Metal metabolism in the red alga Cyanidium caldarium and its relationship to metal tolerance

The unicellular red alga Cyanidium caldarium is tolerant to high levels of various metal ions. Cells of this alga cultured with divalent metal ions at 5 mM contained an elevated concentration of each metal, with the highest level for Zn followed by Mn > Ni > Cu. This order is in fair agreement with the toxicity levels reported previously, with the exception of Mn, which shows a toxicity level comparable to that of Ni. Transmission electron microscopy indicated the presence of electron-dense bodies in the algal cells, and elemental analysis by energy dispersive X-ray spectrometry showed high levels of Fe and P in these bodies. Accumulation of Zn was found in these particles in Zn-treated algal cells, whereas no such deposition was found for Cu, Ni, or Mn in cells treated with the respective metals. Although trapping of Zn in the intracellular bodies may contribute to reduction of metal activity in the cells, this effect can be overcome by high intracellular levels of Zn that result in a high degree of toxicity. The correlation between intracellular concentration and toxic levels of metal ions implies that the reduced incorporation of the metals is a major detoxification mechanism in this alga.     

The ultrastructure and calcification of the scales of Tilapia mossambica (Peters)

Summary The scales of Tilapia are surrounded by an envelope of scleroblasts responsible for the production of layers of collagen that constitute the bulk of the scale. The scleroblasts adjoining the lateral face of the oldest scale region gradually atrophy. New collagen layers are deposited against the inner face of the scale, the adjoining scleroblasts showing evidence of high metabolic activity. Calcification occurs by inotropic deposition of crystals alongside the fibres. There is no sharp demarcation between calcified and non-calcified scale regions, a calcification front gradually moving towards newly formed collagen layers. It is felt that fish scales should be considered as calcified derivatives of dermal collagen layers.     

Proton-induced X-ray emission analysis of atherosclerotic plaques of the carotid bifurcation

The trace elements of both calcified atherosclerotic plaques and plaque-free vessel walls of the carotid bifurcation from 31 autopsies were investigated using the proton-induced X-ray emission (PIXE) method. The trace elements studied were phosphorus (P), calcium (Ca), chrome (Cr), iron (Fe), copper (Cu), zinc (Zn), lead (Pb), selenium (Se), bromine (Br), strontium (Sr), and rubidium (Rb). All samples contained Fe and Zn. Mercury (Hg) was not detected in any of the samples studied. All plaque-free samples contained Cu and almost all Br and Ca, none Sr. All calcified atherosclerotic plaques contained Ca and almost all Br and Sr. The relative levels of Ca were higher in the calcified plaques than in the plaque-free vessel walls. The relative value of Ca in calcified and uncalcified samples was greatest in the group who had died because of cardiovascular disorders and smallest in the group who had died from other causes. There was a strong positive correlation between the Ca and Sr of the plaque samples and between the P and Br of the plaque-free samples.     

Common carp (Cyprinus carpio L.) scales as a bioindicator reflecting its exposure to heavy metals throughout life

The aim of the present study was to demonstrate the usefulness of fish scales as a bioindicator reflecting the concentrations of heavy metals in the environment as compared to typical recognized bioindicators such as the liver or kidney. Heavy metal (Zn, Mn, Cu, Cd, Fe, Ni, Pb) concentration analysis (with the atomic absorption technique) was performed in different annuli on the fish scale to characterize the metals’ accumulation in the consecutive years of the fish's life corresponding to individual scale increments. The results confirmed the usefulness of fish scales as a bioindicative tissue reflecting the condition of the environment in subsequent growing seasons. The results of the present study demonstrated that fish scales are more sensitive to the accumulation of Mn, Ni, and Pb, whose concentrations in different parts of the scales were even 10 times higher than in soft tissues, and significantly correlated with their levels in liver or kidney. In light of this finding, further studies involving controlled exposure of fish to different metals could be performed. Their results could allow for obtaining an excellent tool for monitoring the environment inhabited by fish in the present and past without the need to kill the animals.     

The humoral immune response of Salmo trutta L. and Cyprinus carpio L. exposed to heavy metals

Salmo trutta L. and Cyprinus carpio L. were exposed to low levels of waterborne heavy metals, 0.75 mg Ni dm^-3, 1.06mg Zn dm^-3, 0.29 mg Cu dm^-3 and 1.01 mg Cr dm^-3, at pH 7.83, water hardness 206.9 mg CaCO, dm^-3, and water temperature 15.5° C. During a 38 week exposure period, the humoral antibody response to MS2 bacteriophage was followed using a 50% viral neutralization assay (SD₅₀) method. A suppression of the immune response was observed in fish exposed to the four heavy metals. Total suppression of the humoral antibody response was found only in C. carpio exposed to Cu or Cr, and these fish exhibited symptoms of acute toxicosis. The time for the primary blood clearance of live bacteriophage was increased in S. trutta exposed to the heavy metals, with the exception of Zn-exposed fish, and in C. carpio exposed to Cu. Following the suppressed primary responses, the Ni-exposed S. trutta and Zn-exposed C. carpio exhibited an adjuvant-like response to the second bacteriophage challenge.     

Zinc binding to the gills of rainbow trout: the effect of long-term exposure to sublethal zinc

The effects of sublethal waterborne Zn (2·28 μmol l⁻¹) on Zn binding kinetics to the apical gill surface were studied in juvenile rainbow trout ( Oncorhynchus mykiss ). Two separate radiotracer techniques were employed to ascertain this information. First, in vitro binding kinetic experiments were performed at extremely elevated zinc concentrations (up to 20 mmol l⁻¹) to measure relatively low-affinity binding sites at the gill epithelium. There were no differences in Zn binding parameters ( K_m and B _max) for fish sublethally exposed to Zn for 21 days and their simultaneous controls. Nevertheless, Ca did have an increased inhibitory effect on Zn binding in Zn-exposed fish suggesting that the anionic groups on the gill epithelium of these fish had been altered in some manner. Additionally, in vivo Zn binding kinetics were investigated using environmentally relevant waterborne Zn concentrations (low μmol l⁻¹ range) to isolate high-affinity Zn binding sites (Ca transporters). No appreciable alterations in the Km and B _max values for Zn binding were seen between the Zn-exposed group and its simultaneous control following 15 days of exposure. Furthermore, no significant differences in CC morphometry were observed between treatments. Despite these lack of treatment effects, there were temporal alterations in K_m , B _max and CC fractional surface area in both groups. It is proposed that these fluctuations are controlled by hormonal factors (such as stanniocalcin), believed to play a role in Ca influx.     

Heavy metal localisation in mycorrhizas ofEpipactis atrorubens (Hoffm.) Besser (Orchidaceae) from zinc mine tailings

Summary The metal distribution within mycorrhizal and nonmycorrhizal roots ofEpipactis atrorubens collected from zinc mine tailings and an area rich in heavy metal ores (both located in southern Poland) was investigated. The tailings, consisting of postflotation material, were characterised by high levels of toxic elements such as Zn, Pb, and Cd, while soil outside the tailings was also strongly enriched in heavy metals. Atomic absorption spectrometry and proton-induced X-ray emission analysis revealed that heavy metals were mostly accumulated within orchid roots. Elemental maps from proton-induced X-ray emission showed that plant root epidermis and fungal coils which had developed within cortical cells of roots collected from the zinc mine tailings were the main places of Zn and Pb accumulation, associated with increased concentrations of Fe, Cd, Ti, Mn, Si, Ca, and S. The mean content of Pb and Zn in the coils was 4 to 5 times higher than in the root epidermis. In mycorrhizal roots from the tailings a statistically significant decrease in Pb and Zn content towards the inside of the root was observed. The mean content of Pb in coils from roots of plants growing outside the tailings was about 1% of the concentration in root coils from the tailings. Coils selected from orchid roots originating from a site outside the tailings contained comparatively high concentrations of Zn, Cd, and Cu, which was probably due to the high content of these elements in the soil. The results presented suggest a biofiltering effect against heavy metals by orchid mycorrhizal fungi.     

Environmental dependence of manganese and zinc in the scales of Atlantic salmon, Salmo salar (L) and Brown trout, Salmo trutta (L)

Analysis of manganese and zinc concentrations in the scales of salmon and trout from a number of sites showed significant dependence of the levels of these metals in the scales on the levels in the environment. There is some evidence to suggest that in trout scales there is a minimum concentration of zinc above which there is a direct proportion between environmental and scale concentration. It is also possible that the response of trout to environmental zinc differs from that of salmon.     

Cadmium and zinc content of fish from an industrially contaminated lake

Eleven species of fish from an industrially-contaminated lake were analysed for whole body cadmium and zinc content by atomic absorption spectrophotometry. Cadmium and zinc content of fish were species related, and most species accumulated these trace metals to levels significantly higher than background. Maximum concentrations detected were 13.60 μg Cd g⁻¹ (dry wt) in a bluegill and 820 μg Zn g⁻¹ in a redear sunfish. Cadmium content was much more variable than zinc content. Distributions of concentrations of both cadmium and zinc in fish were lognormal, and concentrations of both metals tended to decrease in higher trophic levels. Zinc concentrations significantly decreased as total length increased in three species.     

Zinc induced apoptosis in HEP-2 cancer cells: the role of oxidative stress and mitochondria

Induction of apoptosis by zinc sulfate was investigated during 96 h exposure on the cancer Hep-2 cell line. During 48 h of exposure, zinc translocated into mitochondria and stimulated production of reactive oxygen species (ROS), affected cellular GSH management and induced moderate activation of p53 and dissipation of mitochondrial membrane potential. In Zn-exposed cells, mitochondria released cytochrome c and AIF, whose translocation to the cytoplasm or the nucleus coincided with the activation of apoptosis. The use of various pharmacological inhibitors inhibiting particular apoptotic targets (antioxidants such as N-acetyl-cysteine and coenzyme Q, the caspase inhibitors z-DEVD-fmk and z-VAD-fmk, cyclosporin A and bonkgrekic acid) proved that Zn acts both directly and indirectly on mitochondria and observed apoptosis is executed by caspase-dependent and caspase-independent pathways.     

Metallothionein-like proteins and zinc-copper interaction in the hindgut of Porcellio scaber (crustacea: Isopoda) exposed to zinc

Metallothioneins (MTs) are ubiquitous low-molecular-weight metalbinding proteins, with a variety of functions in metal metabolism ascribed to them. Among terrestrial invertebrates, MTs have been studied in nematodes, insects, snails, and earthworms. The aim of this study was the characterization of MT-like proteins in the terrestrial isopod crustacean Porcellio scaber in order to analyze their probable role in the metaboliss of copper (Cu) and zinc (Zn). Dietary Zn supplementation (793 μg Zn/g dry food, 6 d) was applied to stimulate MT synthesis. After separation of the hindgut postmicrosomic supernatant (cytosol) of Zn-exposed animals by gel filtration on a Sephadex G-75 column, a Cu- and Zn-containing peak was detected in the position of V _c/V_o≈2, where MTs are expected to elute. Rechromatography of these fractions by size-exclusion chromatography-high-performance liquid chromatography revealed that the 215-nm absorbance peak coincided with the absorbance peak of the rabbit MT II standard. These low-molecular-weight Cu- and Zn-binding compounds, detected in the cytosol of the hindgut cells in Zn-exposed P. scaber. are considered to be Cu, Zn-MT-like proteins. To our knowledge, this is the first report on the characterization of MT-like proteins in isopod crustaceans. These results also indicate that both Zn and Cu dynamics in P. scaber hindgut are affected at the given dietary Zn supplementation and that MT-like proteins are involved in this Zn-Cu interaction.     

Copper,zinc, and selenium in whole blood and thyroid tissue of people with various thyroid diseases

We investigated the possible differences among the concentrations of copper, zinc, and selenium, and their mutual relations in the whole blood and thyroid tissue of patients with various thyroid disorders. Trace elements were determined by total-reflection X-ray fluorescence. The mean levels of these metals in blood as well as the mean Cu/Zn, Cu/Se, and Zn/Se ratios in the patients with thyroid cancer were significantly higher that in other patients and the control groups. However, the mean Zn and Se concentrations in the thyroid cancer tissue were significantly lower than in the thyroid tissue of other patients. In addition, the mean Cu/Zn and Cu/Se ratios in the thyroid cancer tissue were significantly higher than in the patients with other thyroid diseases. We confirm that the highest levels of copper and zinc as well as the Cu/Zn, Cu/Se, and Zn/Se ratios in the whole blood of the patients with thyroid cancer may suggest the progression of the proliferation process in the thyroid gland. We suggest that the low concentrations of zinc and selenium in the thyroid tissue confirm their participation in the carcinogenic process.     

Alterations of tissue glutathione levels and metallothionein mRNA in rainbow trout during single and combined exposure to cadmium and zinc

The objective of this study was to assess the effects of Cd and Zn exposure of rainbow trout (Oncorhynchus mykiss) on (a) hepatic glutathione (GSH) levels; and (b) hepatic and branchial metallothionein (MT) mRNA expression. Juvenile rainbow trout were exposed to waterborne Cd (nominal concentrations: 1.5 or 10 microg Cd l(-1)), Zn (150 or 1000 microg Zn l(-1)) or Cd/Zn mixtures (1.5 microg Cd l(-1) with 200 microg Zn l(-1) or 10 microg Cd l(-1) with 1000 microg Zn l(-1)). After 14 and 28 days of treatment, hepatic concentrations of total glutathione, oxidized glutathione (GSSG) and cysteine were determined by means of fluorometric high performance liquid chromatography (HPLC). Branchial and hepatic expression of MT mRNA was measured by means of semi-quantitative RT-PCR. Exposure of trout to Zn did not result in significantly elevated tissue levels of Zn, whereas Cd accumulation factors changed significantly with time and concentration. Despite of the absence of Zn accumulation, hepatic GSH but not MT mRNA levels were significantly altered in Zn-exposed fish. Cd, on the contrary, affected mainly the MT response but not GSH. Also tissue specific differences in the regulation of the two thiol pools were expressed. The thiol response after exposure to metal mixtures could not be explained by simple addition of the effects of the individual metals. The results indicate that cellular thiol pools show different reaction patterns with respect to specific metals and metal mixtures. Under conditions of long-term, low dose metal exposure, the function of GSH appears to go beyond that of a transitory, first line defense.     

Effects of different levels of dietary zinc on the gilthead,Sparus aurata during the growing season

Gilthead were fed three diets. Diet A was the control diet, whereas diets B and C were supplemented with 300 and 900 mg Zn/kg, respectively. Fish fed with diet C, at the end of the experiment, showed the lowest weight. Zinc concentrations presented the higher values in gills, liver, and kidney. Muscle and brain had the lower mean values and showed a tight control of zinc levels. These results reinforce the hypothesis that zinc in the CNS should be strictly controlled in order to maintain the functional role of the metal. Significant differences in tissue zinc concentrations were obtained between fish fed different amounts of zinc, the metal concentrations being higher in tissues of fish fed diet C. The tissue decrease of zinc, found at the end of the experiment, may depend on a lower feed consumption or on different zinc requirements during the cold season. These changes, even if not univocal among the three diets, may be associated with the life cycle of fish. Furthermore, copper concentrations were little affected by the different concentrations of zinc in the three diets; liver and kidney presented the highest concentrations; liver showed a significant decrease in copper content at the end of the experiment. We conclude that: zinc concentrations of the diet may affect the gilthead weights and the tissual metal content; and zinc concentrations in the diets, depending on the growth rate, may be varied depending on the season.     

Structure and composition of teleost scales from snakehead Channa argus (Cantor) (Perciformes: Channidae)

The structure of teleost scales from snakehead Channa argus was investigated using thermogravimetric analysis (TG), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive analysis of X-rays (EDAX), Fourier transform infra-red spectroscopy (FTIR) and X-ray diffraction (XRD). Thermal treatment of fish scales indicates that the fibrillary plate is partially calcified. SEM shows two kinds of scale denticles, arranged along the circuli in the anterior field and the lateral fields, respectively. TEM indicates the stratum laxum with abundant fibrils, chromatophores and capillary blood vessels within the scale covering, and shows the fibrillary plate as an 'orthogonal plywood structure' of stratified lamellae, consisting of 80–100 nm diameter collagen fibres co-aligned in individual lamellae and alternated by c. 90° of the fibre alignment between adjacent lamellae. EDAX, FTIR and XRD show that the mineral phase of the scales is a carbonated hydroxyapatite with a Ca:P molar ratio of 1·85.     

Differential Hepatic Metal and Metallothionein Levels in Three Feral Fish Species along a Metal Pollution Gradient

The accumulation of cadmium, copper and zinc and the induction of metallothioneins (MT) in liver of three freshwater fish species was studied. Gudgeon (Gobio gobio), roach (Rutilus rutilus) and perch (Perca fluviatilis) were captured at 6 sampling sites along a cadmium and zinc gradient and one reference site in a tributary of the Scheldt River in Flanders (Belgium).At each site up to 10 individuals per species were collected and analyzed on their general condition factor (K), hepatosomatic index (HSI) and gonadosomatic index (GSI). From each individual fish the liver was dissected and analyzed on Cd, Cu and Zn and MT-content. Although not all species were present at each site, hepatic Cd and Zn levels generally followed the pollution gradient and highest levels were measured in perch, followed by roach and gudgeon. Nevertheless also an effect of site was observed on this order. MT-levels appeared to be the highest in gudgeon although differences with the other species were not very pronounced and depended on the site. Significant relationships were found between hepatic zinc accumulation and MT levels. For each species the ratio MT_theoretical/ MT_measured was calculated, which gives an indication of the relative capacity to induce MTs and thus immobilize the metals. Perch had the lowest capacity in inducing MTs (highest ratio). Relationships between hepatic metal levels and fish condition indices were absent or very weak.     

Dietary and water-borne Zn exposures affect energy reserves and subsequent Zn tolerance of Daphnia magna

Enhanced tolerance of aquatic organisms to metal toxicity is one of the important issues of environmental monitoring programs. Determination of dominant uptake route(s) of metals may help to better predict the toxic effects posed by metals. This study aimed to investigate the importance of Zn uptake routes on tolerance and energy reserves of Daphnia magna. Neonates of D. magna were exposed to water-borne zinc, dietary zinc (algae Pseudokichneriella subcapitata loaded with Zn) or to combination of both for 4 days. LC50 (48 h) values of Zn were considerably different from different zinc pre-exposures. Four-day pre-exposure of D. magna neonates produced enhanced tolerance to Zn toxicity. The lowest LC50 values were found in controls (48.2 microM) (no Zn added to their exposure medium and food) and after water-borne Zn pre-exposures (46.2 microM). The level of tolerance increased when dietary Zn was included in pre-exposures, reaching the highest level of LC50 value (70.8 microM) in the highest pre-exposure concentration of diet and water-borne combination experiment. The energy reserves of D. magna also varied significantly under different pre-exposure routes of zinc. In all cases, control animals contained lowest levels of protein, sugar and lipid. Likewise, they represented the lowest energy reserves. Protein levels were highest in the highest dietary Zn exposure, and lowest in the water-borne exposures. Highest and lowest sugar levels were measured in the lowest and highest water-borne Zn exposures, respectively. In contrast, lipid levels were higher in the higher Zn exposure of all exposure routes, the combination exposure resulting in highest lipid levels. The highest total energy reserve was measured in animals that lived in the highest Zn exposure of diet and water-borne combination experiment, mainly due to greater lipid reserves in algae reared in Zn containing media. Results suggest that the dietary exposure route should be considered carefully in natural monitoring studies, and be considered in regulatory assessments of zinc and population dynamics of cladocerans.     

Zinc distribution and excretion in the leaves of the grey mangrove,Avicennia marina (Forsk.) Vierh

Mangroves are important as primary producers in estuarine food chains. Zinc is often a major anthropogenic contaminant in estuarine ecosystems and has potential ecotoxicological consequences for mangrove communities. Accumulation, distribution and excretion of zinc in the leaf tissue of the grey mangrove, Avicennia marina was studied using SEM X-ray microanalysis and Atomic Absorption Spectroscopy. The first leaves of A. marina grown in 500 μg Zn as ZnCl₂ per g of dry soil were found to accumulate 106.3±18.5 μg Zn per g dry tissue, significantly higher than control plants, after a 7-month period. Washings from first leaves contained significantly higher amounts of zinc (0.30±0.14 μg/cm² Zn) than control plants after 1 month, suggesting excretion of zinc from glandular trichomes. SEM X-ray microanalysis revealed salt crystals exuded from glandular tissue on the adaxial surface of first leaves to be composed of alkaline metals and zinc in zinc treated plants. SEM X-ray microanalysis of seedlings dosed with 4 g/l Zn as Zn Cl₂ revealed a decreasing Zn gradient from xylem tissue, through photosynthetic mesophyll, to hypodermal (water) tissue. A subsequent increase in Zn concentration was observed in glandular tissue. Cell wall Zn concentrations were consistently higher than intracellular Zn concentrations.     

Concentrations of Thirteen Trace Metals in Scales of Three Nototheniid Fishes from Antarctica (James Ross Island,Antarctic Peninsula)

In this study, we assessed concentrations of 13 trace metals in the scales of Notothenia coriiceps, Trematomus bernacchii and Gobionotothen gibberifrons caught off the coast of James Ross Island (Antarctic Peninsula). Overall, our results for scales broadly match those of previous studies using different fish and different organs, with most metals found at trace levels and manganese, aluminium, iron and zinc occurring at high levels in all species. This suggests that scales can serve as a useful, non-invasive bioindicator of long-term contamination in Antarctic fishes. High accumulation of manganese, aluminium, iron and zinc is largely due to high levels in sediments associated with nearby active volcanic sites. Manganese, vanadium and aluminium showed significant positive bioaccumulation in T. bernacchii (along with non-significant positive accumulation of iron, zinc, cobalt and chromium), most likely due to greater dietary specialisation on sediment feeding benthic prey and higher trophic species. Levels of significance in bioaccumulation regressions were strongly affected by large-scale variation in the data, driven largely by individual differences in diet and/or changes in habitat use and sex differences associated with life stage and reproductive status. Increased levels of both airborne deposition and precipitation and meltwater runoff associated with climate change may be further adding to the already high levels of manganese, aluminium, iron and zinc in Antarctic Peninsula sediments. Further long-term studies are encouraged to elucidate mechanisms of uptake (especially for aluminium and iron) and possible intra- and interspecific impacts of climate change on the delicate Antarctic food web.