Aldo Viarengo

Mixtures of Chemical Pollutants at European Legislation Safety Concentrations: How Safe Are They?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations.

The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach

Within the frame of the 2nd Iberian Congress of Environmental Contamination and Toxicology (University of the Basque Country, Leioa, June 1998) a workshop was held about the use of biomarkers in marine pollution monitoring. Among others, the following biomarkers received special attention: metallothionein induction, acetylcholinesterase inhibition, cytochrome P450 system induction, imposex, lysosomal enlargement and lysosomal membrane destabilisation, and peroxisome proliferation. These biomarkers can be used to evaluate exposure to and effect of different contaminants (metals, organic xenobiotics and organometallic compounds) and they can be measured using different methodological approaches (biochemistry, cytochemistry, immunochemical methods based on the use of biotechnology). Before the application of a set of biomarkers in pollution monitoring programmes, well-defined protocols of Quality Assurance have to be established to allow adequate comparison of results. It is also necessary to include analysis of standard reference materials and to obtain detailed knowledge of basal values and seasonal variations of the biomarkers in various species, as well as to integrate the information obtained with the different biomarkers. Marine bivalve molluscs such as mussels are appropriate sentinel species for most of the biomarkers proposed except for the induction of the cytochrome P450 system, which should be measured in fish, and the degree of imposex, which is a biomarker of exposure to TBT specifically measured in certain gastropod molluscs. As a result of the workshop, a battery of biomarkers of contaminant exposure and effects are proposed that could be incorporated into programmes monitoring the quality of the coastal environment in the Iberian Peninsula. These measures would be undertaken in conjunction with chemical measures of contaminant burdens in selected sentinel species.

A SIMPLE SPECTROPHOTOMETRIC METHOD FOR METALLOTHIONEIN EVALUATION IN MARINE ORGANISMS : AN APPLICATION TO MEDITERRANEAN AND ANTARCTIC MOLLUSCS

Abstract A spectrophotometric method to evaluate the concentration of metallothioneins in the tissues of marine organisms has been developed. Metallothionein concentration was evaluated utilizing a partially purified metalloprotein fraction obtained by acidic ethanol/chloroform fractionation of the tissue homogenate. The procedure takes into account precautions to obtain a complete metallothionein precipitation and to avoid the oxidation of sulphydryl groups (SH), the contamination by soluble low molecular weight thiols and enzymatic protein degradation which can occur during sample preparation. In the extracts the concentration of metallothioneins, denatured by low pH and high ionic strength, was quantified spectrophotometrically utilizing the Ellmans SH reagent. The SDS-PAGE separation and the fluorimetric analysis of the proteins, labelled with the fluorescent SH reagent Thiolyte, indicated the presence of minimal amounts of SH-proteins different from metallothioneins in the preparation. The spectrophotometric method was applied to quantify the metallothionein concentration in both Mediterranean (M. galloprovincialisis and Antarctic (A. colbecki) molluscs either wild or experimentally exposed to heavy metals (Cu and Cd). Moreover, Zn and metallothionein content in the digestive gland of wild mussels during an annual cycle was evaluated indicating that metallothionein concentration varies with the Zn concentration in the tissues. In conclusion, this spectrophotometric method allows the simple, repeatable and low cost detection of minimal concentrations (nmoles) of metallothionein in biological samples and therefore it is suggested as a tool for metallothionein quantification in eco-toxicological investigations and biomonitoring programmes.

Seasonal variations in the antioxidant defence systems and lipid peroxidation of the digestive gland of mussels

1. The seasonal variations in the level of antioxidant compounds (glutathione (GSH), vitamin E, carotenoids) and in the activity of antioxidant enzymes, superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), GSH-peroxidase (EC 1.11.1.9) in the digestive gland of mussels (Mytilus sp.) were evaluated. The lipid peroxidation process was also measured by determining the tissue concentration of malondialdehyde (MDA). 2. The physiological fluctuations of the antioxidant defence systems were inversely related to the accumulation of lipid peroxidation products (MDA) in the tissue. The observed seasonal variations are presumably related to the changing metabolic status of the animals, itself dependent on such factors as gonad ripening and food availability. 3. In particular, the obtained data indicate that a reduction of the antioxidant defence systems, occurring during winter, could be directly responsible for an enhanced susceptibility of mussels tissues to oxidative stress, as indicated by the high MDA concentration observed in this period.

Heavy metal effects on lipid peroxidation in the tissues of mytilus gallopro vincialis lam.

Abstract 1. The effects of heavy metals on lipid peroxidation in the gills and digestive gland of mussels exposed for six days to Cu 2+ , Cd 2+ or Zn 2+ (40 μg/l/animal) were investigated. 2. In the tissues of Cu-exposed mussels a significant increase of the level of malondialdehyde (MDA), which is indicative of the peroxidative process, and a decrease of the concentration of glutathione were observed. 3. Moreover, in the digestive gland of mussels, copper exposure results in an increase of other carbonyl compounds and in the lysosomal accumulation of lipofuscin granules. 4. The exposure of mussels to Zn or to Cd did not elicit any of the above effects. 5. The results are discussed in relation to the possible role that Cu-induced lysosomal lipofuscin accumulation may play in heavy metal detoxification.

Heavy metals and glutathione metabolism in mussel tissues

Abstract Data are presented on the tissue glutathione content and on the activity of two of the main enzymes involved in GSH metabolism (γ-glutamyl-cysteine synthetase (GCS), GSH transferase (GST)) in the gills and digestive gland of mussels (Mytilus galloprovincialis Lam.) exposed for 1, 4 and 7 days to sublethal concentrations of copper and of mercury, both in the inorganic form of HgCl2 and in the organic form of CH3HgCl. The results indicate that Cu2+ (0.6 μM) and CH3Hg+ (0.2 μM) can affect, although to a different extent, glutathione metabolism in mussel tissues; common traits were a decrease in tissue glutathione content, stimulation of GST activity and inhibition of GSH synthesis rate; on the other hand, inorganic Hg2+ (0.2 μM) did not significantly affect glutathione content and metabolism in mussel tissues. Exposure to copper (0.6 μM) resulted in a decrease in total glutathione content in both gills and the digestive gland that was maximal after the first day, followed by a tendency to recover at longer exposure times. Such a decrease seemed to be mainly related to a stimulation of GST activity, which was transient in gills and persistent in the digestive gland. The tissue glutathione content was also decreased in mussel exposed to methylmercury (0.2 μM); however, such a decrease seemed to be mainly due to inhibition of the GSH synthesis rate and, to a lesser extent, to an increased GST activity. On the contrary, inorganic Hg2+ (0.2 μM) did not significantly affect glutathione metabolism, although mussel exposure to the same concentration of organic and inorganic Hg2+ resulted in similar total metal tissue load in mussel tissues. The role of glutathione as a potential defense against heavy metals is discussed in relation to different routes of metal toxicity and homeostasis in mussel tissues.

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.

Genotoxicity biomarkers in the assessment of heavy metal effects in mussels: experimental studies.

Heavy metals are stable and persistent environmental contaminants. The range of metal concentrations is generally below acute thresholds in coastal areas, where recognition of chronic sublethal effects is more relevant. Evidence of long‐term adverse effects, such as cancer, due to heavy metals in marine animals comes from a number of field and experimental studies. The mechanism of metal carcinogenicity remains largely unknown, although several lines of experimental evidence suggest that a genotoxic effect may be involved. The aim of our study was to evaluate the sensitivity of genotoxicity tests, alkaline elution and micronucleus test, as biomarkers for the detection of heavy metals in mussels as the sentinel species. Experimental studies were carried out on Mytilus galloprovincialis exposed in aquarium (5 days) to different concentrations of three selected metal salts, CuCl2 (5, 10, 20, 40, 80 µg/l/a), CdCl2 (1.84, 18.4, 184 µg/l/a), and HgCl2 (32 µg/l/a), and to a mixture of equimolar doses of the three metals to study the results of their joint action. Metallothionein quantitation was used as a marker of metal exposure. Lysosomal membrane stability was applied to evaluate the influence of physiological status on genotoxic damage. The ranking of genotoxic potential was in decreasing order: Hg > Cu > Cd. Cu and Hg caused an increase of DNA single‐strand breaks and micronuclei frequency. Cd induced a statistical increase of DNA damage, but gave negative results with the micronucleus test. A relationship between genotoxic effects and metallothionein content was observed. Reduction in lysosomal membrane stability with the increasing concentration of heavy metals was also evident. Environ. Mol. Mutagen. 33:287–292, 1999