Lysosomal responses are widely accepted cellular effect biomarkers of general stress. Up to now, these biomarkers have been analysed by means of conventional techniques based on enzyme histochemical ...methods, where lysosomal enzymes such as acid phosphatase and β-glucuronidase (β-GUS) have been employed as markers of lysosomes. The aim of the present work was to develop more advanced and sensitive methods based on the use of polyclonal antibodies to measure lysosomal enzymes in different sentinel organisms. For this purpose, we have studied the cross-reactivity of two commercial polyclonal antibodies against the lysosomal enzymes acid phosphatase and β-GUS with molluscan digestive gland by means of immunoblotting and immunohistochemistry. The antibody against acid phosphatase cross-reacted specifically with the lysosomal fraction of the digestive gland, while unspecific immunoreaction occurred with digestive gland whole homogenates and tissue sections. The antibody against β-GUS cross-reacted specifically with digestive gland whole homogenates and tissue sections. The cross-reactivity of this antibody was tested also in crab hepatopancreas and mullet liver where the same successful results were obtained. The second aim of the present study was to test if the immuno-based approach was sensitive enough to detect lysosomal alterations provoked by contaminants. For this purpose two experiments were carried out with mussels treated with cadmium in two ways: in vivo treatment by injection and in vitro treatment using digestive gland explants. Afterwards immunoblotting studies with the antibody against β-GUS were applied and immunoreactive bands were quantified by means of a gel analysis programme. We found that β-GUS protein levels were higher in treated mussels when compared with controls in either in vivo or in vitro treatments. All these data suggest that the polyclonal antibody against β-GUS is adequate to be used in immuno-based approaches to detect contaminant-induced lysosomal alterations.
Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors involved in embryo development and differentiation of several tissues in mammals. The ...aim of the present study was to investigate the possible differential expression of the three PPAR subtypes (PPARalpha, PPARbeta, and PPARgamma) in relation to gender and developmental stage in zebrafish. For this purpose PPAR expression was assessed by immunohistochemistry in 7-day-old larvae, 1-month-old juveniles, and 1-year-old adults. Additionally, the activity of peroxisomal acyl-CoA oxidase (AOX), a gene regulated by PPARs, and the volume density of catalase-immunolabeled liver peroxisomes (V(VP)) was examined. No significant gender-related differences were detected in the tissue distribution of the three PPAR subtypes or in peroxisomal AOX activity and V(VP). The percentage of PPARbeta-positive hepatocytes was significantly higher in females than in males suggesting a specific regulatory role of this subtype in female zebrafish. The three PPAR subtypes were already expressed at the larval stage, with a similar tissue distribution pattern to that found in adults. For all stages, PPARalpha and PPARgamma were expressed at higher levels than PPARbeta, and PPARbeta immunolabeling was stronger in juveniles than in larval or adult stages. The percentages of hepatocyte nuclei immunolabeled for PPARs was higher in early developmental stages than in adults, similarly to AOX activity and V(VP). In conclusion, our results indicate that PPAR expression, the activity of its target gene AOX, and peroxisomal biogenesis are developmentally modulated in zebrafish.
Environmental estrogenic compounds or xenoestrogens can mimic natural estrogens and cause a variety of adverse effects on aquatic wildlife. The purpose of the present work was to investigate if ...xenoestrogens are able to cause proliferation of liver peroxisomes using zebrafish (
Danio rerio) as a model. Adult male zebrafish were exposed for 15 days to 17
β-estradiol (E2) and the xenoestrogens dibutylphthalate (DBP), methoxychlor (MXC), 4-tert-octylphenol (OP) and 17
α-ethynylestradiol (EE2). All five tested compounds caused significant proliferation of liver peroxisomes (
p
<
0.05) as indicated by increased peroxisomal surface and numerical densities and elevated activities of the peroxisomal
β-oxidation enzyme acyl-CoA oxidase (AOX). In the case of DBP, MXC and E2, positive significant correlations between peroxisomal density parameters and AOX were found. The treatments did not produce gross alterations in testis histology, but spermatogenic cell proliferation was disturbed in E2 and EE2-treated groups and vitellogenin levels increased significantly in fish exposed to MXC, OP, EE2 and E2 with respect to controls. Furthermore, a significant correlation between vitellogenin levels and AOX activity was found for MXC, OP and EE2 treatments, suggesting that for the latter xenoestrogens early estrogenic effects are associated with liver peroxisome proliferation. No such association occurred with typical peroxisome proliferators such as DBP.
Our aim was to contribute to the understanding of the synthesis, maturation and activation of lysosomal enzymes in an invertebrate cellular model: the endo-lysosomal system (ELS) of mussel digestive ...cells. The activities of 5'-nucleotidase (AMPase), arylsulphatase (ASase) and acid phosphatase (AcPase), which are transported towards acidic compartments as membrane proteins, were localised by enzyme cytochemistry. AcPase activity was found within large heterolysosomes and residual bodies. ASase was located in endosomes, endolysosomes and heterolysosomes. AcPase and ASase activities were recorded within small vesicles and cisterns of the trans-Golgi network. Conversely, AMPase activity was primarily found in microvilli and apical vesicles and, less conspicuously, in lysosomes and the cis-side of the Golgi and the cis-Golgi network (CGN). In order to understand the processes of synthesis and maturation of these lysosomal enzymes, selected glycoconjugates were localised after lectin cytochemistry. N-acetylglucosamine, mannose and fucose residues were almost ubiquitous in the ELS, as were galactose residues, which were apparently less abundant. N-acetylglucosamine residues occurred in the inner membrane co-localised with mannose residues within the lysosomal and pre-lysosomal acidic compartments. Based on these results, glycosylation and sorting pathways are proposed for both soluble and membrane enzymes. Unlike in mammalian cells, O-glycosylation is fully completed in the CGN, mannose addition in N-glycosylation extends beyond the CGN and galactose addition is fully achieved at the intermediate side. Sorting of soluble lysosomal enzymes, as in crustaceans, is mediated by the indirect transport of membrane-linked proteins with GlcNAc1-P6Man residues that are removed in endolysosomes and heterolysosomes.
Mussels, Mytilus galloprovincialis, were collected from six coastal sites of dissimilar water quality (Zierbena, Santurtzi, Arrigunaga, Galea, Meñakoz, and Plentzia) at Biscay Bay in September 1991, ...January 1992, June 1992, and September 1992. The extent of hemocyte infiltration in connective tissue of the digestive gland was quantified by stereology on histological sections in terms of volume density of hemocytes (HVD). HVD was elevated in mussels collected from Plentzia (the less polluted site) in January 1992 and September 1992, while such increases occurred in January 1992 in Santurtzi and Arrigunaga and in September 1991 and September 1992 in Galea. Conversely, HVD was reduced in Arrigunaga in September 1991 and in Galea in January 1992. Moreover, HVD was kept unchanged through the year in mussels collected from Meñakoz and Zierbena. On the basis of this preliminary in vivo study, hemocytic activities of mussels collected in September 1994 from Arrigunaga and Plentzia were further investigated by means of four in vitro immunotoxicity assays: (a) the trypan blue exclusion assay, indicative of cell viability; (b) the zymosan phagocytosis assay, indicative of phagocytic activity; (c) the diaminobenzidine-manganese (DAB-Mn2+) oxidation assay for estimating reactive oxygen intermediate (ROI) production; and (d) the neutral red (NR) uptake assay, indicative of endocytic ability. These in vitro tests indicated some significant differences between Plentzia and Arrigunaga. Hemocytes from mussels collected in Plentzia exhibited a higher capability to phagocytose zymosan while, conversely, hemocytes from mussels collected in Arrigunaga endocytosed more NR and produced more ROI under nonstimulated conditions. These differences in the in vitro hemocytic activities of mussels from Plentzia (nonpolluted) and Arrigunaga (moderately polluted) suggest that in vitro assays may be used as biomarkers of environmental quality in coastal and estuarine areas.
In November 2002 the tanker ‘Prestige’ sunk in front of the Galician coast (NW Iberian Peninsula). As a result, >60 000 t of heavy fuel oil leaked into the sea, affecting >1000 km of coastline. In ...order to assess the effects of the oil spill on coastal ecosystems, musselsMytilus galloprovincialiswere sampled (April, July and September 2003) in 17 locations along the Galician and Bay of Biscay coasts. In this study, 3 biomarkers were assessed: lysosomal responses as changes in the lysosomal structure and in the lysosomal membrane stability, accumulation of intracellular neutral lipids and peroxisome proliferation as induction of acyl-CoA oxidase (AOX) activity. Mussel flesh condition index and gonad developmental stages were assessed as supporting parameters. Lysosomal membrane stability was reduced in mussels from all locations, indicating disturbed health, especially in mussels from all Galician locations. Similarly, lysosomal enlargement was observed in most locations, as shown by relatively low values of the surface-to-volume ratio, although the volume density of lysosomes was low due to decreased lysosomal numbers. Overall, intracellular accumulation of neutral lipids was conspicuous in digestive tubules of mussels collected in July and was increased further in September. AOX induction was detectable in mussels sampled in April, except for those collected in Galicia. In July mussels from the most impacted stations in Galicia, Caldebarcos and Camelle, showed the highest AOX values. In conclusion, the biomarkers employed detected exposure to toxic chemicals and a disturbed status of health in mussels from the northern Iberian Peninsula after the ‘Prestige’ oil spill and will allow assessment of the long-term effects of the spill on the coastal ecosystems.
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.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
In an attempt to improve the detection of peroxisome proliferation as a biomarker in environmental pollution assessment, we have applied a novel approach based on peroxisomal proteomics. Peroxisomal ...proteins from digestive glands of mussels Mytilus galloprovincialis were analyzed using 2‐DE and MS. We have generated a reference 2‐DE map from samples obtained in a well‐studied reference area and compared this with peroxisomal proteomes from other sequenced genomes. In addition, by comparing 2‐DE maps from control samples with samples obtained in a polluted area, we have characterized the peroxisome proliferation expression pattern associated with exposure to a polluted environment. Over 100 spots were reproducibly resolved per 2‐DE map; 55 differentially expressed spots were quantitatively detected and analyzed, and 14 of these showed an increase in protein expression of more than fourfold. Epoxide hydrolase, peroxisomal antioxidant enzyme, and sarcosine oxidase (SOX) have been identified by ESI MS/MS, and acyl‐CoA oxidase, multifunctional protein, and Cu,Zn‐superoxide dismutase were immunolocalized by Western blotting. Our results indicate that a peroxisomal protein pattern associated to marine pollutant exposure can be generated, and this approach may have a greater potential as biomarker than traditional, single‐protein markers.
Abstract
Metallothionein (MT) induction is widely used as a biomarker of exposure to metals in mussels. The aims of the present work were first to compare the suitability of spectrophotometry and ...differential pulse polarography (DPP) for MT detection in mussels exposed to 200 ppb cadmium for 9 days in a laboratory experiment and in mussels sampled in different seasons from expected pollution gradients along the Mediterranean Sea; second, to intercalibrate the widely used spectrophotometric method using mussels from Saronikos Gulf. In the intercalibration of the spectrophotometric method, similar results (p>0.05) were obtained by two different research teams indicating a good reproducibility of the technique. However, polarographic and spectrophotometric methods gave significantly (p<0.05) different results in laboratory and field studies. In the laboratory experiment, MT values detected with DPP were nine times higher than with spectrophotometry. The results obtained by the two methods were significantly correlated. Both methods could discriminate between control and exposed mussels. In field studies, MT values obtained by DPP were 34-38-fold higher than with spectrophotometry, and MT concentrations measured by both methods were not correlated. This discrepancy could be due to several factors, including the low levels of bioavailable metals in the studied areas and the possibility that the different methods can measure MT isoforms differentially. Further work is needed to decipher the functions of MT isoforms in mussels. This information is relevant for the application of MT as a biomarker in biomonitoring programmes.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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