA oxidase activity, micronucleus test and transcription levels of vitellogenin and MT20 genes. Health status of mussels was impaired after 3 days of caging at the untreated outfall discharge and at the waste water treatment plant effluent discharge to the estuary. The most relevant finding was the significant up-regulation of vitellogenin gene transcription in male mussels exposed to the untreated outfall discharge. Metals and xenoestrogenic endocrine disruptors were bioavailable in some discharges and disturbed the health status of mussels. Biomarkers were effective in the assessment of effects of urban discharges and could be implemented in operative controls required to assess the risks associated to effluent discharges.