Francesco Regoli

Glutathione, glutathione-dependent and antioxidant enzymes in mussel, Mytilus galloprovincialis, exposed to metals under field and laboratory conditions: implications for the use of biochemical biomarkers

Abstract The effects of exposure to metals under and laboratory conditions were investigated in the Mediterranean mussel Mytilus galloprovincialis. The examined biological responses included the concentrations of heavy metals, the level of glutathione, and the activity of several enzymes selected among glutathione-dependent oxidoreductases and hydrolases: glutathione reductase. EC1.6.4.2; glyoxalase I, EC4.4.1.5; glyoxalase II, EC3.1.2.6; glutathione S-transferases, EC2.5.1.18; Se-dependent, EC1 11.1.9 and Se-independent, EC2.5.1.18 glutathione peroxidases; catalase, EC1.11.1.6; superoxide dismutase, EC1.15.1.1; alkaline phosphatase, EC3.1.3.1; cholinesterase, EC3.1.1.8; arylesterases, EC3.1.1.2. The analyses were carried out on digestive gland and gills of mussels from two populations, respectively from a polluted and a clean site. The same parameters were measured in control mussels transplanted to the polluted environment, and in molluscs exposed to copper under laboratory conditions. The comparison between different field and laboratory exposures was expected to give useful indications for a proper use of biochemical responses as biomarkers in monitoring trace metal pollution. Compared to control mussels, the polluted ones (native, transplanted and copper-exposed) showed significantly lower levels of glutathione and higher activities of the glyoxalases (which detoxify reactive α-ketoaldehydes formed in cellular oxidative processes). On the other hand, native mussels from both the polluted and control populations exhibited similar enzymatic activities of glutathione reductase, glutathione peroxidases, catalase, superoxide dismutase and alkaline phosphatase, whereas, in both transplanted and copper-exposed mussels, these enzymes showed significant variations. This finding could suggest the occurrence of some adaptation or compensatory mechanism in chronically polluted organisms. No clear results were obtained with glutathione S-transferases, whereas arylesterases and cholinesterases appeared not to be affected by metal pollution. From the results, three different kind of biological responses were identified and the implications for ecotoxicological studies discussed.

A rapid gas chromatographic assay for determining oxyradical scavenging capacity of antioxidants and biological fluids

Herein, we report a new, rapid,and reliable method for measuring the protective antioxidant potential of pure antioxidant solutions or biological tissues. Peroxyl radicals generated by thermal homolysis of 2,2-azobis-amidinopropane (ABAP) cause the oxidation of alpha-keto-gamma-methiolbutyric acid (KMBA) to ethylene; ethylene formation is monitored by gas chromatographic analysis of head space from the reaction vessel. The partial inhibition of ethylene formation in the presence of antioxidants that compete with KMBA for oxyradicals is the basis of the Total Oxyradical Scavenging Capacity Assay (TOSCA). The assay is shown to be reliable for quantifying ROS scavenging potential. The quantifiable parameters are consistent with the relative order of those predicted by the fluorescence- and oxygen electrode-based assays reported in the literature. Antioxidants competing for peroxyl radicals influenced the rate of KMBA oxidation in different ways, but the calculation of TOSC was not affected by such variations. Responses were linear over a wide range of sample concentrations and the TOSC values of classical soluble antioxidants showed the following relative order: Trolox > uric acid > ascorbic acid > GSH. The KMBA method was reliable for biological tissues; the TOSC for 1 microg rat liver cytosolic protein was 0.40 +/- 0.02 and for the microsomal membrane, 0.15 +/- 0.03. Soluble antioxidants accounted for 77% of the protective antioxidant potential in rat liver cytosol. When incorporated into the microsomal membrane, alpha-tocopherol markedly enhances antioxidant protection against peroxyl radical; thus, the assay is suitable for the assessment of fat-soluble antioxidants.

Oxidative stress in ecotoxicology: from the analysis of individual antioxidants to a more integrated approach.

An integrate approach will be discussed for investigations on oxidative stress in xenobiotic toxicity. While the analysis of individual antioxidants is useful for their sensitivity and to understand the mode of action of a stressor, the integration with the analysis of the total antioxidant capacity provides a more holistic assessment of the overall biological significance of such variations. TOSC has a greater predictive value on the health condition of the organisms and allows to discriminate the different role of specific ROS in oxidative stress syndrome.

Lysosomal and antioxidant responses to metals in the Antarctic scallop Adamussium colbecki

Lysosomal and antioxidant responses were investigated in the Antarctic scallop Adamussium colbecki exposed under laboratory conditions to metals as well as in specimens transplanted from a pristine area to the Bay receiving the discharges of the Italian Base. Exposure to copper and mercury resulted in a significant reduction of lysosomal membrane stability and in the appearance of severe morphological alterations varying from the presence of enlarged lysosomes to the loss of lysosomal structures. More limited effects were observed in Pb-exposed scallops. Moreover, laboratory exposures to Cu and Hg greatly depleted the levels of total glutathione and the activities of catalase and glutathione S-transferases were significantly reduced. More variable were the responses of glutathione peroxidases while superoxide dismutase remained almost constant. The interaction between lysosomal alterations and antioxidant defences is discussed and the results interpreted in view of the extreme environmental conditions for the Antarctic organisms. The results obtained from the transplantation experiment indicated only very limited effects on scallops translocated near the effluents of the Italian Base suggesting that the biological impact of the Base should not have major deleterious consequences on benthic marine communities.

DNA integrity and total oxyradical scavenging capacity in the Mediterranean mussel, Mytilus galloprovincialis: a field study in a highly eutrophicated coastal lagoon

In Mediterranean coastal lagoons, the combination of human impact and wide variability of natural environmental factors can lead to upsets in ecosystem homeostasis resulting in biodiversity decline. Oxidative damage has been causally linked to various kinds of environmental stress, both natural and artificial, the result being impairment of cellular functions. DNA damage and the efficiency of antioxidant defences in Mytilus galloprovincialis from the highly eutrophicated Orbetello Lagoon (Tuscany, Italy) were investigated, respectively by the single cell gel electrophoresis (or Comet test) and the total oxyradical scavenging capacity assay. Results showed significantly higher levels of DNA damage in mussels collected from the inner parts of the lagoon compared to specimens from more external sites. Specimens with the lower genetic integrity also exhibited a reduced efficiency in neutralizing three potent cellular oxidizing species, namely peroxyl radicals (ROO*), hydroxyl radicals (*OH) and peroxynitrite (HOONO), suggesting the involvement of reactive oxygen species in mediating the genetic damage. The analyzed biological parameters also showed a seasonal variability with a minimum of both DNA integrity and antioxidant scavenging efficiency during the warm months and an opposite trend in winter. The potential of analyzed techniques is discussed for the assessment of both anthropogenic and natural disturbance.

Biochemical characterization of the antioxidant system in the scallop Adamussium colbecki, a sentinel organism for monitoring the Antarctic environment

Abstractu2002The scallop Adamussium colbecki can be profitably used for monitoring Antarctic coastal environments but its utility would be increased if chemical analyses of pollutants were integrated with data on their biological effects. Since oxidative stress is a common pathway of toxicity induced by xenobiotics, a preliminary biochemical characterization was carried out on the antioxidant system of this species and baseline data collected for future assessment of the anthropogenic impact in this remote area. The digestive gland and gills were investigated for levels of glutathione and the activity of several glutathione-dependent and antioxidant enzymes: gluthathione reductase, EC 1.6.4.2; glyoxalase I, EC 4.4.1.5; glyoxalase II, EC 3.1.2.6; gluthathione S-transferases, EC 2.5.1.18; Se-dependent, EC 1.11.1.9 and Se-independent, EC 2.5.1.18 gluta-thione peroxidases; catalase, EC 1.11.1.6; and super-oxide dismutase, EC 1.15.1.1. The same enzymatic activities were measured for comparison in the Mediterranean molluscs Mytilus galloprovincialis and Pecten jacobaeus. Very high levels of glutathione S-transferases were found in the digestive gland of both species of scallop compared to mussels, suggesting the importance of different feeding behaviour among these molluscs. However, catalase activity, much higher in Adamussium colbecki than in the Mediterranean molluscs, may represent a biochemical adaptation to the Antarctic marine environment with high levels of dissolved oxygen. Enzymes from the Antarctic species appeared to be generally more active at low temperatures but, with a few exceptions, their activities increased at higher temperatures.

Accumulation and subcellular distribution of metals (Cu, Fe, Mn, Pb and Zn) in the Mediterranean mussel Mytilus galloprovincialis during a field transplant experiment

Abstract Marine mussels are generally used as biomonitors of heavy metal pollution in coastal areas. However, the time-integration capacity of these molluscs may vary from different metals and this fact should be considered when interpreting heavy metal concentrations in natural mussel populations. The aim of this work was to investigate the uptake-excretion kinetics and the subcellular distribution of metals in field conditions: the results were expected to provide useful information on the different time-integration capacities of mussels for various elements. Mussels from a clean site were transplanted to a heavy metal polluted environment where they accumulated Pb, Fe, and Mn. The accumulation was not linear but a steady state was reached after only 2 weeks, indicating that mussels can rapidly equilibrate with enhanced environmental levels of these pollutants. Copper and zinc were not accumulated by transplanted mussels, suggesting a similar bioavailability in the two environments or a certain regulation capability of molluscs for these essential elements. Polluted mussels, allowed to depurate in clean seawater, showed excretion rates which were different between elements. Moreover the subcellular distribution was characteristic for each metal but the patterns were similar in wild populations as well as in transplanted and depurating mussels, indicating that the same subcellular fractions are involved in the uptake, storage and excretion of metals. From the results, the different time-integration capacities of mussels for various elements appear to be related to the cellular mechanisms involved in their detoxification and/or excretion.

Induction of DNA strand breakage and apoptosis in the eel Anguilla anguilla

The ability of benzo[a]pyrene, Aroclor 1254, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone to induce DNA strand breaks (SB) and apoptosis in erythrocytes of the European eel (Anguilla anguilla) was investigated following by in vivo exposure. DNA damage was evaluated by the Comet assay, while the diffusion assay was used to investigate the induction of apoptosis 7 days after a single intraperitoneal administration. 2-3-7-8-Tetrachlorodibenzo-p-dioxin induced the highest genotoxic effect, followed by benzo[a]pyrene, while the other two substances had limited effects. A significant induction of apoptosis was observed at the highest doses after exposure to benzo[a]pyrene, when DNA damage was also elevated. The occurrence of apoptotic cells after exposure to Aroclor, 2-3-7-8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone was quite variable and did not show clear dose-related responses. The role of oxidative stress in mediating DNA damage was also discussed.

Seasonal variations of susceptibility to oxidative stress in Adamussium colbecki, a key bioindicator species for the Antarctic marine environment.

The area of free radical biology is of increasing interest for marine organisms since the enhanced formation of reactive oxygen species (ROS) is a common pathway of toxicity induced by stressful environmental conditions. In polar environments responses of the antioxidant system could be useful as an early detection biomarkers of unforeseen effects of human activities which are progressively increasing in these remote areas. However, the characterization of antioxidant defences in appropriate sentinel species is of particular value also in terms of a possible adaptation to this extreme environment. The scallop, Adamussium colbecki, is a key species for monitoring the Antarctic environment and, besides single antioxidants, the total oxyradical scavenging capacity (TOSC) assay has been recently used for quantifying the overall ability of this organism to neutralize peroxyl radicals (ROO*), hydroxyl radicals (*OH) and peroxynitrite (HOONO). The aim of this work was to obtain a better characterization of these biological responses which can indicate the occurrence of biological disturbance; in this study the total oxyradical scavenging capacity was further analyzed to assess the presence of seasonal fluctuations in the susceptibility to oxidative stress in this species. The capability to neutralize peroxyl radicals and hydroxyl radicals increased at the end of December, while resistance towards peroxynitrite did not show any significant variations during the Antarctic summer. These results suggest the occurrence of metabolic changes which mainly influence intracellular formation of ROO* and *OH, with more limited effects on HOONO. Despite the limited time window analyzed, as a typical constraint in Antarctic research at Terra Nova Bay, an increased resistance to these specific oxyradicals might be related to the period of highest feeding activity, or to other intrinsic factors in the animals physiology such as the phase of reproductive cycle.

Mytilus galloprovincialis as a bioindicator of lead pollution : biological variables and cellular responses

Abstract The mussel Mytilus galloproaincialis was used as a bioindicator species in a lead-polluted area of the North Tyrrhenian Sea. A pronounced seasonality was observed in Pb concentration within the digestive gland, whereas the total Pb content of the organ appeared less variable. This difference is mainly due to the influence of gonadal development on digestive gland weight. Control mussels transplanted for 6 months into the polluted area showed a strong increase of Pb, which reached a steady state after 4 weeks. Subcellular responses were investigated in mussels sampled from the polluted environment. A severe disturbance of the lysosomal system was demonstrated, indicating the utility of stress indices based on the responses of these organelles to pollutants. Our results are discussed in terms of their implications for biomonitoring programs.