Biological activity of pentachlorophenol on the digestive gland cells of the freshwater mussel Unio tumidus

Many chlorinated phenols and their derivatives are used extensively as insecticides, fungicides and herbicides by industrial and agricultural users throughout the world. Among these substances, pentachlorophenol (PCP) is a broad-spectrum biocide, which is still used as a wood preservative. In this paper, the digestive gland cells were used to assess the effect of PCP in the range of concentrations 3.75-75 microM (0.01-0.2 ppm) on oxidative DNA damage, fluidity changes and peroxidation activity in the plasma membrane. The toxic property of PCP on DNA strand breakage was studied using the comet assay. The results showed that pentachlorophenol in the range of 37.5-75 microM contributed to these lesions. To demonstrate the changes in the fluidity of plasma membrane we used the spectrofluorimetric method using two fluorescence probes: 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 12-(9-anthroyloxy) stearic acid (12-AS). It was shown that PC did not influence the surface of plasma membrane but contributed to the increase in the fluidity of the internal region of the lipid bilayer in the range of concentrations 18.75-75 microM (0.05-0.2 ppm). We also examined the effect of PCP on the lipid peroxidation. To imply its peroxidation properties the spectrophotometry method was used to measure the level of malondialdehyde (MDA), one of the endpoints of the peroxidation of polyunsaturated fatty acids. The obtained results showed that PCP in the used doses did not initiate the formation of lipid peroxides. Thus, our investigation indicates that PCP can behave as a prooxidant agent but its action depends on the used doses and parameters chosen for the research.     

Response of digestive gland cells of freshwater mussel Unio tumidus to phenolic compound exposure in vivo

The exposure of freshwater mussels Unio tumidus to phenolic compounds (tannic, ellagic and gallic acid) in vivo caused changes in proteins and DNA function of digestive gland cells. The mussels were exposed to various concentrations of tested polyphenols (60, 200 and 500 microM) for 24 and 48 h and their antioxidant and pro-oxidant effects were determined. The number of SH-groups was quantified spectrophotometrically using Ellman's reagent. Oxidative modification of proteins increased in the digestive gland cells in a dose- and time-dependent manner. The level of nuclear DNA damage was investigated using the comet assay. The results revealed that polyphenolic acids induce single and double-strand breaks in DNA. The highest changes were observed for tannic and gallic acids and the smallest ones for ellagic acid. 1h of DNA repair process was also studied using the same method. The data obtained in this experiment demonstrate that the most effective DNA repair occurs in the cells exposed to phenolic compounds for 24h. A longer incubation (up to 48 h) does not decrease the capacity of the repair mechanism. The antioxidant activity of the tested phenols was analyzed spectrofluorimetrically using a fluorescence probe DCFH-DA (dichlorofluorescein-diacetate). The experimental data showed that the tested acids can act as antioxidants when used at higher doses (200 and 500 microM) against the reactive oxygen species present in the digestive gland cells. The most effective was ellagic acid, also applied at the smallest dose of 60 microM, in comparison with tannic and gallic acids. In conclusion, our results demonstrate that chosen water-soluble polyphenols, which are located in various plant tissues and are also found in the aquatic environment, can influence organisms living in the water. They can be exposed to these chemicals that cause morphological alterations and changes in certain physiological processes in their organs (i.e. digestive gland cells of bivalve molluscs).     

Antioxidant and pro-oxidant effects of tannins in digestive cells of the freshwater mussel Unio tumidus

Bivalve molluscs, particularly mussels, are sensitive biomarkers of aquatic ecosystem pollution. The tannins, water-soluble plant polyphenols, may play an important role in this environment and, mainly as a consequence of interaction with pollutants, their toxicity may change. We studied three naturally occurring compounds, tannic acid, ellagic acid and gallic acid, for their ability to modulate DNA damage produced by these tannins alone and in the presence of the oxidative stress inducer H(2)O(2), in cells of the digestive gland of mussels (Unio tumidus). After the treatment of the cells with polyphenols at different concentrations (1, 5, 15, 30, 60, 80, 100, 120, 180, 240 microM) and with hydrogen peroxide in the range of 0.04 and 0.1mM, single-strand breaks (ssb) in DNA were investigated, using the comet assay. The ability of phenolic acids to decrease DNA damage through their antioxidant properties was also assessed. The results show that the phenols, which are known as antioxidative agents, could also act as pro-oxidants. They induced ssb in DNA of the digestive gland at concentrations higher that 10 microM, but lower doses (1 and 5 microM) did not contribute to the DNA damage. This study was also designed to evaluate the protective effect of these tannins against H(2)O(2)-mediated DNA damage in the cells. In this treatment, the two concentrations (1 and 5 microM) significantly decreased the amount of lesions induced by H(2)O(2) (0.04 and 0.1mM). In conclusion, our results demonstrate that antioxidative properties of tannins may change to pro-oxidative activities at the higher concentrations. This suggests that the biologic actions of these compounds may be rather complicated.     

DNA damage in digestive gland and mantle tissue of the mussel Perna perna

Data concerning the susceptibility of DNA to damage by reactive oxygen and nitrogen species and other endogenous compounds produced by physiological stress in marine organisms is lacking, especially in bivalve mollusks. In this article, we analyzed the background levels of lipid peroxidation (malondialdehyde, MDA), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 1,N2-etheno-2'-deoxyguanosine (1,N2-epsilon dGuo) in digestive gland and mantle tissue of mussels Perna perna collected at a cultivation zone in Florianópolis (Santa Catarina, Brazil). The present data point to the possibility of the use of both 8-oxodGuo and 1,N2-epsilon dGuo as complementary indicators of oxidative stress processes in mussels. A sensitive method coupling high performance liquid chromatography to mass spectrometry was applied for the detection of 1,N2-epsilon dGuo in mussel tissues.     

Immunochemical localisation of proliferating cells in mussel digestive gland tissue

The distribution of proliferating cells in the digestive gland of the common marine mussel, Mytilus galloprovincialis Lmk, was investigated by means of immunochemical techniques employing PC10, a commercial monoclonal antibody to the proliferating cell nuclear antigen (PCNA). Immunoblot analysis of digestive gland whole homogenates revealed a single crossreactive band of 36–37kDa, identical to the corresponding protein of rat liver and murine melanoma cells. A band of slightly higher electrophoretic mobility (34–35kDa) was found in fish liver. In mussel digestive gland, the samples obtained from young specimens presented a more intense signal for PCNA than in those obtained from old mussels, suggesting that the digestive gland cells of young mussels exhibit a higher proliferative activity. In paraffin sections, PC10 specifically labelled nuclei of all cell types, but only a smaller number of cells lining the different digestive epithelia. PCNA expression was more intense in digestive cells than in basophilic cells. Hemocytes circulating along the interdiverticular spaces also presented immunoreactive nuclei. Electron microscopy revealed a specific and moderate PC10 labelling in nuclei. Thus, single gold particles appeared disseminated throughout the nuclei with accumulations of particles in the sites of DNA replication. Taken together, these data reveal that the capacity to proliferate resides within all cell types in the digestive diverticula and do not support the hypothesis of the existence of one stem cell in this epithelium. As opposed to the hepatopancreas of the crab, Carcinus maenas, where mitotic figures and PCNA immunoreactivity are only observed in the embryonic cells within the distal portions of the digestive diverticula, apparently there are not discrete regions of cell proliferation in the digestive gland of mussels.     

Circatidal variation in epithelial cell proliferation in the mussel digestive gland and stomach

Epithelial cell renewal in mussel (Mytilus galloprovincialis, Lmk) digestive gland and stomach was investigated by bromodeoxyuridine (BrdU) immunohistochemistry. Mussels were exposed to 4 mg BrdU/l seawater continuously. Starting at 6 h after treatment, samples were collected every 2 h for 2 days and BrdU labelling was estimated by direct counting at the light microscope, with values being noted per thousand BrdU-positive cells. BrdU-positive reaction was observed in the nuclei of digestive, basophilic, duct and stomach cells, and in haemocytes. Cell renewal in digestive diverticula was synchronised following a circatidal pattern: BrdU labelling increased during low tide and decreased during high tide. Clearcut mitotic figures were identified in digestive cells, thereby confirming that mature cell types proliferate, in agreement with results from immunohistochemistry for proliferating cell nuclear antigen and BrdU. Epithelial cell renewal in the stomach also appeared to be synchronised.This investigation was funded by the Basque Government (GVPI95-36 and GVP99-1) and by a grant to Consolidated Research Groups (UPV/EHU)     

Subcellular distribution of cadmium and its cellular ligands in mussel digestive gland cells as revealed by combined autometallography and X-ray microprobe analysis

Autometallography (AMG) and electron probe X-ray microanalysis (EPXMA) were applied in combination to determine the subcellular distribution of Cd and its subcellular ligands in the digestive gland cells of Cd-exposed mussels Mytilus galloprovincialis. Black silver deposits (BSD), which reveal the presence of metals when AMG is applied, were only localized in digestive cell lysosomes. Digestive cell cytoplasm and basophilic cells were devoid of BSD. EPXMA (static probe and X-ray mapping) indicated that Cd, S (possibly associated with metallothioneins or metallothionein-like proteins) and autometallographical Ag ions are co-localized within digestive cell lysosomes. In addition, Cd and S co-occur in the absence of Ag in the cytosol of digestive cells. AMG does not reveal the presence of the Cd pool strongly bound to cytosolic Cd-metallothionein complexes; only free Cd or Cd supposedly loosely bound to (semi)digested metallothionein within lysosomes was revealed. The levels of lysosomal Cd were indirectly quantified by stereology as the volume density of BSD (Vv_BSD). Significantly higher values were recorded in Cd-exposed mussels compared with controls at all exposure times. However, Vv_BSD values were lower at days 7 and 21 than at day 1. This relative decrease in Vv_BSD reflected another (and confounding) response elicited by Cd-exposure in the digestive epithelium: the volume density of basophilic cells (Vv_BAS) increased significantly as exposure progressed. Due to this cell-type replacement, the net accumulative capacity of the digestive epithelium decreases at long exposure times.     

Anti-oxidant, pro-oxidant properties of tannic acid and its binding to DNA

Tannic acid has numerous food and pharmacological applications. It is an additive in medicinal products, and is used as a flavouring agent and as an anti-oxidant in various foods and beverages. We have previously shown that tannic acid in the presence of Cu(II) causes DNA degradation through generation of reactive oxygen species. On the other hand, it exhibits antimutagenic and anticarcinogenic activities, and induces apoptosis in animal cells. It is known that most plant-derived polyphenolic anti-oxidants also act as pro-oxidants under certain conditions. In this paper, we compare the anti-oxidant and pro-oxidant properties of tannic acid and its structural component gallic acid. It is shown that tannic acid is the most efficient generator of the hydroxyl radical in the presence of Cu(II), as compared with gallic acid and its analogues syringic acid and pyrogallol. The anti-oxidant activity of tannic acid was studied by its effect on hydroxyl radical and singlet oxygen mediated cleavage of plasmid DNA. Again, tannic acid provided the maximum protection against cleavage, while gallic acid and its structural analogues were found to be non-inhibitory or partially inhibitory. The results suggest that the structural features of tannic acid that are important for its anti-oxidant action are also those that contribute to the generation of hydroxyl radicals in the presence of Cu(II). Restriction analysis of treated phage DNA and thermal melting profiles of calf thymus DNA indicated that tannic acid strongly binds to DNA. Indirect evidence indicates that modification of DNA bases may also occur.     

Tributyltin-driven enhancement of the DCCD insensitive Mg-ATPase activity in mussel digestive gland mitochondria

The lipophilic pollutant tributyltin (TBT), other than inhibiting the DCCD (N,N′-dicyclohexylcarbodiimide) and oligomycin-sensitive Mg-ATPase activities in digestive gland mitochondria from the Mediterranean mussel Mytilus galloprovincialis, at higher than 1.0 μM concentrations in vitro promotes an increase in the ATPase activity fraction refractory to inhibitors of F_O moiety, namely oligomycin and DCCD. By exploring the mechanisms involved in the TBT promoted enzyme desensitization to DCCD, we pointed out intriguing differences in the enzyme desensitization to the two inhibitors. Differently from oligomycin, the TBT promoted enzyme desensitization to DCCD is independent of the redox state of thiol groups of the enzyme complex and strongly temperature dependent, being significantly elicited only at temperatures above the break of the Arrhenius plots (around 18 °C). Such differences may cast light on multiple TBT interaction modes with the enzyme complex. The TBT-driven increase in the activation energy of the Mg-ATPase activities insensitive to inhibitors of F_O sector suggests that the temperature-dependent incorporation of the lipophilic toxicant within the lipid bilayer may deeply affect the membrane-bound complex functionality. Accordingly, incorporated TBT may cause structural changes in the intramembrane F_O subunits, thus weakening or even preventing DCCD binding to the enzyme complex.     

Peroxisomes in digestive gland cells of the mussel Mytilus galloprovincialis Lmk. Biochemical, ultrastructural and immunocytochemical characterization.

The present study was undertaken because of the paucity of information on peroxisomes in molluscs and the increasing importance of these organisms as sensitive indicators of environmental pollution. Peroxisomes were identified by electron microscopy in all three main cell types of the digestive gland of the bivalve mollusc Mytilus galloprovincialis Lmk. They stained weakly with the alkaline diaminobenzidine reaction but showed distinct immunolabeling with an antibody against mammalian catalase by the postembedding protein A-gold procedure. In addition, mussel digestive gland peroxisomes were isolated by differential and metrizamide-density gradient centrifugation, and a 30-fold enrichment of catalase and a 20-fold enrichment of palmitoyl-CoA oxidase was obtained over the initial homogenate. By Western blotting, isolated peroxisomes crossreacted with antibodies to catalase and, furthermore, specific and prominent labeling of isolated peroxisomes was also demonstrated in thin sections incubated with anti-catalase antibodies. These observations establish that peroxisomes in molluscan digestive gland contain the peroxisomal marker enzymes catalase and acyl-CoA oxidase and that they can be labeled by cytochemical and immunocytochemical techniques. Further studies of alterations of molluscan peroxisomes by environmentally relevant xenobiotics are warranted.     

Influence of some metal concentrations on the activity of antioxidant enzymes and concentrations of vitamin E and SH-groups in the digestive gland and gills of the freshwater bivalve Unio tumidus from the Serbian part of Sava River

We examined whether the freshwater bivalve Unio tumidus from the Sava River can serve as a bioindicator organism for long-term biomonitoring of river ecosystems for the presence of metal pollutants. To this end, we assessed in the digestive glands and gills of mussels, changes in activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), the phase II biotransformation enzyme glutathione-S-transferase (GST)), and changes in the concentrations of the non-enzymatic components of the antioxidant system (vitamin E and sulfhydryl groups (-SH), after exposure to metals in the environment. Mussels were collected at four sites where the concentrations of dissolved metals (Cu, Cd, Zn, Fe, Mn, Hg, Ni, As, Pb) were quantified. Cu, Ni and As exerted concentration-dependent inhibitory effects on CAT and GST activities. Increasing concentrations of Cd promoted increases in GSH-Px activity and -SH concentration. In response to increased Zn concentration GR activity increased whereas Fe promoted decreased enzymatic activity. Negative correlations between the concentrations of Cu and Cd and vitamin E, and a positive correlation between Mn and vitamin E concentrations were detected. The described correlations between components of the antioxidant system and metal levels in the environment reveal a high physiological sensitivity of freshwater mussels to pollution, supporting their use in biomonitoring of metal contamination in river ecosystems.     

DNA damage (comet assay) and 8-oxodGuo (HPLC-EC) in relation to oxidative stress in the freshwater bivalve Unio tumidus

The relationships between DNA damage and oxidative stress in the digestive gland, gills and haemocytes of the freshwater bivalve Unio tumidus were investigated. Two markers of genotoxicity were measured: DNA breaks by means of the comet assay, and oxidative DNA lesions by means of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) measured using high-performance liquid chromatography (HPLC) coupled to electrochemical detection. Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) tissue levels. Effects were studied after exposure of bivalves for 6 days to benzo[a]pyrene (B[a]P) (50 and 100 μg?l^?1) and ferric iron (20 and 40 mg?l^?1), applied alone or in combination. Lipid peroxidation in the digestive gland and gills resulted from exposure to Fe³⁺ or B[a]P whatever the concentrations tested. DNA oxidatively formed lesions were induced in the two tissues at a higher level after B[a]P exposure than after Fe³⁺ treatment. No significant dose–response relationship was found with the two compounds and no synergistic effect was observed between Fe³⁺ and B[a]P. The gills appeared less sensitive than the digestive gland to DNA lesions expressed as 8-oxodGuo and comet results. Good correlations were noted between 8-oxodGuo and comet. MDA and DNA damage did not correlate as well, although it was stronger in the digestive gland than in the gills. Production of mucus by the gills likely served to prevent lesions by reducing the bioavailability of the chemicals tested, which could explain that dose–effect relationships and synergistic effects were not observed.     

Glycosylation and sorting pathways of lysosomal enzymes in mussel digestive cells

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.This work was funded by projects UPV 075.327–EA033/92 and UPV 075.327–EA053/93 of the University of the Basque Country and by a grant to Consolidated Research Groups (UPV/EHU). Y.R. was the recipient of a MEC–DGCYT pre-doctoral fellowship.     

DNA damage (comet assay) and 8-oxodGuo (HPLC-EC) in relation to oxidative stress in the freshwater bivalve Unio tumidus.

The relationships between DNA damage and oxidative stress in the digestive gland, gills and haemocytes of the freshwater bivalve Unio tumidus were investigated. Two markers of genotoxicity were measured: DNA breaks by means of the comet assay, and oxidative DNA lesions by means of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) measured using high-performance liquid chromatography (HPLC) coupled to electrochemical detection. Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) tissue levels. Effects were studied after exposure of bivalves for 6 days to benzo[a]pyrene (B[a]P) (50 and 100 microg l(-1)) and ferric iron (20 and 40 mg l(-1)), applied alone or in combination. Lipid peroxidation in the digestive gland and gills resulted from exposure to Fe3+ or B[a]P whatever the concentrations tested. DNA oxidatively formed lesions were induced in the two tissues at a higher level after B[a]P exposure than after Fe3+ treatment. No significant dose-response relationship was found with the two compounds and no synergistic effect was observed between Fe3+ and B[a]P. The gills appeared less sensitive than the digestive gland to DNA lesions expressed as 8-oxodGuo and comet results. Good correlations were noted between 8-oxodGuo and comet. MDA and DNA damage did not correlate as well, although it was stronger in the digestive gland than in the gills. Production of mucus by the gills likely served to prevent lesions by reducing the bioavailability of the chemicals tested, which could explain that dose-effect relationships and synergistic effects were not observed.     

Isolation and biochemical characterization of the microsomal fraction from the digestive gland of mussel Mytilus galloprovincialis Lam

A procedure to prepare microsomes from the mussel digestive gland is proposed. The data concerning the biochemical characterization of this subcellular fraction shows a typical RNA:protein ratio, but the presence of hydrolytic enzymes was also found; therefore a mixture of hydrolase inhibitors to study the different biochemical characteristics was used. The biochemical data demonstrate that glucose-6-phosphatase activity (G6Pase), a typical microsomal marker in mammalian cells, is not present in mussel digestive gland microsomes but a high non-specific phosphatase activity was detected. Benzo[a]pyrene hydroxylase activity was found to be present although in a minimal amount. The evaluation of the molecular weight of the rRNA demonstrates that the larger ribosomal subunit contains RNA of Mr 1.40 X 10(-6) (approximately 26S) and the smaller subunit is composed of RNA of Mr 0.65 X 10(-6) (18S). The data from mussel digestive gland microsomes was compared with that experimentally obtained from rat liver microsomes and discussed from a functional or an evolutionary point of view.