Gloria Isani

Increased acetylcholinesterase activities in specimens of Sparus auratus exposed to sublethal copper concentrations

The present study looks at possible changes in the activity of acetylcholinesterase (AChE) in tissues (brain and white muscle) of the Mediterranean bony fish Sparus auratus after a 20 days exposure to sublethal concentrations (0.1 or 0.5 ppm) of copper in the marine water and on control untreated animals. The trials also included measurements of Cu concentration in the tissues to evaluate possible metal accumulation. Moreover, sedimentation analysis as well as V(max) and K(m) determination were carried out in tissue extracts of Cu-exposed or control animals. V(max) and K(m) were also determined with or without addition of Cu(2+) in the assay. No Cu accumulation occurred in brain and muscle after Cu exposure. AChE showed in both tissues a molecular polymorphism with putative globular (G) and asymmetric (A) forms. Cu exposition led to an increased specific activity and improved catalytic efficiency of AChE in brain and muscle, seemingly regarding G forms. The increase in catalytic efficiency also resulted from the in vitro assay with tissue extracts and Cu(2+) addition. The higher AChE activity and catalytic efficiency in both tissues after Cu exposition and without metal accumulation, suggests an increase of free Cu aliquot into the cells, likely due to mechanisms of metal homeostasis.

Correlation between metallothionein and energy metabolism in sea bass, Dicentrarchus labrax, exposed to cadmium☆

Specimens of sea bass (Dicentrarchus labrax) were exposed to two different cadmium concentrations (0.5 and 5 μg Cd2+/ml seawater) for a period of 7 days. Cadmium accumulated in the tissues of D. labrax in the following order: kidney > liver > gills at both concentrations. Accumulation patterns in fish exposed to 0.5 μg Cd2+/ml seawater were different with respect to 5.0 μg Cd2+/ml seawater. At both Cd concentrations a similar stress situation occurred during the first 4 hr as noted by the depletion of glycogen stores and the increase in free glucose in the muscle; metallothionein was induced in the liver, but failed to bind all the cytosolic Cd, which was in part bound to high-molecular-weight ligands. Fish recovered from this initial stress situation within 24 hr as indicated by the increase in glycogen and the decrease of glucose. Long-term effects were clearly dependent upon metal concentration: at lower Cd exposure, metallothionein induction increased linearly with time and counteracted the toxic effect of the metal; on the other hand, when fish were exposed to 5.0 μg Cd2+/ml seawater a clear stress occurred at the end of the exposure, as indicated by the notable decrease of glycogen stores, the increase of free glucose, the decrease of AEC in the muscle and the increase of Cd bound to high-molecular-weight ligands in the liver.

Energy metabolism of the mussel, Mytilus galloprovincialis, during long-term anoxia

Abstract In the mussel, Mytilus galloprovincialis , the pattern of metabolic adaptations to long-term anoxia was clearly biphasic. The first phase (transition stage) lasted 24 hr and was characterized by the decrease of adenylate energy charge, the consumption of aspartate and the accumulation of succinate and alanine. The second phase (stationary stage) was characterized by the depletion of glycogen stores, the production of propionate, which was subsequently excreted into incubation water, and the metabolic depression. The ATP turnover rate was reduced by a factor of 3.9 from short-term anoxia to long-term anoxia.

Zinc-bound metallothioneins as potential biological markers of ageing

Metallothioneins (MTs) (I+II) play pivotal roles in metal-related cell homeostasis because of their high affinity for metals forming clusters. The main functional role of MTs is to sequester and/or dispense zinc participating in zinc homeostasis. Consistent with this role, MT gene expression is transcriptionally induced by a variety of stressing agents to protect cells from reactive oxygen species. In order to accomplish this task, MTs induce the secretion of pro-inflammatory cytokines by immune and brain cells, such as astrocytes, for a prompt response against oxidative stress. These cytokines are in turn involved in new synthesis of MTs in the liver and brain. Such protective mechanism occurs in the young-adult age, when stresses are transient. Stress-like condition is instead constant in the old age, and this causes continuous stealing of intracellular zinc by MTs and consequent low bioavailability of zinc ions for immune, endocrine, and cerebral functions. Therefore, a protective role of zinc-bound MTs (I+II) during ageing can be questioned. Because free zinc ions are required for optimal efficiency of the immune-endocrine-nervous network, zinc-bound MTs (I+II) may play a different role during ageing, switching from a protective to a deleterious one in immune, endocrine, and cerebral activities. Physiological zinc supply, performed cautiously, can correct deficiencies in the immune-neuroendocrine network and can improve cognitive performances during ageing and accelerated ageing. Altogether these data indicate that zinc-bound MTs (I+II) can be considered as novel potential markers of ageing.

Seasonal dependence of cadmium accumulation and Cd-binding proteins in Mytilus galloprovincialis exposed to cadmium.

At different periods of the year specimens of Mytilus galloprovincialis were exposed to 0.5 microg Cd/ml seawater for 7 days. Concentrations of trace elements (Cd, Zn, Cu and Fe) and Cd-binding proteins in gills, viscera, muscle and mantle were determined after 1 weeks exposure. Cadmium accumulation was higher in May and June and was tissue dependent; it was highest in the gills and decreased in the order: gills > viscera > mantle and adductor muscle. Significant seasonal variations of zinc, copper and iron, were also found, in both exposed and control molluscs. The percentage of Cd distribution between cytosol and pellet changed during the year; a clear shift from the particulate fractions to the cytosolic fractions was measured during May and June, especially in the gills. Metallothionein (MT) was the main ligand responsible for Cd accumulation, and this protein reached a maximum between May and June. Inclusion of mercaptoethanol during the purification procedure was found to improve MT isolation by gel chromatography. In the absence of mercaptoethanol, MT showed polymerization patterns which were season dependent and temperature independent, whereas its concentration was increased in mussels kept at higher temperature.

Comparative toxicity of CuO nanoparticles and CuSO4 in rainbow trout.

This study compared the toxicity and accumulation of two different Cu compounds, CuO nanoparticles (NPs) and soluble CuSO4, in erythrocytes and different tissues in rainbow trout (Oncorhynchus mykiss). The crystal structure of CuO NP analysed by XRD indicates that the NP are Tenorite, a monoclinic CuO. The in vitro toxicity results indicate that both Cu compounds increase the haemolysis rate in a dose-dependent way, but the effect was reduced treating cells with CuO NP. Moreover, both Cu compounds induce DNA damage and the entity of the damage, similarly to haemolysis, was more marked in cells treated with CuSO4. In vivo results, obtained after intraperitoneal injection, showed that Cu concentrations were significantly higher in gills (p<0.0001), kidney (p=0.007) and liver (p<0.05) of exposed fish with a significant increase in plasma Cu concentration 15h after CuSO4 treatment. Cu concentrations were significantly higher in fish exposed to CuSO4 than CuO in kidney (p<0.05) and gills (p<0.0001). Significant DNA damage with respect to controls was detected only when Cu was injected as CuSO4. The present data could serve to evaluate environmental Cu toxicity in fish depending on Cu speciation.

Metallothioneins, Unconventional Proteins from Unconventional Animals: A Long Journey from Nematodes to Mammals

Metallothioneins (MTs) are ubiquitous low molecular weight cysteine-rich proteins characterized by high affinity for d10 electron configuration metals, including essential (Zn and Cu) and non-essential (Cd and Hg) trace elements. The biological role of these ancient and well-conserved multifunctional proteins has been debated since MTs were first discovered in 1957. Their main hypothesized functions are: (1) homeostasis of Zn and Cu; (2) detoxification of Cd, and Hg; and (3) free radical scavenging. This review will focus on MTs in unconventional animals, those not traditionally studied in veterinary medicine but of increasing interest in this field of research. Living in different environments, these animals represent an incredible source of physiological and biochemical adaptations still partly unexplored. The study of metal-MT interactions is of great interest for clinicians and researchers working in veterinary medicine, food quality and endangered species conservation.

Metallothioneins (I+II) and thyroid-thymus axis efficiency in old mice: role of corticosterone and zinc supply.

Abstract Thymic atrophy or thymus absence causes depressed thyroid–thymus axis (TTA) efficiency in old, young propyl-thiouracil (PTU) (experimental hypothyroidism) and in young-adult thymectomised (Tx) mice, respectively. Altered zinc turnover may be also involved in depressed TTA efficiency. Zinc turnover is under the control of zinc-bound metallothioneins (Zn-MTs) synthesis. Thyroid hormones, corticosterone and nutritional zinc affect Zn-MT induction. Zn-MT releases zinc in young-adult age during transient oxidative stress for prompt immune response. In constant oxidative stress (ageing and liver regeneration after partial hepatectomy), high liver Zn-MTs, low zinc ion bioavailability and depressed TTA efficiency appear. This last finding suggested that MT might not release zinc during constant oxidative stress leading to impaired TTA efficiency. The aim of this work/study is to clarify the role of Zn-MTs (I+II) in TTA efficiency during development and ageing. The main results are (1) Old and PTU mice display high corticosterone, enhanced liver MTmRNA, low zinc and depressed TTA efficiency restored by zinc supply. Increased survival and no significant increments in basal liver Zn-MTs proteins occur in old and PTU mice after zinc supply. (2) Lot of zinc ions bound with MT in the liver from old mice than young (HPLC). (3) Young-adult Tx mice, evaluated at 15 days from thymectomy, display high MTmRNA and nutritional–endocrine–immune damage restored by zinc supply or by thymus grafts from old zinc-treated mice. (4) Young-adult Tx mice, but evaluated at 40 days from thymectomy, display natural normalisation in MTmRNA and nutritional–endocrine–immune profile with survival similar to normal mice. (5) Stressed (constant dark for 10 days) mice overexpressing MT display low zinc, depressed immunity, reduced thymic cortex, high corticosterone, altered thyroid hormones turnover showing a likeness with old mice. These findings, taken altogether, show that corticosterone is pivotal in MTs induction under stress. MTs bind preferentially zinc ions in constant oxidative stress, but with no release of zinc from MT leading to impaired TTA efficiency. Zinc supply restores the defect because zinc has no interference in affecting pre-existing Zn-MTs protein concentrations in old and PTU mice. Therefore, free zinc ions are available for TTA efficiency after zinc supply. Thymus from old zinc-treated mice induces the same restoring effect when transplanted in Tx recipients. However, Tx mice display natural normalisation in MTmRNA and in nutritional–endocrine–immune profile in the long run. Therefore, Zn-MTs (I+II) are crucial in zinc homeostasis for endocrine–immune efficiency during the entire life assuming a role of potential and novel ‘biological clock of ageing’.

Effect of Copper Exposure on the Antioxidant Enzymes in Bivalve Mollusc Scapharca inaequivalvis

G. Isani, M. Monari, G. Andreani, M. Fabbri and E. Carpenè* Department of Biochemistry, Veterinary Section, Alma Mater Studiorum, University of Bologna, V ia T olara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy *Correspondence: Dipartimento di Biochimica ‘G. Moruzzi ’, Sezione di Biochimica Veterinaria, Facoltà di Medicina Veterinaria, Alma Mater Studiorum, Università di Bologna, V ia T olara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy E-mail: carpene@vet.unibo.it

Energy metabolism during anaerobiosis and recovery in the posterior adductor muscle of the bivalve Scapharca inaequivalvis (Bruguiere)

Abstract 1. 1. In the posterior adductor muscle of Scapharca inaequivalvis both aspartate and glycogen were substrates for anaerobic metabolism. ATP and arginine phosphate only contributed to a small extent. Alanine and succinate were major end products, whereas opines were minor end products. Lactate production was not observed. 2. 2. The ATP turnover rate was reduced by a factor of 6 in the later anoxic period. Glycolysis and aspartate contributed about 90% either during the initial step or during the prolonged anoxia. 3. 3. Recovery of 24 hr was not enough for a complete restoration of energy reserves, whereas anaerobic end products were eliminated over this period. There was no strombine or lactate accumulation.