M. Orunesu

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.

Role of metallothioneins in Cu and Cd accumulation and elimination in the g gill and digestive gland cells of mytilus galloprovincialis lam.

Abstract The data presented in this paper demonstrate that exposure of mussels to Cu (40 μg/liter) or Cd (200 μg/liter) for a period of 2 days is sufficient to induce Cu or Cd thioneins in the gills of the metal-exposed animals. The concentrations of Cu and Cd thioneins increase during the period of metal exposure (3–4 weeks), confirming the fact that metallothioneins play a fundamental role in the accumulation of these metals in the tissues examined (gills and digestive gland). Moreover, it has been demonstrated that when metal-loaded mussels are returned to the field for the recovery period, Cu is rapidly eliminated from the gill and digestive gland cells, showing a biological half-life of 9–10 days, whereas Cd is released much more slowly from the tissues, only about 50% of the total metal being lost after 4 months of detoxification. Interestingly enough, during the detoxification period the concentration of the metal bound to thioneins also follows the same pattern as the total metal present in the tissues, in fact the concentration of Cu thioneins decreases near the control level in about 24 days but, in contrast, the Cd-thionein concentration in the cytisol of the gill and digestive gland cells remaains high, decreasing only by about 40% at the end of the 4 month recovery period. The data presented also demonstrate that when Cd-loaded, detoxified mussels are exposed to Cu (100 μg/liter) for 6 h, the metal taken up by the gill cells is able to displace the Zn which is always present in the Cd-thionein fraction, but it does not, however, displace the Cd present. In this case also, period (20 days), whereas the Cd-thionein content decreases slightly. Such data seem to indicate that the metabolic characteristics of thioneins are closely related to the metal bound to them, particularly where the elimination rate of the metals from the cells is concerned.

Stress on stress response: A simple monitoring tool in the assessment of a general stress syndrome in mussels

Abstract Data are reported demonstrating that short term exposure to sublethal concentrations (less than μm) of pollutants, such as Cu2+, DMBA, Aroclor 1254, significantly reduced mussel capacity to survive in air. This effect was markedly dose-dependent, and was strongly increased by pollutant mixtures. The results of this study indicate that the reduction of survival in air, or stress on stress response, could be measured as an index of a general stress syndrome in mussels. This parameter shows a sensitivity which is in the same range of other commonly used general stress indices at the cellular level (i.e. lysosomal membrane stability). Moreover, the methodology is simple, low in cost and does not require sophisticated equipment. Therefore, the stress on stress response can be integrated in monitoring programmmes as a general stress index for the assessment of contaminated coastal areas.

Rapid induction of copper-binding proteins in the gills of metal exposed mussels

Abstract The exposure of the mussel Mytilus galloprovincialis Lam. to sublethal concentrations of Cu 2+ (0.08 ppm) for 24–48 hr results in the appearance of low molecular weight (about 12,000), heat stable copper-binding proteins in the soluble extracts obtained from the gills. 2. The soluble low molecular weight copper-binding proteins do not occur in the gill extracts from the controls and from the mussels submitted to a short intoxication (4 hr). 3. The incorporation of 35 S cysteine into the 12,000 molecular weight copper-rich fraction from the gills of mussels exposed for 48 hr to Cu 2+ is 7–10 fold greater than in controls. This result strongly supports the inducible nature of these proteins. 4. The low molecular weight proteins synthesized in response to the uptake of Cu 2+ show many of the characteristics of metallothioneins, having a high SH content, few aromatic amino acids and a high metal binding capacity.

Lipid peroxidation and level of antioxidant compounds (GSH, vitamin E) in the digestive glands of mussels of three different age groups exposed to anaerobic and aerobic conditions

Abstract The effects of anaerobiosis and subsequent re-oxygenation on lipid peroxidation and on the levels of the two major antioxidant compounds (GSH and vitamin E) were evaluated in the digestive gland of mussels (Mytilus edulis L.) of three different age groups (I = 2–4 years; II = 6–8 years; III = > 10 years). The lipid peroxidation level, as evaluated by malondialdehyde (MDA) concentration is significantly higher in the digestive gland of the > 10 year old mussels with respect to the younger animals. This fact appears to be related to the lower levels of GSH and vitamin E found in the tissue of the aged mussels. A decrease both in the MDA concentration and in the level of GSH and vitamin E was observed in mussels of the three age groups exposed for 24 h to anaerobic conditions. After re-immersion in aerated seawater the MDA level greatly increased only in the > 10 year old mussels. The results demonstrate that, during anaerobiosis, a reduction of the cellular antioxidant compounds occurs in the digestive gland of mussels. In the > 10 year old animals, such a decrease in the cellular defence systems would result in a major oxidative stress, as evidenced by the high MDA level observed in the older mussels after re-oxygenation.

Effects of copper on the uptake of amino acids, on protein synthesis and on ATP content in different tissues of Mytilus galloprovincialis Lam.

Abstract The effects of sublethal concentrations of Cu ++ (0·08 ppm) on the uptake of amino acids, protein synthesis and ATP content in different tissues of Mytilus galloprovincialis have been evaluated. It has been shown that the uptake of amino acids by the gills, digestive gland and mantle is reduced to 5–10% of the control value after 7 days of exposure to the metal. In the mussel tissues examined the rate of protein synthesis, as measured by following the incorporation of labelled amino acids into trichloroacetic acid-insoluble material, is also significantly reduced (to about 50–70%). Finally, evidence has also been presented showing that, within 7 days of exposure to Cu ++ ions, the ATP concentration in the mussel tissues is reduced to about 70–75%. From these results it is assumed that the decrease in protein metabolism in the copper-exposed mussels may represent a biochemical index of stress exerting a detrimental effect on growth and survival.

Purification and biochemical characterization of a lysosomal copper-rich thionein-like protein involved in metal detoxification in the digestive gland of mussels

1. The lysosomal insoluble Cu-rich protein present in the digestive gland of metal-exposed mussels (Mytilus galloprovincialis Lam.) was extracted and biochemically characterized. 2. The solubilized Cu-protein shows Chromatographic and electrophoretic properties similar to those of mussel metallothioneins. 3. The UV spectrum of the purified lysosomal Cu-protein is typlcal of Cu-thioneins; it consistently shows an amino acid composition similar to that of previously characterized cytosolic metallothioneins. It differs, however, in that it has a low glycine content as well as a high level of aspartic and glutamic acid. 4. The possible role of lysosomes in metallothionein metabolism is discussed.

Changes in activity and mRNA levels of poly(ADP-ribose) polymerase during rat liver regeneration

ADP-ribosylation of nuclear proteins, catalysed by the enzyme poly(ADP-ribose) polymerase, is involved in the regulation of different cellular processes of DNA metabolism. To further clarify the role of the enzyme during proliferating activity of mammalian cells, we have studied the control of gene expression in regenerating rat liver. The changes in activity and mRNA levels were analysed during the early and late phases of the compensatory model. When enzyme activity was measured in isolated liver nuclei obtained at different times after hepatectomy, two different phases were observed: an early wave occurring before the onset of DNA synthesis, and a second one, starting several hours after the onset of DNA synthesis and returning to control values at later times. The evaluation of the enzymatic level in nuclear extracts and by activity gel analysis showed a more gradual increase starting 1 day after hepatectomy, in concomitance with the peak of DNA synthesis. By using a specific murine cDNA probe, a significant enhancement of mRNA levels for poly(ADP-ribose) polymerase was observed during liver regeneration, slightly preceding the onset of DNA synthesis. The results obtained show that changes in poly(ADP-ribose) polymerase activity, during liver regeneration, are associated both to early events preceding the increase in DNA synthesis and to later phases of the cell proliferation process.

Differences in lipid composition of cell membranes from Antarctic and Mediterranean scallops

The molecular mechanisms evolved in adaptation of cellular membranes to low temperatures have been studied. The lipid composition of membranes from digestive gland cells of two Pectinidae living at different temperatures such as Adamussium colbecki (Smith, 1902), an Antarctic mollusc, and Pecten jacobaeus L., from the Mediterranean Sea, has been compared. Our results demonstrate that cold adaptation of the membranes from Pectinidae digestive gland cells mainly implies the regulation of the ratio of short/long and straight/branched fatty acid chains and of the cholesterol content of membranes

Effects of sublethal copper concentrations, temperature, salinity and oxygen levels on calcium content and on cellular distribution of copper in the gills of Mytilus galloprovincialis lam.: A multifactorial experiment

Abstract The paper presents the results of a multifactorial experiment performed to evaluate the effects of simultaneous variations of temperature, salinity, oxygen and Cu 2+ , on copper ‘metabolism’ and calcium content in mussel gills. Our results demonstrate that the factors studied have main and interaction effects not only on the copper level, but also on the ratios between the cytosolic Cu and Cu bound to thioneins, probably resulting in different degrees of toxicity. In addition, it has been shown that the calcium levels are affected by simultaneous variations of the experimental factors. In particular the gills of mussels exposed to Cu 2+ exhibit a significant increase in calcium concentration, thus indicating that copper may alter Ca 2+ homeostasis.