Biochemical responses to cadmium exposure in Oncorhynchus mykiss erythrocytes.

Cd is known for its carcinogenic effects, however its mechanism of toxicity and in particular its ability to promote oxidative stress is debated. In fact, although it is considered a redox-inactive metal, at high concentration Cd was shown to promote indirectly oxidative stress. In this study we investigated metal accumulation in ex vivo exposed trout (Oncorhynchus mykiss) erythrocytes and Cd dose-dependent effect in terms of RBC viability, cytosolic and mitochondrial ROS levels as well as its effects on mitochondrial membrane depolarization, hemoglobin stability and precipitation. In the concentration range used, Cd did not affect cell viability. However, metal accumulation was associated with an increase in all oxidative indexes evaluated, except mitochondrial superoxide anion production that, on the contrary, was significantly decreased, probably due to a lowered respiration rate associated with interference of Cd with complex I, II and III, as suggested by the observed Cd-dependent mitochondrial membrane depolarization. On the other hand, hemoglobin destabilisation seems to be the major trigger of oxidative stress in this cell type.

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.

Effects of waterborne Cu exposure in gilthead sea bream (Sparus aurata): a proteomic approach.

Aquatic organisms may suffer from exposure to high Cu concentrations, since this metal is widely used in feed supplementation, in pesticide formulation and as antifouling. Chronic exposure to Cu, even at sub-lethal doses, may strongly affect fish physiology. To date, several biomarkers have been used to detect Cu exposure in fish producing contrasting results. Therefore, we used a proteomic approach to clarify how Cu exposure may affect the serum proteome of gilthead sea bream (Sparus aurata), since serum could be considered a good source of early-biomarkers of Cu toxicosis. For this purpose we exposed juvenile gilthead sea bream to waterborne Cu (0.5 mg/L). Our results indicate that fish tightly regulate circulating Cu levels, which are not affected by metal exposure. This homeostatic control is mainly achieved by the liver, able to excrete high amounts of the metal via bile. Cu exposure caused differential expression of several serum proteins, 10 of which were identified by Mascot and BLAST search. All these proteins, with the exception of growth hormone receptor and γ-glutamyl-carboxylase, can be related to: 1) Cu-induced hepatotoxicity (cytochrome oxidase subunit I, alanine aminotransferase, glutathione S-transferase); 2) potential immunosuppression due to interference of Cu with the inflammation/immunity network (α-1 antitrypsin, angiotensinogen, complement component C3, recombination-activating protein-1 and warm temperature acclimation-related 65 kDa protein).

Metallothionein cDNA cloning, metallothionein expression and heavy metals in Scapharca inaequivalvis along the Northern Adriatic coast of Italy.

The aims of this work were: (1) identification of the metallothionein (MT) gene coding sequence in order to prepare an MT probe in Scapharca inaequivalvis and (2) quantification of Cd, Zn, Cu, MT and MTmRNA expression in tissues of molluscs from three areas along the Northern Adriatic coast of Italy. By RT-PCR we cloned the MTcDNA of S. inaequivalvis using the RNA extracted from hepatopancreas of specimens exposed to Cd. The 61 amino acids sequence of MT was deduced and was 70% identical to S. brughtonii MT. Cd concentration in molluscs from the wild was significantly higher in gills from specimens sampled near Ravenna. Zn concentration in the same tissue was significantly higher in Ravenna with respect to Porto Garibaldi while no difference with respect to Cesenatico was detected. Cu levels showed significant differences among sites in gills and mantle whereas values in the hepatopancreas were similar in all sites. The low MT levels were indicative of a low metal exposure; few differences were found in MTmRNA concentrations, which resulted significantly higher in hepatopancreas of molluscs from Porto Garibaldi.

Trace Elements in Home-Processed Food Obtained from Unconventional Animals.

Wild animals have been used as food since ancient times and, currently, the consumption of unconventional animals is increasing worldwide. The process of cooking meat using traditional recipes includes a variety of ingredients, which can influence the total metal intake from the diet. In this study, the concentrations of eight essential (Fe, Zn, Cu, Mn, Se, Ni, Mo, and Co) and six non-essential (Pb, Cd, Hg, Al, As, and Cr) trace elements were determined in home-processed food obtained from snails and from three common species of game animals (woodcock, pheasant, and hare), seasoned with anchovies, mushrooms, and different vegetables using inductively coupled plasma mass spectrometry (ICP-MS). In general, Fe was the most abundant trace element, ranging from 18 ± 8 µg/g in pheasant to 99 ± 76 µg/g in snail, and Co was the least abundant, ranging from 0.007 ± 0.003 µg/g in hare to 0.093 ± 0.048 µg/g in snail. Regarding the non-essential trace elements, Pb concentrations showed wide variations, reaching a concentration of 17.30 µg/g in hare, while Cd concentrations were higher in snail, ranging from 0.18 to 0.46 µg/g. These alternative food sources can offer an important contribution to the human nutritional requirements of essential trace elements, in particular of Fe. The high concentrations of Pb and Cd present in some samples should be considered as potentially dangerous for the consumers.

Environmental exposure to non-essential trace elements in two bat species from urbanised (Tadarida teniotis) and open land (Miniopterus schreibersii) areas in Italy.

Bats are particularly suited as bioindicators of trace element pollution due to their longevity and their position in the trophic chain. In this study, the concentrations of ten non-essential trace elements (Al, As, Ba, Cd, Hg, Pb, Sb, Sr, Th, Tl) were determined in the tissues (whole body, skin-fur, skinned body, liver, kidney and bone) of lactant Tadarida teniotis from a nursery colony in Rome. A large number of bats from this nursery died before fledging and had bone deformities and fractures. The concentrations of non-essential trace elements in bone and whole body were also analysed in adult specimens of Miniopterus schreibersii from a colony located in a natural park in Northern Italy. In lactant T. teniotis, the Pb concentration decreased in the following order: bone>liver>skinned body>whole body>skin-fur>kidney, and exceeded the toxic threshold associated with negative effects reported for different mammalian species. The levels of the other non-essential trace elements were within a range indicative of low environmental contamination in both species. Significant interspecies differences (P < 0.05) were observed for concentrations of Pb and Ba, higher in the bones of T. teniotis, and of Cd, Hg and Sr, higher in the bones of M. schreibersii. In lactant T. teniotis, the different sources of Pb exposure, through inhalation and/or food, may represent a potential threat to the colony of this synanthropic European bat.

Metal distribution and metallothionein in loggerhead (Caretta caretta) and green (Chelonia mydas) sea turtles.

The first aim of our study was to determine the concentrations of selected trace elements (Zn, Cu, Fe, Mn, Cd and Pb) in tissues of green turtles from Tortuguero National Park on the North Caribbean coast of Costa Rica and of loggerheads from the Mediterranean Sea. Zn, Cu, Fe, Mn and Cd were present at detectable concentrations in all samples and showed clear organotropism, whereas Pb was not always over the detection limit and did not show any particular tissue distribution. The two species presented significant differences: Cu and Cd in liver and kidney of Chelonia mydas were significantly higher with respect to the concentrations found in Caretta caretta. The second and major goal of our study was to evaluate hepatic and renal metallothionein (MT) as a biomarker of environmental metal exposure. The present paper is the first to describe and quantify MT in kidney and liver of loggerhead turtles and in kidney of green turtles. MT concentrations were higher in green than in loggerhead turtles. In addition, positive correlations were found between Cu and Cd concentrations and Cu-MT and Cd-MT in liver and kidney in both species, suggesting a pivotal role of MT in metal storage and detoxification. The quantification of metals and MT in liver and kidney may be a valid biomarker of metal exposure in the aquatic environment to assess the health of marine sea turtles as long as accurate analytical methods are adopted.

Metallothionein functions and structural characteristics.

Metallothioneins (MTs) are low molecular weight proteins characterized by a high cysteine content and give rise to metal-thiolate clusters. Most MTs have two metal clusters containing three and four bivalent metal ions, respectively. The MT gene family in mammals consists of four subfamilies designated MT-1 through MT-4. MT-3 is expressed predominantly in brain and MT-4 in differentiating stratified squamous epithelial cells. Many reports have addressed MT structure and function, but despite the increasing experimental data several topics remain to be clarified, and the true function of this elusive protein has yet to be disclosed. Owing to their induction by a variety of stimuli, MTs are considered valid biomarkers in medicine and environmental studies. Here, we will discuss only a few topics taken from the latest literature. Special emphasis will be placed on MT antioxidant functions, the related oxidation of cysteines, which can give rise to intra/intermolecular bridges, and the relations between MTs and diseases which could be originated by metal dysregulation.

Trace elements in unconventional animals: A 40-year experience.

The role of trace elements in animal health has attracted increasing interest in recent years. The essentiality and toxicity of these elements have been extensively investigated in humans, laboratory animal models and partially in domestic animals, whereas little is known about trace element metabolism in most living organisms. Forty years ago our research started on Cd metabolism in molluscs, thereafter expanding to Zn, Cu, and Fe metabolism in many unconventional animal species of veterinary interest. This review summarizes the main results obtained over this long period of time: some of the findings are original and have not been published to date. They are discussed in more detail and compared with data obtained in conventional animals, including man.

Distribution of Cd, Zn, Cu and Fe among selected tissues of the earthworm (Allolobophora caliginosa) and Eurasian woodcock (Scolopax rusticola).

We have measured the concentrations of heavy metals in soils, earthworms and tissues of woodcocks in Quaderna Valley, northern Italy. The soil concentration of metals analysed in this research is consistent with data reported by other authors for uncontaminated or slightly contaminated soils. In earthworms, metals were mostly accumulated in the encapsulating chloragogenous tissue; the positive correlation between Cu concentration in the soil and in earthworms is noteworthy. Heavy metal distribution in the tissues of woodcock showed that Cd accumulation in the kidney was linked to the diet. Cu and Fe were preferentially concentrated in the liver and Zn in the testis. Kidney Cd and Zn concentrations were higher in adults than in juveniles. In addition, a main kidney metallothionein isoform, containing Cd and Zn, was isolated. In the kidney, Cd levels were linearly correlated with the concentration of metallothionein. Of the investigated metals, Cd raises the greatest concern, due to the increasing soil contamination by human activities.

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

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'.

Effects of chronic treatment with sodium tetrachloroaurate(III) in mice and membrane models.

Gold is a nonessential element with a variety of applications in medicine. A few gold(I) compounds are used in the clinics for treatment of rheumatoid arthritis and of discoid lupus. Some novel gold(III) compounds are under evaluation as anticancer agents. It is known that gold compounds generally produce toxic effects on the kidneys and characteristic lesions in the brain. However, information concerning the neurotoxicity of gold derivatives in humans as well as in experimental toxicology is rather scarce. For this reason we tried to shed some further light on this aspect of gold neurotoxicity by chronic treatment of mice with sodium tetrachloroaurate(III) in order to observe possible biophysical and morphological alterations that may occur in the brain. Chronic gold treatment resulted in a markedly decreased expression of metallothioneins and of glial fibrillary acidic protein in astrocytes of different brain areas. To examine its effects on cell membranes, interactions of sodium tetrachloroaurate(III) with molecular models were also evaluated. The models consisted in bilayers built-up of classes of phospholipids located in the outer and inner monolayers of biological membranes. Structural perturbation of cell membrane models was observed only at concentrations 10(5) times higher than those detected in the brains of animals after three months' treatment. These results show that toxic effects on animal brain upon treatment with sodium tetrachloroaurate develop with difficulty and may be observed only at high doses.

Metal concentrations (Cu, Zn and Cd) and metallothionein expression in Sparus aurata exposed to waterborne copper.

Metal concentrations in different tissues of Sparus aurata are linked to the functional role of the investigated organ: in the liver, Zn and Cu are highly concentrated, due to the detoxification and storage processes typical of the hepatic tissue. Comparison between control specimens of farmed and wild gilthead seabream resulted in significant differences in Zn and Cu concentrations between the tissue metal levels of the two populations. The cadmium levels, always less than 1 microg/g wet weight, were indicative of a low exposure to the non essential element. In Cu-exposed fish the metal was mostly accumulated in the liver and to a lesser extent in the gills in a dose-dependent manner, whereas zinc concentrations were unaffected by the metal treatment. In the liver a 1.6-fold and 4-fold increase was observed in fish exposed to 0.1 microg/mL and 0.5 microg/mL respectively; moreover, Cu exposure caused an increase of MTmRNA and CuMT. On this basis, the two molecular parameters could be considered as useful biomarkers of an early cellular response against Cu toxicity.

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.

Reference values for hematology and plasma biochemistry variables, and protein electrophoresis of healthy Hermann's tortoises (Testudo hermanni ssp.).

BACKGROUND: Hermann's tortoise, Testudo hermanni, is currently on the International Union for Conservation of Nature (IUCN) red list of endangered species. Reptile medicine relies also on laboratory analyses to evaluate health status, but reference ranges for hematology and biochemistry variables and protein electrophoresis in plasma of healthy tortoises are not available. OBJECTIVE: The purposes of this study were to establish reference ranges for select hematologic and biochemical variables in clinically healthy Hermann's tortoises, and evaluate the impact of sex and season. METHODS: Blood samples were collected from 34 healthy tortoises at the end of September and beginning of July. Blood smears, HCT, concentrations of HGB and select plasma biochemical analytes, select enzyme activities, and plasma protein fractions were evaluated. Reference ranges were determined and checked for influence of sex and sampling time point. RESULTS: Typical reptilian RBC and WBC were observed in blood smears. HCT and concentrations of HGB, uric acid and urea, and ALT and AST activities were significantly higher in males than in females. Concentrations of glucose, uric acid, and phosphate, and AST activity were significantly higher at the beginning of July, whereas concentrations of urea and Cl were higher at the end of September prior to hibernation. The electrophoretic protein fractions included albumin, and α, ß, and γ globulins. CONCLUSIONS: The reference ranges defined in the present study are useful for clinical tortoise medicine and conservation. Sex and seasonal sampling were identified as factors significantly affecting hematology and blood chemistry analytes; they should be taken into consideration when assessing tortoise health status.

Trace elements and metallothionein in liver and kidney of Felis catus.

Trace metals such as Zn, Cu, and Fe are essential for life; differently, no biochemical function is known for Cd. Changes in dietary metal concentrations can cause deficiency or toxicity. Studies on trace elements in cat are lacking. This paper aimed to analyze Zn, Cu, Fe and Cd concentrations in liver and kidney of pathological domestic cat and to isolate metallothionein (MT) in these tissues. It was not possible to explore a possible correlation between metal concentrations and pathologies because the incidence for each of them was too low. Fe was the most abundant metal; in particular, the liver accumulates average Fe concentrations one order of magnitude higher than Zn and Cu, ranging from 66.75 and 1,444.23 microg/g. Significantly, higher levels of Fe were found in the liver of elder animals. Zn concentrations varied between 26.31 and 84.78 microg/g in the liver whereas in the kidney, ranged between 7.69 and 71.15 microg/g. Cu concentrations were between 2.37 and 112.91 microg/g in liver and between 2.12 and 9.85 microg/g in kidney. Cd was the least abundant metal with the exception of the kidney of the oldest cats where it reached a maximum of 13.71 microg/g. Gel-filtration metal distribution profiles from cytosolic extracts revealed the presence of Cd, Cu, Zn thioneins either in the liver or in the kidney. Because tissue samples were taken from pathological cats from different breed and age, care must be taken to use these data as a baseline profile of trace elements in healthy animals. Our results are indicative that for some specimens the feed levels of Fe and Cu could be higher than the optimal dietary intake and in few cats, there was also an exposure to Cd that was counteracted by MT biosynthesis.

Multiple scattering x-ray absorption studies of Zn2+ binding sites in bacterial photosynthetic reaction centers.

Binding of transition metal ions to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides has been previously shown to slow light-induced electron and proton transfer to the secondary quinone acceptor molecule, Q(B). On the basis of x-ray diffraction at 2.5 angstroms resolution a site, formed by AspH124, HisH126, and HisH128, has been identified at the protein surface which binds Cd(2+) or Zn(2+). Using Zn K-edge x-ray absorption fine structure spectroscopy we report here on the local structure of Zn(2+) ions bound to purified RC complexes embedded into polyvinyl alcohol films. X-ray absorption fine structure data were analyzed by combining ab initio simulations and multiparameter fitting; structural contributions up to the fourth coordination shell and multiple scattering paths (involving three atoms) have been included. Results for complexes characterized by a Zn to RC stoichiometry close to one indicate that Zn(2+) binds two O and two N atoms in the first coordination shell. Higher shell contributions are consistent with a binding cluster formed by two His, one Asp residue, and a water molecule. Analysis of complexes characterized by approximately 2 Zn ions per RC reveals a second structurally distinct binding site, involving one O and three N atoms, not belonging to a His residue. The local structure obtained for the higher affinity site nicely fits the coordination geometry proposed on the basis of x-ray diffraction data, but detects a significant contraction of the first shell. Two possible locations of the second new binding site at the cytoplasmic surface of the RC are proposed.