Bruno Burlando

Role of metallothionein against oxidative stress in the mussel Mytilus galloprovincialis

Metallothionein (MT) is a sulfhydryl-rich protein involved mainly in heavy metal homeostasis and detoxification. In this study, the use of the mussel as an experimental model allowed us to test MT antioxidant properties at the molecular, cellular, and organism level. MT induction was achieved by mussel exposure to Cd (200 μg/l) in aquaria for 7 days followed by detoxification in the sea for 28 days. Cd-preexposed and nonexposed mussels were then treated with Fe (300-600 μg/l) in aquaria for 3 days. Biochemical assays on digestive gland tissue showed that treatment with Fe led to a significant increase in oxyradical production and malondialdehyde level only in mussels not preexposed to Cd. The Cd-dependent resistance to oxidative stress was ascribed to MT induction, as Cd produced no significant variation of reduced glutathione and major antioxidant enzymes. Digital imaging of isolated digestive gland cells showed lower oxyradical rise and higher viability in cells from Cd-preexposed mussels after treatments with 0.5-5 mM H2O2. Analyses on whole organisms showed that anoxic survival was lowered in mussels that had been treated with Fe, but such an effect was less pronounced in Cd-preexposed mussels compared with nonpreexposed ones. In conclusion, data suggest an antioxidant role for MT, which seems to occur through oxyradical scavenging and is able to protect both isolated cells and the entire organism from oxidative stress.

Exposure to elevated temperatures and hydrogen peroxide elicits oxidative stress and antioxidant response in the Antarctic intertidal limpet Nacella concinna

This study deals with the occurrence of oxidative stress and antioxidant response in the Antarctic intertidal limpet Nacella concinna, as an effect of temperature increments and H2O2 exposure under controlled laboratory conditions. Experiments were designed to simulate transient conditions of increased T and:or H2O2 accumulation met by the limpets in intertidal rockpool habitats [5]. Specimens were collected at Jubany Station, South Shetland Islands, transferred to the Alfred-Wegener Institute, Bremerhaven and maintained in seawater aquaria at 0°C. Different groups of animals were acclimated at 4 and 9°C for 24‐48 h (controls at 0°C). The effect of starvation was studied at 0°C and of H2O2 exposure at 4°C. Temperature acclimation above 0°C resulted in a progressive alteration of the lysosomal compartment in digestive gland cells, as shown by cytochemical analyses (lipofuscin and neutral lipid accumulation and lysosomal membrane destabilization). Concurrently, real activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (i.e. measured at the respective experimental temperature or calculated by means of previously determined Q10 values) increased in gills and digestive gland tissues. Measurements of intracellular pH at the different temperatures showed a rise from pH 7.21 at 0°C to 7.36 at 9°C. These changes in pH are indicated to increase SOD activity by approximately 10% in both kinds of tissue at 9° as compared to 0°C. H2O2 exposure at 4°C produced physiological alterations at the systemic (lowered O2 consumption) and at the cellular levels (enhanced lysosome damage). Starvation induced lysosomal alterations in animals kept at 0°C and inhibited CAT activation under H2O2 exposure at 4°C. The complex of data suggests that when Nacella migrates to intertidal levels during the Austral Spring it experiences oxidative stress which induces an antioxidant response, which is facilitated by higher temperatures and increasing intracellular pH and the exploitation of intertidal food resources. Yet, the occurrence of cellular damage and systemic alterations shows that the limpets approach their boundaries of physiological tolerance during prolonged exposure to higher temperatures and H2O2 in intertidal habitats.

Metallothionein as a tool in biomonitoring programmes

The biochemical features of metallothioneins and their functional role in the cell are described. On this basis, the potential role of MTs as a biomarker of exposure in aquatic organisms, such as fishes and molluscs, is evaluated in the light of recent knowledge about MT gene regulation and inducibility. It appears that in fish MTs should be considered as a kind of stress protein which is particularly responsive to heavy metals. In molluscs, in particular in mussels, MTs seem more specifically involved in responses to heavy metals and they should therefore be considered a biomarker of exposure to heavy metal pollution. Common techniques for MT evaluation are listed and a simple spectrophotometric method recently developed is also reported. Finally, the correct approach to the use of MTs as a biomarker of exposure in biomonitoring programmes for an assessment of the physiological status of aquatic organisms is discussed.The biochemical features of metallothioneins and their functional role in the cell are described. On this basis, the potential role of MTs as a biomarker of exposure in aquatic organisms, such as fishes and molluscs, is evaluated in the light of recent knowledge about MT gene regulation and inducibility. It appears that in fish MTs should be considered as a kind of stress protein which is particularly responsive to heavy metals. In molluscs, in particular in mussels, MTs seem more specifically involved in responses to heavy metals and they should therefore be considered a biomarker of exposure to heavy metal pollution. Common techniques for MT evaluation are listed and a simple spectrophotometric method recently developed is also reported. Finally, the correct approach to the use of MTs as a biomarker of exposure in biomonitoring programmes for an assessment of the physiological status of aquatic organisms is discussed.

Heavy metal inhibition of EROD activity in liver microsomes from the bass Dicentrarchus labrax exposed to organic xenobiotics: Role of GSH in the reduction of heavy metal effects

Abstract In vitro effects of Cu 2+ , Hg 2+ and of CH 3 Hg + on fish liver microsomal EROD activity have been studied. Liver microsomes from the fish Dicentrarchus labrax , treated with β-naphthoflavone (50 mg/kg) or benzo-a-pyrene (20 mg/kg), showed a 4 to 15-fold increase of EROD activity. Addition to the reaction mixture of nmolar concentrations of Cu 2+ , Hg 2+ and CH 3 Hg + significantly decreased EROD activity, micromolar concentrations totally inhibited the enzyme, while treatments with mixtures of nanomolar Cu 2+ , Hg 2+ and CH 3 Hg + exerted an additive inhibitory effect. Addition of physiological concentrations of GSH drastically reduced the heavy metal inhibitory effects. These results demonstrate that fish liver EROD activity is highly sensitive to extremely low concentrations of heavy metals, also suggesting that GSH plays a fundamental role as a first-line defense against heavy-metal cytotoxicity.

The fractal dimension of taxonomic systems

The hypothesis that biological diversity has a fractal geometry is tested through the examination of size-frequency distributions of taxa with different numbers of subtaxa. Data used derive from 44 checklists and catalogues of species concerning protists, fungi, plants and animals, and from three synoptic classifications of protists, plants and animals. Distributions give hyperbolic curves whose log-log plots are almost linear, with negative slopes. Long tails of distribution curves due to very large taxa call for skew variants of hyperbolic curves. The positive values of log-log regression line slopes correspond to the fractal dimensions D nof the taxonomic assemblages, characterizing their diversity. Non-random occurrence ofn D nvalues among groups suggests a relationship with true biologic diversity patterns, rather than an effect of taxonomic criteria. Differences in fractal dimension among the examined lists are discussed, the more relevant being the higher differentiation of marine groups with respect to continental ones. The fractal geometry of diversity is viewed as an evolutionary pattern possibly related to scaling evolutionary processes, suggested by the finding of hyperbolic trends at different taxonomic levels.

Metabolic integration between symbiotic cyanobacteria and sponges: a possible mechanism

Metabolic relationships between symbiotic cyanobacteria and host sponge have been investigated in the marine species Chondrilla nucula and Petrosia ficiformis (collected in the Ligurian Sea in 1992). DNA, RNA, total protein, cytosolic protein, total sugar, cytosolic sugar, total lipid, nonprotein sulfhydryl groups, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were assayed in cortex-free sponge tissue, where cyanobacteria are all but absent. For both species, biochemical parameters were determined in specimens living in illuminated habitats and in dark caves, where sponges are virtually aposymbiotic for cyanobacteria. As C. nucula is unable to colonize dark sites, specimens of this species were artificially transferred to a cave and maintained in dark conditions for 6 mo. Results showed that in the absence of light (i.e., in the absence of cyanobacteria) C. nucula undergo metabolic collapse and thiol depletion. In contrast, P. ficiformis activates heterotrophic metabolism and mechanisms which balance the loss of cell reducing power. This suggests that cyanobacteria effectively participate in controlling the redox potential of the host cells by the transfer of reducing equivalents. Only P. ficiformis is capable of counteracting, by means of heterotrophic metabolism, the loss of the contribution from symbionts which is caused by dark conditions. This explains the differences in the ecological requirements of the two species. Because cyanobacterial symbionts release fixed carbon in the form of glycerol and other small organic phosphate (Wilkinson 1979), a model based on the glycerol 3-phosphate shuttle (typically occurring in chloroplasts and mitochondria) is suggested. The mechanism proposed appears to be an ancient biochemical adaptation which arose among ancestral symbiotic systems, and further developed in the relationships between endosymbiotic organelles and cytoplasm.

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

Abstract The pro-oxidant effect of H 2 O 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 2 O 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 2 O 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.

Antiproliferative Effects on Tumour Cells and Promotion of Keratinocyte Wound Healing by Different Lichen Compounds

Five compounds representative of major structural classes of lichen polyketides, VIZ. (+)-usnic (1), salazinic (2), vulpinic (3), gyrophoric (4), and evernic acids (5), were investigated for their ability to affect cell proliferation or wound healing, two functional targets of relevance for research on cancer or tissue regeneration. The experiments were carried out on MM98 malignant mesothelioma cells, A431 vulvar carcinoma cells, and HaCaT keratinocytes. The NRU and CV cytotoxicity assays showed high toxicity for (+)-usnic acid, intermediate toxicity for vulpinic acid, and low toxicity for salazinic, gyrophoric and evernic acids. Scratch wounding experiments on HaCaT monolayers, in the presence of subtoxic doses of lichen compounds, showed strong wound closure effects by (+)-usnic and gyrophoric acid, an intermediate effect by vulpinic and salazinic acids, and no effect by evernic acid. A combination of (+)-usnic and gyrophoric acids gave a further increase in the wound closure rates. The results of a cell migration test correlated with the wound healing data. In conclusion, (+)-usnic acid might be a particularly interesting compound for the prevention of hyperproliferation syndromes, while (+)-usnic and gyrophoric acids qualify as interesting leads in the promotion of tissue regeneration.

Hmgb1 Promotes Wound Healing of 3T3 Mouse Fibroblasts via Rage-Dependent ERK1/2 Activation

HMGb1 is a nuclear protein playing a role in DNA architecture and transcription. This protein has also been shown to function as a cytokine and to stimulate keratinocyte scratch wound healing. Due to the importance of finding new wound healing molecules, we have studied the effects of HMGb1 on fibroblasts, another major skin cell type, using the NIH 3T3 line. HMGb1 expression in these cells was assessed by Western blot, while its nuclear localization was pointed out by confocal immunofluorescence. HMGb1-induced cell proliferation with a maximum at a concentration of 10 nM, and such a dose also stimulated cell migration and scratch wound healing. Western blot analysis showed that HMGb1 activates ERK1/2, while the use of an anti-RAGE receptor-blocking antibody and of the selective MEK1/2 inhibitor PD98059 blocked ERK1/2 activation and wound healing responses to HMGb1. Taken together data show that HMGb1 promotes 3T3 fibroblast wound healing by inducing cell proliferation and migration, and that this occurs through the activation of the RAGE/MEK/ERK pathway. In conclusion, HMGb1 seems a good candidate for the development of medical treatments to be used on chronic or severe wounds.

Biogeographic traits and checklist of Antarctic demosponges

SummaryThe biogeography of Antarctic demosponges has been studied by dividing Antarctic and circumantarctic areas into geographic entities, and then assigning to these entities all recorded species according to literature reports. Correspondence analysis ordination based on the presence or absence of species shows the existence of a distinct Antarctic Faunistic Complex (AFC), including continental Antarctica, most of the Antarctic and circumantarctic islands and the Magellan area. Such a result has enabled us to drawup a checklist of 352 Antarctic demosponge species. Investigation of within-AFC patterns indicates that the continent is a highly homogeneous area, establishing closer relationships with the Scotia Arc and to a lesser extent with the Magellan region. The AFC has low specific affinities with the other circumantarctic regions (South Africa, temperate Australia and New Zealand), whereas at the generic level relationships appear more pronounced. This biogeographic pattern may lead us to suppose a common Gondwanian origin for Antarctic and circumantarctic sponge faunas, followed by differentiation due to Gondwana fragmentation. Antarctica moved towards polar latitudes and became progressively isolated, only maintaining active interchanges with South America. Climatic changes possibly induced intense processes of speciation in the Antarctic demosponge fauna, thus contributing to its differentiation.