Mytilus Galloprovincialis Digestive Gland Research Articles

Differential HSP70 gene expression in the Mediterranean mussel exposed to various stressors

HSP70 gene expression was studied by quantitative RT-PCR after cloning and sequencing of two different HSP70 gene fragments from the digestive gland of Mytilus galloprovincialis, called MgHSP70 and MgHSC70. Heat shock (1 h at 35 °C) caused rapid induction of MgHSP70, while no change was observed for MgHSC70. Hg 2+ (150 μg/L for different time periods) significantly induced MgHSP70 expression that reached maximum levels after 24 h, decreasing thereafter. MgHSC70 expression was inhibited after 1 day and induced after a 6-day exposure to Hg 2+. A 1-week exposure to Cr 6+ (1, 10, and 50 ng/L) induced and inhibited MgHSC70 and MgHSP70 transcript levels, respectively. MgHSC70 and MgHSP70 appear to play different roles in cell protection; the former is induced after acute stress and/or during the earlier phase of the response while the latter is induced by chronic stress. The present results provide new insights into mechanisms used by mussels to adapt to stressful environments.

Cadmium effects on ROS production and DNA damage via adrenergic receptors stimulation: Role of Na+/H+ exchanger and PKC

The objective of the present study was to elucidate the events that are involved in reactive oxygen species (ROS) production and DNA damage after adrenergic receptors stimulation by cadmium, in relation to cAMP, protein kinase C (PKC) and Na+/H+ exchanger (NHE). Cadmium (50 μM) caused increased levels of ROS with a concomitant increase in DNA damage in digestive gland of Mytilus galloprovincialis. Either the use of EIPA, a NHE blocker, or calphostin C, the inhibitor of PKC, reduced cadmium effects. Cells treated with α1-, α2-, β- and β1- adrenergic antagonists together with cadmium reversed cadmium alone effects, while the respective adrenergic agonists, phenylephrine and isoprenaline, mimic cadmium effects. Moreover, cadmium caused an increase in the levels of cAMP in digestive gland cells that were reversed after NHE and PKC inhibition as well as in the presence of each type of adrenergic antagonist. The different sensitivity of α1-, α2-, β-, β1- adrenergic receptors on ROS, cAMP production and DNA damage possibly leads to the induction of two signaling pathways that may be interacting or to the presence of a compensatory pathway that acts in concert with the α- and β- adrenergic receptors. In these signaling pathways PKC and NHE play significant role.

Comparison of two SH-based methods for estimation of metallothionein level in the digestive gland of naturally occurring mussels, Mytilus galloprovincialis

Two methods for the evaluation of metallothionein (MT) level in Mytilus galloprovincialis digestive gland homogenate extract, polarographic and spectrophotometric, were compared in combination with sample subjected to heat treatment (at 70 and 85°C) as well as to solvent precipitation. Spectrophotometric measurements ranging within 0.081–0.144 mg MTg−1 were at least one order of magnitude lower in comparison with the polarographic ones (2.21–2.88 mg MTg−1) depending on the particular sample treatment as well as two specific reagents and reference standards applied.Sephadex G-75 column chromatography of the differently treated samples and subsequent polarographic analysis in the pooled elution fractions indicated that a level of nonMT interfering thiolic component in homogenate extract may be accounted for more than 50% of the total polarographic signal, irrespective of a method o sample treatment applied. Consequently, the large discrepancy between methods could not be attributed only to the sample treatment procedures but more likely to certain not entirely explained analytical problems, particularly calibration with appropriate reference standard.

Effect of hydrogen peroxide on antioxidant enzymes and metallothionein level in the digestive gland of Mytilus galloprovincialis

The pro-oxidant effect of H 2O 2 at a concentration of 20 μM was examined in the digestive gland of Mytilus galloprovincialis, a bivalve mollusc frequently used in biomonitoring programs. The oxidative stress caused by H 2O 2 has been evaluated in terms of lipid peroxidation and lysosomal system alteration. Complex cellular antioxidant defence mechanisms of the mussel were investigated at the enzymatic and non-enzymatic level in order to explain their relative role in reducing the risk of oxidative injury. Metallothionein, glutathione, superoxide dismutase, catalase and glutathione peroxidase were assayed after 1, 4 and 7 days of exposure to H 2O 2. The metallothionein content showed an increase by 43% after 4 days of exposure, followed by a decrease back to control values at 7 days. Antioxidant enzyme activities followed a similar pattern with a moderate increase after 1 or 4 days of treatment and a return to control values at 7 days. All data indicate a ‘transient’ oxidative stress response, after which mussel cells restore the redox balance.

Enzymatic activity in Mytilus galloprovincialis (Mollusca: Bivalvia) digestive gland

Mitochondrial dehydrogenases, antioxidant enzymes, transaminases and cytochrome P450 activity have been studied from the digestive gland of Mytilus galloprovincialis, reared in a non-polluted area of the Mar Grande of Taranto (Ionian Sea, Mediterranean).

Mitochondrial contribution to metabolic changes in the digestive gland of Mytilus galloprovincialis during anaerobiosis.

The activities of several enzymes and the levels of metabolites have been measured in the digestive gland of Mytilus galloprovincialis exposed to increasing hypoxia from 7 to 168 hr. A sharp decrease of pyruvate kinase was observed after 7 hr. The anoxic enzyme showed increased Km for phosphoenolpyruvate and decreased apparent Ki for alanine. Glyoxalase I was constant after up to 72 hr of exposure and then decreased. Phosphoenolpyruvate carboxykinase and alanopine dehydrogenase decreased. The metabolites alanine and succinate increased with hypoxia time, whereas D- and L-lactic and aspartic acids were undetectable and constant respectively. Mitochondrial formation of pyruvate from D-lactate was demonstrated in intact mitochondria isolated from the digestive gland of Mytilus galloprovincialis. The significance of the observed enzyme and metabolite changes in hypoxia is discussed in comparison with other invertebrate organisms. The role of mitochondria in the overall adaptive strategy of Mytilus galloprovincialis is discussed. J. Exp. Zool. 286:107-113, 2000.

Variation in levels of cytochrome P4501A, 2B, 2E, 3A and 4A-immunopositive proteins in digestive gland of indigenous and transplanted mussel Mytilus galloprovincialis in Venice Lagoon, Italy

Mytilus galloprovincialis digestive gland microsomes were prepared from (i) indigenous populations sampled from a clean reference (Lido) and an urban-contaminated site (Salute) and (ii) mussels transplanted for up to 3 weeks from Lido to an industrial-contaminated site (CVE) in Venice Lagoon, Italy. Western blot analysis was performed using antibodies to five mammalian or fish CYP forms (1A, 2B, 2E, 3A, 4A). Simultaneously run M. edulis digestive gland partially purified CYP aided identification of immunopositive bands. Levels of CYP1A, CYP2E, and CYP4A-immunopositive proteins were 50 to 300% higher in indigenous M. galloprovincialis from Salute compared to Lido ( p < 0.05). Three weeks after transplantation to CVE, levels of only the CYP1A-immunopositive protein were determined to be higher (63%) than levels for Lido ( p < 0.05), indicating that anti-CYP1A shows greater specificity for a contaminant-inducible CYP form than the other antibodies.

Determination of the cadmium and copper content inherent to metallothionein

The reliability of the voltammetric determination of the cadmium and copper content (at pH 1.0), inherent to metallothionein (MT) isolated from the digestive gland of Mytilus galloprovincialis, was investigated. An artifact signal enhancement of copper, caused by the cupric-thionein complex adsorption at the mercury electrode, was established. This artifact was removed by UV-digestion of the sample for 15–20 h prior to analysis. A similar artifact was not detected for cadmium, because at this pH the cadmium-thionein complex has dissociated, and cadmium exists in the ionic form. Therefore, the voltammetric analysis of the cadmium content can be performed directly at pH 1.0, without prior UV-digestion of the sample.

Mussel glyoxalase I as a possible marker for ecotoxicological studies: Purification and preliminary characterization

Alpha-ketoaldehydes may be formed in cells during oxidative processes and glyoxalase I is the main enzyme involved in the detoxification pathway for these highly toxic compounds. Increased glyoxalase I activity has been observed in mussels exposed to high environmental levels of pollutants and a role for this enzyme as a protection mechanism against peroxidation damage has been hypothesized. In this paper, glyoxalase I from mussel tissue has been purified and a preliminary investigation of its molecular properties carried out. A two step purification procedure for glyoxalase I from digestive gland of Mytilus galloprovincialis is described. The pure enzyme is a 48 kDa protein with an heterodimeric quaternary structure composed of 24 and 25 kDa subunits. The isoelectric point of the native enzyme is at pH 5.0 and there is a divalent cation (probably Zn ++) requirement for activity. The series of alkyl-S-glutathiones, from methyl- to decyl-, are competitive inhibitors of glyoxalase I. Ki values exponentially decrease from 1.15 mM to 2.65 μM with increasing chain length. Mussel glyoxalase I exhibits molecular properties similar to those of the mammalian enzyme. The possible role of glyoxalase I in the detoxification of α-ketoaldehydes formed during oxidative stress is discussed.

Studies on isoenzymes of glutathione S-transferase in the digestive gland of Mytilus galloprovincialis with exposure to pollution

The possible utility of glutathione S-transferase isoenzymes in affinity purified extracts of digestive gland from Mytilus galloprovincialis as indicators of exposure to organic pollution has been assessed by fast protein liquid chromatography (FPLC) using a Mono-Q ion exchange column. Four main peaks of protein (isoenzymes 1–4) were detected in these chromatographic studies all of which displayed detectable catalytic activity with 1-chloro-2,4-dinitrobenzene and glutathione as substrates. The bulk of the activity with these substrates was associated with isoenzyme 1. The specific activity of cytosolic extracts from six sites in Venice Lagoon and the Adriatic Sea (UNESCO-MURST Venice Lagoon Ecosystem Project) decreased slightly with increasing tissue levels of chemical contaminants (hydrocarbons, PCBs and organochlorines). FPLC of affinity purified extracts from three of these sites was used to assess the possible variation in the levels of individual isoenzymes relative to pollution status. No difference was observed in the levels of these isoenzymes.

Quantitative determination of metallothionein-like proteins in mussels. Methodological approach and field evaluation

Electrochemical quantitation of metallothionin-like proteins (MLP) in mussels was based on the determination of their constituent cysteinyl residues according to Brdicka's catalytic reaction. Calibration was performed by an internal MLP standard isolated from the digestive gland of Mytilus galloprovincialis for which protein concentration had been estimated by Bradford's spectrophotometric method. For that purpose three metal-binding proteins [MLP-I, MLP-II and Cu-BP (binding protein)] were separated by DEAE-Sephadex A-25 chromatography from the digestive gland of mussels previously exposed to Cd. The most negatively changed MLP-II fraction was characterized by the fact that it contained the largest amount of both total metal and sulphydryl (-SH) content per mass of protein, although this was approximately two times lower than the-SH level of commercially available MT from rabbit liver. Exposure of mussels to a relatively low level of cadmium (0.2 μg Cd l-1) added into the seawater either by itself or as a mixture with other metals (2 μg Cu l-1 and 1.6 μg Pb l-1) resulted in a measurable level of MLP induction within 14 d in comparison to the control specimens. The effect of the metal mixture on MLP synthesis appears to be less than additive, suggesting a competitive interaction between metal ions for uptake and binding sites as well as differing potentials for MLP induction. Variations in the MLP content observed in the digestive gland of mussels seasonally collected from the same location are in the range 2.1±0.4 mg g-1 on a wet weight basis. The methodological and conceptual aspects of the application of MLP induction in the Mytilus sp. as a biomarker in seawater trace metal monitoring are critically evaluated.

Possible biological reference material for environmental control analyses — cadmium induced proteins from Mytilus galloprovincialis

Electrochemical methods have been applied for chemical characterization of metallothionein-like proteins (MLPs) isolated from the digestive gland of Mytilus galloprovincialis, chronically exposed to cadmium. Previous results support the view that the induction of MLPs in the digestive gland of this edible marine organism represents a measurable biological response to the chronic exposure of cadmium and therefore the development of a possible reference material from the digestive gland is proposed.

Selective activation of carcinogenic aromatic amines to bacterial mutagens in the marine mussel Mytilus gallopro vincialis

1. 1. The postmitochondrial fraction of the marine mussel Mytilus galloprovincialis digestive gland activates selectively precarcinogenic aromatic amines, but not precarcinogenic benzo( a]pyrene, to Salmonella typhimurium TA 98 mutagens. 2. 2. This activation potential is NADPH-dependent, is not inducible by exposure to Diesel 2 oil and a polluted environment, and is inhibited by methimazole. 3. 3. The characteristics of this activation potential are consistent with the recent finding of the presence of FAD-containing-, and lack ofcytochrome P-450 dependent-, monooxygenase activity in Mytilus edulis. 4. 4. The presence of such selective potential in marine invertebrate(s) may bring new insight into our understanding of the fate and the effects of carcinogens in the marine environment.

Purification and properties of 6-phosphogluconate dehydrogenase fromMytilus galloprovincialis digestive gland

1. The enzyme 6-phosphogluconate dehydrogenase from digestive gland of Mytilus galloprovincialis was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis. 2. The enzyme was purified 229-fold with a final specific activity of 2.3 μmol of NADP + reduced/min per mg of protein and overall yield of 10%. 3. The molecular weight of the native enzyme is estimated to be 100, 000 from gel-filtration studies. 4. The influence of pH and MgCl 2 concentration on enzyme activity have been studied and the results have been compared to those reported by other authors for enzymes from different sources. 5. The K m values for 6-phosphogluconate and NADP + at room temperature (20°C) are approx. 20 and 40 μM respectively. 6. NADPH was an inhibitor strictly competitive with respect to NADP + ( K i = 14 μM) and non-competitive with respect to 6-phosphogluconate ( K i = 45 μM).