Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses

The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves (Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson's r ranging from -0.63 to -0.78). On the other hand, theoretical maximum shell lengths (L( infinity )) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most frequently and most strongly correlated with the population variables (Pearson's r ranging from -0.58 to -0.80). Our findings demonstrate, however, the difficulty of currently assigning to sub-cellular metal partitioning measurements (mainly Cd bound to the HMW fraction) any predictive role for population health, notably because of the influence of ecological confounding variables (e.g., the cumulative number of degree-days in the littoral zone, as is the case here). Metal contamination of our lakes has decreased markedly in the past 10 years and consequently we believe that the toxic effects of metals may have been replaced by some natural factors as the main agent for structuring the clam populations in these lakes.     

The relationships of urinary metallothionein with other indicators of renal dysfunction in people living in a cadmium-polluted area in Japan

An epidemiologic investigation was carried out to study the significance of urinary excretion of metallothionein (MT) in people aged 50 years and over living in a cadmium (Cd)-polluted area in Japan. The urinary level of MT was compared with various parameters (age, urinary alpha 1-microglobulin (alpha 1-MG), beta 2-microglobulin (beta 2-MG), total protein, Cd, copper (Cu), and zinc (Zn), and relative clearances to creatinine of alpha 1-MG, beta 2-MG, phosphate and uric acid). It was found that the urinary excretion of MT is closely associated with Cd and the indices of renal dysfunction listed above. This observation was more remarkable in women than men. When subjects with signs of renal dysfunction were compared as a group to those with normal renal functions, the excreted amount of MT in the former is significantly greater. The results support the notion that the urinary excretion of MT reflects not only Cd exposure levels but also renal dysfunction caused by long-term Cd exposure.     

Sub-cellular partitioning of cadmium, copper, nickel and zinc in indigenous yellow perch (Perca flavescens) sampled along a polymetallic gradient

Sub-cellular metal distributions were studied in indigenous yellow perch (Perca flavescens) collected from eight lakes located along a cadmium (Cd), copper (Cu), nickel (Ni) and zinc (Zn) concentration gradient. Ambient dissolved metal concentrations were measured to evaluate exposure and total hepatic metal concentrations were determined as a measure of metal bioaccumulation. Metal partitioning among potentially metal-sensitive fractions (cytosolic enzymes, organelles) and detoxified metal fractions (metallothionein) was determined after differential centrifugation of fish liver homogenates. Major proportions of hepatic Cd and Cu were found in the heat-stable cytosolic peptides and proteins fraction (HSP), a fraction including metallothioneins, whereas the potentially metal-sensitive heat-denaturable proteins fraction (HDP) was the largest contributor to the total Ni and Zn burdens. The concentrations of Cd, Cu and Ni (but not Zn) in each sub-cellular fraction increased along the metal contamination gradient, but the relative contributions of each fraction to the total burden of each of these metals remained generally constant. For these chronically exposed fish there was no threshold exposure concentration below which binding of Cd or Ni to the heat-denaturable protein fraction did not occur. The presence of Cd and Ni in the HDP fraction, even for low chronic exposure concentrations, suggests that metal detoxification was imperfect, i.e. that P. flavescens was subject to some metal-related stress even under these conditions.     

Comparison of tissue distribution of cadmium in mice intravenously injected with metallothionein and a polymer of metallothionein

Polymers of cadmium metallothionein I and II were prepared and used for distribution studies. Mice were intravenously injected with comparable doses of cadmium as chloride, metallothionein and metallothionein-polymer and were killed at 3 h and 24 h after injection. A considerably higher concentration of cadmium was found in the plasma and spleen of animals exposed to metallothionein-polymer than other groups, while the cadmium concentration in the kidney was less than, but in the liver was greater than those injected with cadmium-metallothionein. Gel filtration of liver and kidney cytosols showed that metallothionein is the most stable form of cadmium in the tissues. The injected polymer was degraded into the monomer within 24 h in tissues. The high concentration of polymer in plasma indicates a slow clearance of cadmium in this form. The increased antigenicity of metallothionein-polymer may be related to its slow clearance from blood and increased deposition in spleen.     

Use of a simulated gizzard to measure bioavailability of metals and other elements to waterfowl

We used a simulated gizzard environment to examine the bioavailability of barium, cadmium, nobreak copper, lead and zinc to wild and sentinel mallards (Anas platyrhynchos) using a seasonally-flooded wetland. This impoundment was originally constructed as a containment area for dredged sediments which were contaminated through previous smelting operations. Extraction of elements from gizzard contents indicated that sentinel mallards experienced greater exposure to contaminants than their wild counterparts. Concentrations of the selected elements extracted from ingesta were lower than in seeds and sediments from the study site. Notable exceptions were barium and lead, which were not detected in seeds of some plant species, though they were present in the ingesta of one or more treatment groups. This technique may provide a more realistic means of estimating bioavailability and exposure than methods which measure total concentrations of elements in dietary components. The use of sentinel mallards may result in inflated estimates of risk to wild ducks using seasonally-available wetlands.An erratum to this article can be found at     

The induced synthesis of metallothionein in various tissues of rats in response to metals. II. Influence of zinc status and specific effect on pancreatic metallothionein

Metallothionein (MT) of various tissues contains bound zinc (Zn) and any change in Zn status can alter its synthesis and tissue deposition. The changes in MT levels and its inducibility in Zn-injected and Zn-deficient (Zn-D) rats were studied. MT levels in 11 tissues (brain, lung, heart, liver, kidney, stomach, small intestine, pancreas, spleen, testes and muscle) of control and rats injected with different doses of ZnSO₄ (20 mg Zn/kg for 2, 4 or 7 times) were measured by the cadmium-hemolysate (Cd-hem) method. A dose dependent increase in MT levels was observed only in the pancreas, liver, small intestine and kidney after ZnSO₄ injection — the highest level being in the pancreas. A positive correlation was found between Zn and MT concentrations and also the relative inducibility of MT was similar in these 4 tissues (slopes of regression equations were 12.6–15.5). In order to study the effect of Zn-D in MT induction, rats were fed a diet containing 1 ppm Zn for 18 days and CdCl₂ (1 mg Cd/kg) was injected subcutaneously 3 times at 48-h intervals to control and Zn-D rats. Although the tissue distribution of Cd was similar in both the groups, MT concentrations in pancreas and kidney were significantly decreased in Zn-D. The plasma and tissue levels of Zn were also decreased in Zn-D rats injected with CdCl₂. The decrease in both Zn and MT levels was more prominent in pancreas than other organs of Zn-D rats. The results suggest that of all the organs studied, the induction of pancreatic MT is sensitive to Zn status and Zn may be a primary inducer of MT.     

Cytogenetic markers and their spatial distribution in a population living in proximity to areas sprayed with pesticides

Human populations are in contact with potentially toxic substances in varying amounts, if the exposure is work-related or direct, generally the amount of toxin is usually greater than if the exposure is environmental through the drifts that occur. It was proposed to determine the existence of genotoxic damage evaluated through Chromosomal Aberrations and Micronuclei assays and their spatial distribution pattern, as well as the possible relationship between that damage and the values found in biochemical biomarkers, in groups of individuals environmental exposure (respiratory exposure) to mixtures of pesticides, in the province of Córdoba-Argentina. Biochemical and hematological determinations were made in each samples. The results reveal that the monitoring of human populations through the analysis of cytogenetic markers enabled the detection of direct damage in man caused by polluting substances and the results were obtained rapidly. The disadvantage of this type of study is the inability to estimate the degree of exposure.     

Sensitivity to cadmium along a salinity gradient in populations of the periwinkle, Littorina littorea, using time-to-death analysis

In this study, we assessed the combined effect of Cd concentration and salinity, on Cd uptake and mortality rate of Littorina littorea, collected along a salinity and pollution gradient in the Western Scheldt estuary (The Netherlands). Animals kept at their field salinity levels were exposed to three Cd concentrations (i.e. 10, 40 and 320 microM), while animals kept in 10 microM of Cd were subjected to five salinity treatments (i.e. 15, 20, 25, 30 and 35 per thousand). Mortality was recorded every 24h and Cd body burdens were measured with ICP-AES. Time-to-death data were analysed via Cox proportional hazard models, including the co-variates "site-Cd treatment" in the Cd experiment and "site-salinity treatment" in the salinity experiment. "Cd-treatment" and "field-salinity" affected mortality rates significantly in the Cd experiment, such that the mortality risk increased by 2.3 times when salinity was lowered from 35 to 15 per thousand, while it decreased by 19.7 times when Cd dropped from 320 to 10 microM. "Site" did not significantly affect the mortality risk in the salinity experiment but affected time-to-death via its interaction with the "salinity-treatment". Generally, mortality did not occur at a given threshold Cd tissue level, but changed over time and treatments, in function of the site. The results demonstrate the importance of the animals' environmental history and illustrate the usefulness of time-to-death analyses in ecotoxicological experiments.     

Phagocytic activity of marine and freshwater bivalves: in vitro exposure of hemocytes to metals (Ag,Cd, Hg and Zn)

We measured non-specific immune function of various bivalves from marine (Cyrtodaria siliqua, Mactromeris polynyma, Mesosdesma arctatum, Mya arenaria, Mya truncata, Mytilus edulis, Serripes groenlandicus, Siliqua costata) and freshwater environments (Dreissena polymorpha and Elliptio complanata). We used flow cytometry to quantify the phagocytosis of fluorescent microspheres by hemocytes exposed in vitro to increasing levels of various metal compounds (AgNO(3), CdCl(2), CH(3)HgCl, HgCl(2) and ZnCl(2)). In some species, low doses of mercury (organic and inorganic) and Zn suggest a hormesis-like stimulation of phagocytic activity. At higher levels of exposure, all metals tested induced a significant dose-related inhibition of hemocyte phagocytosis. The species-specific sensitivity of the assay was determined by comparing the in vitro exposure using the metal concentration inducing a 50% suppression (EC(50)) of the phagocytic activity. Different species expressed different levels of sensitivity. Our results show the variability of the toxic response of different species within a group of similar organisms. It also highlights the need to consider species-species differences in ecotoxicological risk assessment.     

Regional distribution of metals and biogenic amines in the brain of monkeys exposed to manganese

Manganese chloride (20 mg/kg/day) was administered orally to male Rhesus monkeys. The levels of copper, calcium, manganese and biogenic amines m different regions of brain were determined after 18 months of manganese exposure. The levels of manganese were increased above control values in the cerebral cortex, cerebellum, diencephalon, corpus striatum, mid-brain and pons, but not in the medulla oblongata. Copper was increased only in the pons and medulla oblongata while calcium declined in pons, medulla oblongata and corpus striatum. Alterations in copper and calcium did not appear to be related to the altered metabolism of catecholamines in the brain.     

Disposition of metals in rats: a comparative study of fecal, urinary, and biliary excretion and tissue distribution of eighteen metals

Fecal (0-4 days), urinary (0-4 days), and biliary (0-2 hr) excretion and tissue distribution of 18 metals were examined in rats after iv administration. Total (fecal + urinary) excretion was relatively rapid (over 50% of dose in 4 days) for cobalt, silver, and manganese; was between 50 and 20% for copper, thallium, bismuth, lead, cesium, gold, zinc, mercury, selenium, and chromium; and was below 20% for arsenic, cadmium, iron methyl mercury, and tin. Feces was the predominant route of excretion for silver, manganese, copper, thallium, lead, zinc, cadmium, iron, and methyl mercury whereas urine was the predominant route of excretion for cobalt, cesium, gold, selenium, and chromium; while both excretion routes were equally important for bismuth, mercury, arsenic, and tin. Biliary excretion seems to be an important determinant for the fecal excretion of silver, arsenic, manganese, copper, selenium, cadmium, lead, bismuth, cobalt, and methyl mercury. Between 45 (silver) and 0.8% (methyl mercury) of the dosages administered of these metals was excreted into bile in 2 hr, and they exhibited high bile/plasma concentration ratios. The biliary excretion of copper, selenium, lead, and chromium did not increase proportionally with dosage, suggesting that the hepatobiliary transport of these metals is saturable. The fraction of dosage excreted into bile was independent of the dosage for silver, arsenic, manganese, bismuth, methyl mercury, mercury, gold, cesium, thallium, and tin, but markedly increased with increase in dosage of cadmium, cobalt, zinc, and iron. The latter phenomenon is probably due to saturation of hepatic (cadmium, zinc) or extrahepatic (iron) metal-binding sites. Comparison of biliary and fecal excretion rates indicates that arsenic and selenium undergo intestinal reabsorption, whereas thallium and zinc enter the feces also by non-biliary routes. Most of the metals reached the highest concentration in liver and kidney. However, there was no direct relationship between the distribution of metals to these excretory organs and their primary route of excretion.     

Cloning of a metallothionein gene and characterization of two other cDNA sequences in the Pacific oyster Crassostrea gigas (CgMT1)

Metallothionein (MT) genes encode essential metal-binding proteins involved in metallic homeostasis and detoxification in living organisms. Here, we describe the structure of the first Pacific oyster Crassostrea gigas metallothionein (CgMT1) gene and the sequences of two other MT cDNA. The CgMT1 gene sequence contains three coding exons plus a 5' entirely non-coding exon, and the predicted protein contains 21 cysteine residues organized in Cys-X-Cys motifs as classically described for MTs. The three cDNA sequences present few substitutions in either coding sequence or UTRs. Induction of these MT-mRNA in heavy metal-treated oysters (i.e. cadmium) was confirmed by Northern blot analysis and RT-PCR and suggests a potential specific tissue expression rate. Southern blot analysis suggested the presence of multiple CgMT genes, and allowed the detection of restriction fragment length polymorphisms (RFLPs). Although the CgMT1 coding sequence showed 30-73% nucleotide identities with known sequences in other mollusks, it included the specific motif Cys-X-Cys-X(3)-Cys-Thr-Gly-X-X-X-Cys-X-Cys-X(5)-Cys-X-Cys-Lys found in Mollusk family 2. Marine bivalves are commonly used as pollution bioindicators, thus the development of genetic markers based on CgMT1 polymorphism will allow a monitoring of heavy metal exposure in anthropogenically disturbed ecosystems.     

Changes in susceptibilities to teicoplanin, vancomycin and other antibiotics among Staphylococcus aureus isolates in a tertiary-care University hospital

Changes in antibiotics susceptibilities were evaluated among 374 nosocomial clinical isolates of Staphylococcus aureus collected from 1986 to 1994 in a tertiary-care University hospital where methicillin-resistant S. aureus (MRSA) is an increasing cause of infection. Significant increases in resistance to ciprofloxacin, clindamycin and rifampicin were observed among MRSA isolates in recent years. No resistance to glycopeptides was observed. However, during the last 2 years a significant trend towards a decreasing susceptibility to teicoplanin was observed among MRSA isolates.     

Assessment of the genotoxicity and cytotoxicity in environmentally exposed human populations to heavy metals using the cytokinesis‐block micronucleus cytome assay

The cytokinesis‐block micronucleus cytome (CBMN‐Cyt) assay was developed as a system for evaluating DNA damage, cytostasis, and cytotoxicity. The aim of the present study was to estimate levels of micronuclei (MNi), nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), cell death (apoptosis/necrosis), nuclear division index, and nuclear division cytotoxicity index values in the peripheral blood lymphocytes of environmentally exposed subjects to heavy metals from five Bosnian regions, characterized by different exposure to heavy metals. The study was performed using CBMN‐Cyt assay, considering factors, such as age, gender and smoking habits and their possible effects on analyzed parameters. In total, 104 healthy subjects were selected (49.04% females and 50.96% males; average age, 35.41 years; 51.92% smokers and 48.08% nonsmokers). There was significant difference between the frequency of NBUDs in Tuzla as compared to the control group. Furthermore, there was observed a statistically significant difference for the frequency of NPBs between Zenica, Olovo, and Kakanj when compared with the controls. Males showed a significantly higher number of apoptotic cells than females in controls. There were significant differences between smokers and nonsmokers in the frequency of NPBs in controls (higher in nonsmokers) and necrotic cells in Olovo (higher in nonsmokers). The pack years of smoking significantly influenced the number of necrotic cells in controls and the frequency of NBUDs in the overall sample. The results of the present study provide evidence of significantly increased frequency of NPBs and NBUDs in exposed subjects, suggesting that these endpoints are highly sensitive markers for measuring genotoxicity. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1331–1342, 2015.     

Activation of the metallothionein IIA promoter and other key stress response elements by ursodeoxycholate in HepG2 cells: relevance to the cytoprotective function of ursodeoxycholate

Ursodeoxycholate, used to treat a variety of pathologies, has the ability to reverse cytotoxic and hepatotoxic conditions. We examined HepG2, a hepatic cell line, treated with increasing levels of ursodeoxycholate, for responses of a range of promoters/response elements responsive to DNA damage, heavy metal ions, protein denaturants, aromatic hydrocarbons, retinoids, changes in intracellular AMP levels, end endoplasmic reticulum stress. The metallothionein IIA promoter was the most highly activated by ursodeoxycholate. Since ursodeoxycholate protects against the cytotoxic effects of deoxycholate, our data, combined with observations made by others, implicate metallothionein IIA as being important in this protective pathway.     

Oxidative stress in response to xenobiotics in the blue mussel Mytilus edulis L.: evidence for variation along a natural salinity gradient of the Baltic Sea

Blue mussels (Mytilus edulis L.) collected at three sampling sites in each of three geographical regions (South, Middle, North) along the permanent longitudinal South-North salinity gradient of the Baltic Sea, were exposed for 10 days to copper (35ppb) or 95 octane petrol (0.3 per thousand). During the experiment, they were maintained at the respective sampling site salinity. Scope for growth (SFG) was determined, and biochemical stress markers (protein carbonyl groups, disulfide bond formation, and glutathione transferase (GST), and catalase (CAT) activities) were investigated in gill tissue upon termination of the experiment. Treatment and regional effects for SFG and protein carbonyl groups were all significant for petrol. The largest increase in protein carbonyl groups was observed in the North. Mussels from the southern, more saline ( approximately 7 per thousand) region had the highest SFG, and displayed the largest SFG decrease in response to treatment, indicating that they had the most energy available for allocation to stress response. They also displayed the least increase in the level of protein carbonyl groups. Mussels from the Northern, less saline ( approximately 5%) region had the highest degree of protein carbonyl groups in response to both treatments, and lowest average SFG. Silver stained diagonal gels for samples from one sampling site in South and North, respectively, demonstrated differences in disulfide bond profiles for both stress treatments. There was also a regional difference in the number of protein disulfides observed on diagonal gels. The most diverse protein disulfide response was found in South. No treatment related effects on GST and CAT activities were observed. We suggest that both SFG and protein carbonyl groups show that geographical difference in stress susceptibility, previously established between the North and the Baltic Seas, also apply on a regional scale within the Baltic Sea, along the salinity gradient.     

Uptake and distribution of a new SSRI, NS2381, studied by PET in living porcine brain.

This study tests the utility of a new selective serotonin reuptake inhibitor (SSRI), [11C]NS2381 {(+/-)-(8-[11C]methyl-3-(4-trifluoromethyl-phenyl)-8-azabicyclo[3.2.1]oc t-2-ene)}, as positron-emitting radioligand for labelling serotonin (5-HT) reuptake sites in living brain. Studies of monoamine uptake were carried out initially in vitro using rat brain synaptosomes. They showed that NS2381 and its precursor NS2435 are selective inhibitors of serotonin (5-HT) uptake. Then, studies were carried out in vivo on the uptake and distribution of [11C]NS2381 in living porcine brain. They showed that the radiotracer accumulates readily in brain, and binds reversibly in regions rich in serotonin uptake sites (e.g. raphe, basal ganglia and thalamus). In addition, [11C]NS2381 was displaced from brain tissue by the potent SSRI citalopram. The enantiomers of [11C]NS2381 were, in general, found to be similar to the racemate in terms of their uptake and distribution in living pig brain. Thus, [11C]NS2381 fulfilled several criteria of a PET radioligand for studying 5-HT uptake sites in the living brain.     

Subcellular distribution of a nitroxide spin-labeled netropsin in living KB cells. Electron paramagnetic resonance and sequence specificity studies

A nitroxide spin-labeled netropsin was studied by EPR spectroscopy with respect to its uptake and localization in living KB cells. Whereas the drug was taken up readily, there was relatively little drug in the cytoplasm, but a significant concentration of the drug in the cell nucleus. The EPR signal in the latter site corresponded to a relatively freely rotating radical. The drug exhibited good intracellular stability up to 25 hr. While a delta Tm of 24 degrees between the spin-labeled netropsin and calf thymus DNA confirmed strong binding, the absence of any DNA elongation by viscometry was consistent with nonintercalative exterior binding which was confirmed to be minor groove specific by binding of the agent to T4 DNA with a delta Tm of 17.5 degrees. The sequence specificity of the DNA binding of the spin-labeled drug was confirmed by methidiumpropyl-EDTA (MPE) footprinting on a fragment of pBR322 DNA to be very similar to that of the parent netropsin, i.e. selective for AT-rich sites, with minor differences of protection afforded by introduction of the nitroxide label.