The effects of some divalent metals on cardiac and branchial Ca-ATPase in a freshwater fish Saccobranchus fossilis

The heart and gill of a freshwater fish Saccobranchus fossilis have been shown to contain a Ca²⁺-activated ATPase involved in Ca²⁺ transport. Enzyme showed optimal activity at 3 mM Ca²⁺ and 3 mM ATP for gills and at 3 mM Ca²⁺ and 1 mM ATP for heart. Mg²⁺ was equally effective in stimulating enzyme activity but was not essential for hydrolysis. Maximum activity was found in heart ventricular muscles as compared to gills. Among all the metals tested Hg²⁺ was the most toxic (IC₅₀, 0.75 and 0.85 μM for heart and gill, respectively) followed by Pb²⁺, Mn²⁺, and Cd²⁺. The inhibition was concentration dependent and reached almost 100% with each metal at the highest concentration. Stimulation of enzyme activity was observed at lower concentrations of Mn²⁺ and Cd²⁺ but not with Pb²⁺ and Hg²⁺. Stimulation was more pronounced with Mn²⁺ than with Cd²⁺ in both heart and gills. The results indicated that the inhibitory effect of these metals might be through the Ca²⁺-ATPase which is a manifestation of the calcium pump in various tissues.     

Toxicity of heavy metals using sperm cell and embryo toxicity bioassays with Paracentrotus lividus (Echinodermata: Echinoidea): comparisons with exposure concentrations in the Lagoon of Venice,Italy

Sperm cell and embryo toxicity tests using the Mediterranean sea urchin Paracentrotus lividus Lmk were performed to assess the toxicity of As3+, Cd2+, Cr3+, Ni2+, Pb2+, Cu2+, Zn2+, and Hg2+. The aim of this study was to improve information about the comparative sensitivity of sea urchin bioassays to the heavy metals, which are an important cause of contamination in the ecosystem of the Lagoon of Venice. Considering the data in mM/L, the order of toxicity is Hg2+ > Cu2+ > Zn2+ > As3+ > or = Cr3+ > or = Cd2+ > or = Pb2+ > or = Ni2+ for the sperm cell test and Hg2+ > or = Pb2+ > Cu2+ > Zn2+ > Cd2+ > Ni2+ > As3+ > or = Cr3+ for the embryo test. New toxicity data for metals expressed as median effective concentration (EC50) and no-observed-effect concentration (NOEC) are reported for the Mediterranean species. Accurate observations of embryotoxic effects at increasing metal concentrations were done, detecting some different behaviors in metal toxicity. Toxicity data compared with water column and pore-water concentrations recorded in the Lagoon of Venice (Italy) demonstrate the potential ability of bioassays using sea urchin to detect important contaminants in this ecosystem.     

A relationship between gill silver accumulation and acute silver toxicity in the freshwater rainbow trout: support for the acute silver biotic ligand model

Rainbow trout were exposed to a range of silver concentrations (as AgNO3) in flowing synthetic soft water (0.05 mM Na+, 0.05 mM Cl-, 0.05 mM Ca2+, 0.02 mM Mg2+, 0.02 mM K+, pH 7.0, approximately 0.7 mg C/L dissolved organic carbon, 10 mg CaCO3/L, 10 +/- 2 degrees C) to investigate a possible relationship between short-term gill silver accumulation (3 h or 24 h) and acute silver toxicity (96-h mortality). We also investigated potential relationships between gill silver accumulation and inhibition of Na+ uptake plus inhibition of gill Na+K(+)-adenosine triphosphatase (ATPase) activity. The 96-h median lethal concentration (LC50) values were 13.3 microg total Ag L(-1) and 3.3 microg dissolved Ag L(-1). A relationship was demonstrated between 3-h and 24-h gill silver accumulation and 96-h mortality. A relationship also was demonstrated between gill silver accumulation and inhibition of Na+ uptake at 24 h of exposure. No relationship between gill silver accumulation and inhibition of gill Na+K(+)-ATPase activity was found. The 96-h median lethal gill accumulation (LA50) values of 129 (at 3 h) and 191 ng g(-1) (at 24 h) and a conditional equilibrium binding constant of 8.0 for Ag+ binding to the gills were calculated. These observations support use of the silver biotic ligand model (BLM) as a regulatory tool to predict acute silver toxicity.     

Metal pollution as health indicator of lake ecosystems

As part of a long-term monitoring study of the anthropogenic pollution of the Lake of Balaton (Hungary) the concentrations of toxic metals (Cd2+, Hg2+, Pb2+), as well as of bioelements (Cu2+ and Zn2+) were measured in the muscle, gill and liver of bream (Abramis brama L.) collected in the open water during the autumn of 1999, The highest Cd, Cu, Pb and Zn concentrations were detected in the gill and liver of fish, whereas the highest Hg concentrations were measured in the muscle. The maximum metal concentrations measured in the muscle of bream were generally below maximum permissible levels for human consumption established by the Hungarian Food Directorate. Depending on the sampling site, significantly higher Cd, Hg and Pb concentrations (p < 0.05) were detected in the muscle of fish samples collected from the Western basin, while no significant differences were observed for Cu and Zn. In the muscle and gill an increasing trend of heavy metal (Cd and Cu) load characterizes the individuals of the 2-4 calendar age group (p < 0.05), while for the older specimens the concentration of these elements decreased significantly (p < 0.05). Positive relationships on the whole age scale were found for Cd in the liver, for Pb in the gill, and for Hg both in the muscle and the liver of fish.     

Protective effects of Ca2+, Mg2+, Cu2+, and Ni2+ on mercury and methylmercury toxicity to a cyanobacterium.

Toxicological investigations of the impact of inorganic mercury (Hg2+) and methylmercury (CH3Hg+) in terms of growth, NH4+ uptake, in vivo glutamine synthetase (transferase) activity, and regulation of toxicity by Ca2+, Mg2+, Cu2+ and Ni2+ in the diazotrophic cyanobacterium Nostoc calcicola Bréb. have been completed. Photoautotrophic growth of the cyanobacterium was extremely sensitive to both mercury compounds, CH3Hg+ being 2.5 times more toxic than Hg2+. Although NH4+ uptake was 6 times more sensitive than in vivo GS activity against the two mercurials, both processes had a greater susceptibility toward CH3Hg+. On the basis of Km and Vmax, it is suggested that both mercury species inhibit such metabolic events noncompetitively. Ca2+, Mg2+, Cu2+, and Ni2+ did not change the nature of inhibition and effectively antagonized the Hg2+ and CH3Hg+ toxicities in the sequence Ca2+ greater than Mg2+ much greater than Cu2+ greater than Ni2+.     

Mechanism and regulation of thiamine pyrophosphokinase from parsely leaf.

Thiamine pyrophosphokinase (EC 2.7.6.2) from parsely leaf showed an absolute requirement for divalent cation such as Mg2+, Mn2+ and Co2+. The activation effect varied with the species and concentrations of such cations. When Mn2+ or Co2+ was used as cofactor, maximal activation was found at a lower level than ATP concentration, whereas the activation by Mg2+ increased hyperbolically with the concentration. Studies of initial velocity and product inhibition led to conclude that the kinase reaction obeys a sequential ordered Bi Bi mechanism; i.e. the enzyme combines in turns with MgATP and thiamine, followed by release of TPP and AMP. The inhibition type revealed for inorganic pyrophosphate was competitive with respect to thiamine with Ki of approximately 2.8 mM. On the other hand, thiamine monophosphate exhibited noncompetitive inhibition with Ki of 0.2 mM. The plots of the reaction rate against MgATP concentrations gave a sigmoidal curve. Addition of either AMP or GMP resulted in restoration of a depressed activity at low concentration of MgATP. The "allosteric" inhibition was also relieved by the addition of an excess amount of magnesium ions. These findings suggest that transphosphorylation is regulated by subcellular concentrations of metal ions relative to ATP or of the products involved in the thiamine biosynthesis.     

Effect of cadmium on transmembrane Na+ and K+ transport systems in human erythrocytes.

The effects of cadmium (Cd2+) on Na+,K(+)-ATPase in disrupted human erythrocyte membranes and on various transmembrane Na+ and K+ transport systems in intact erythrocyte suspensions were studied. Cadmium2+ inhibited the erythrocyte Na+,K(+)-ATPase enzyme with a 50% inhibition at a Cd2+ concentration of 6.25 microM. The Cd2+ inhibition in the human erythrocyte was non-competitive with respect to Na+,K+, and ATP. Cadmium2+ exerted no acute effect, however, on the Na+,K(+)-ATPase pump activity as measured by the ouabain sensitive 86Rb uptake or Na+ efflux in intact red blood cells. Cadmium2+ also inhibited the Ca2+ dependent K+ channels in human red blood cells, whereas it had no effect on Na+,K+ cotransport, Na+,Li+ countertransport, anion carrier, and the number of active Na+ pump units. The data indicate that in human erythrocytes under acute conditions Cd2+ exerts an inhibitory effect on Na+,K(+)-ATPase enzyme in disrupted erythrocytes and the Ca2+ stimulated K+ efflux in intact red blood cells without affecting the Na+ pump, Na+,K+ cotransport, and Na+,Li+ countertransport activity.     

Effect of cadmium on transmembrane Na+ and K+ transport systems in human erythrocytes.

The effects of cadmium (Cd2+) on Na+,K(+)-ATPase in disrupted human erythrocyte membranes and on various transmembrane Na+ and K+ transport systems in intact erythrocyte suspensions were studied. Cadmium2+ inhibited the erythrocyte Na+,K(+)-ATPase enzyme with a 50% inhibition at a Cd2+ concentration of 6.25 microM. The Cd2+ inhibition in the human erythrocyte was non-competitive with respect to Na+,K+, and ATP. Cadmium2+ exerted no acute effect, however, on the Na+,K(+)-ATPase pump activity as measured by the ouabain sensitive 86Rb uptake or Na+ efflux in intact red blood cells. Cadmium2+ also inhibited the Ca2+ dependent K+ channels in human red blood cells, whereas it had no effect on Na+,K+ cotransport, Na+,Li+ countertransport, anion carrier, and the number of active Na+ pump units. The data indicate that in human erythrocytes under acute conditions Cd2+ exerts an inhibitory effect on Na+,K(+)-ATPase enzyme in disrupted erythrocytes and the Ca2+ stimulated K+ efflux in intact red blood cells without affecting the Na+ pump, Na+,K+ cotransport, and Na+,Li+ countertransport activity.     

Divalent metal inhibition of non-haem iron uptake across the rat duodenal brush border membrane

Duodenal Fe2+ uptake is essential to body Fe2+ homeostasis, but the interaction of metals with the uptake process remains unclear. The present study compared the effects of four essential trace metals (Mn2+, Zn2+, Co2+ and Ni2+) with two toxic metals (Pb2+ and Cd2+) on Fe2+ uptake across the brush border membrane of villus-attached duodenal enterocytes. Everted rat duodenum was exposed to buffer containing 0.2 mm-59Fe2+-ascorbate with or without the competing metal (2 mm) and the tissue was then processed for autoradiography allowing Fe2+ uptake to be determined at specific crypt-villus regions. The quantification method ensured that uptake by cells, rather than Fe2+ binding to the tissue surface, was measured. Fe2+ uptake was significantly inhibited by Cd2+ in upper villus enterocytes only and Pb2+ was without effect on Fe2+ uptake. The inhibition by Cd2+ was not due to general cell damage as judged by the release of lactate dehydrogenase from tissue into incubation fluid. Essential divalent trace metals reduced uptake significantly along the whole length of the crypt-villus axis. Cd2+ uptake, measured separately, took place at all regions of the villus-crypt axis, highest uptake being into crypt enterocytes. The very different uptake profiles for Cd2+ and Fe2+ suggests that the divalent metal transporter 1 is not the principal transporter of Cd2+. The addition of Fe2+ to incubation buffer inhibited Cd2+ uptake by both crypt and villus enterocytes. The possibility that the inhibitory actions of Fe2+ and Cd2+ on the uptakes of Cd2+ and Fe2+ respectively can be explained by a non-competitive action or the involvement of an additional metal transporter is discussed.     

土壤中萘降解菌的耐铅性能及机制初步研究

目的探讨土壤中萘降解菌对萘和重金属双重污染的耐受性。方法对油田石油污染土壤中分离到的一株高效萘降解菌——伯克霍尔德菌(Burkholderia sp.)TN的重金属耐受性、铅富集机制及在土壤生物修复中的应用进行研究,以分光光度法测定不同Pb~(2+)浓度(100~500 mg/L)对菌悬液吸光度的影响,以电感耦合等离子体发射光谱仪(ICP-OES)测定培养基中重金属(50 mg/L,Pb~(2+),Zn~(2+),Cu~(2+)和Cd~(3+))在菌体培养液、细胞壁、细胞内富集物中的分布,以扫描电镜(SEM)、能谱仪(EDS)和傅里叶变换红外光谱(FTIR)观察100 mg/L Pb~(2+)对菌体形态、元素及官能团分布的影响。结果菌株TN对Pb~(2+)的最高耐受浓度为500 mg/L并可耐受一定浓度的Zn~(2+)、Cu~(2+)和Cd~(3+),TN对Pb~(2+)富集程度最高,细胞壁及胞内富集率达到98.33%;菌体蓄积Pb~(2+)后细胞壁表面有沉淀物附着并有铅元素检出,对Pb~(2+)的蓄积主要依靠细胞壁上的羧基、羟基、酰胺基及磷酸基团;将TN菌株接种到含有2.5 mg/g萘及200 mg/L Pb~(2+)的灭菌土壤中,经过13 d室温培养之后,萘的去除率为94.29%。结论 TN菌株对Pb~(2+)、Zn~(2+)、Cu~(2+)和Cd~(3+)均具有一定的耐受性,对Pb~(2+)的富集能力强。该菌株能够依靠细胞壁上的活性基团富集Pb~(2+),适用于萘和Pb~(2+)双重胁迫的土壤修复。     

Individual and combined cytotoxic effects of cadmium, copper, and nickel on brown cells of Mercenaria mercenaria

An assay based on the lysosomal incorporation of neutral red dye by brown cells of Mercenaria mercenaria (Bivalvia) was used to measure the cytotoxicity of Cd2+, Cu2+, and Ni2+ singly and in combination. Cytotoxicity was a linear function of Cd2+ concentration 0.1-1.5 mM and Cu2+ concentration between 10 and 100 microM. Nickel was not cytotoxic at concentrations as high as 10 mM. The presence of Cu2+ lessened the cytotoxic effect of Cd2+. Ni2+ did not affect cytotoxicity in combination with either Cu2+ or Cd2+.Ni2+ inflated estimates of cell survival by the neutral red assay in this study. Cells exposed to Ni2+ yielded measured quantities of neutral red dye in excess of those measured in cells from control treatments.     

Heavy Metal Concentrations in Food Chain of Lake Velenjsko jezero, Slovenia: An Artificial Lake from Mining

The concentration of metals (Pb, Cd, Zn, Hg) in different ecosystem components (lake water, sediment, plankton, macrophytes, and fish tissues) has been determined in Velenjsko jezero, an artificial lake resulting from mining activity. The risk to humans from consuming fish has been evaluated from the heavy metal load of fish muscle tissue. Heavy metals are transferred through the food chain at different levels, and Hg is found to be the element of the highest ecotoxicological concern. Although both sediment and plankton contain relatively low concentrations of Hg, this element accumulates in high levels in fish, especially in the benthivorous species Abramis brama danubii and predator species Perca fluviatilis. Moreover, Hg appears to be very mobile in the fish organism. Whereas the other metals remained mostly in liver (Cd) or gills (Zn, Pb), levels of Hg in fish muscle and liver were the same and markedly higher than in gills. However, in muscle, the average concentrations of each metal were below their maximum limits, determined either by Slovenian legislation or by the Food and Agriculture Organization.     

Effects of temperature on the sensitivity of sludge worm Tubifex tubifex Müller to selected heavy metals

The present study evaluates the effect of temperature on the sensitivity of the freshwater tubificid sludge worm Tubifex tubifex Müller to 10 heavy metal ions. Metals used in this study were cadmium, chromium, cobalt, copper, iron, lead, manganese, mercury, nickel, and zinc. The acute toxicity of these heavy metals was studied at 15, 20, 25, and 30 degrees C. The percentage mortality, relative toxicity, and EC50 values and their 95% confidence limits from 24 to 96 h were determined at varying temperatures. The EC50 values (mg/liter) of metal ions at 15 degrees C were Hg2+, 0.034; Cu2+, 0.340; Cr6+, 1.846; Zn2+, 10.99; Ni2+, 25.10; Cd2+, 56; Fe3+, 86.09; Co2+, 239.39; Pb2+, 456.76; and Mn2+, 164.55. At 30 degrees C the values were Hg2+, 0.014; Cu2+, 0.031; Cr6+, 0.872; Zn2+, 3.37; Ni2+, 18; Cd2+, 28.55; Fe3+, 71.26; Co2+, 95.35; Pb2+, 165.22; and Mn2+, 239.39. The results indicate that the acute toxicity of cadmium, chromium, cobalt, copper, lead, mercury, nickel, and zinc increases with temperature increase. The toxicity of manganese was not influenced by temperature, and temperature had little effect on iron toxicity. The rank order of toxicity of metal ions at 15, 20, 25, and 30 degrees C is presented and discussed. It is concluded that temperature is an important factor in short-term acute toxicity tests. The study indicates that seasonal temperature changes are an important variable in determining the amount of heavy metals that may be safely released from metal industries and other similar sources into the aquatic environment. Influence of temperature on the short- and long-term toxicity of chemicals should be considered for establishing appropriate water-quality criteria and standards to protect aquatic flora and fauna and human health.     

Element differences and evaluation of the dietary intake from farmed oysters and mussels collected at different sites along the Croatian coast of the Adriatic Sea

Concentrations of the macroelements Ca, Mg, K and Na, microelements Cr, Cu, Fe, Mn and Zn and toxic metals As, Cd, Hg and Pb were determined in oysters and mussels collected at farming sites in the northern, central and south Adriatic Sea. Mean concentrations in oysters were (mg kg⁻¹): As 4.51, Ca 1551, Cd 1.44, Cu 53.6, Cr 0.23, Fe 45.9, Hg 0.047, K 2476, Mg 895, Mn 2.80, Na 7148, Pb 0.52, Zn 675. Mean values in mussels were (mg kg⁻¹): As 4.71, Ca 601, Cd 0.37, Cu 1.91, Cr 0.29, Fe 49.6, Hg 0.026, K 2246, Mg 1043, Mn 2.49, Na 7992, Pb 0.58, Zn 28.8. Significant differences in the concentrations of toxic metals and elements were found in oysters and mussels among farming sites. Obtained Cd, Hg and Pb concentration in oysters and mussels were lower than the limit levels set by the European Community. However, Cd concentration exceeded the limit level of 1 mg kg⁻¹ in oysters from all locations except Lim Bay. The estimated daily intakes (EDIs) indicate that oysters are a good source of Ca, Cu and Zn. The higher Cu and Zn concentrations than the toxicity reference values suggest a risk for consumers if larger quantities of oysters are consumed frequently.     

Heavy metals in three lakes in West Poland.

Concentrations of heavy metals (Ni, Cr, Co, Zn, Mn, Pb, Cd, Cu, Hg, Fe) as well as macronutrients (P, Ca, Mg) were measured in water, bottom sediments, and plants of three lakes in West Poland (southwest of Poznan). The plants collected were Nymphaea alba, Nuphar luteum, Ceratophyllum demersum, Phragmites communis, Typha latifolia, and Schoenoplectus lacustris. These plants contained elevated levels of Co, Zn, Pb, Cd, Cr, and Hg. Analyses of water and bottom sediments indicated that the lakes were polluted with Zn, Cd, Cu, and Pb and partly with Ni and Hg. Strong positive correlations were found between concentrations of Cd in water and in plants, between concentrations of Cd in bottom sediments and in plants, between concentrations of Cr in water and in plants, between concentrations of Cr in bottom sediments and in plants, and between concentrations of Fe in water and in plants, indicating the potential of plants for pollution monitoring for these metals. A negative correlation was found between biomass production and Pb and Mn content in water and Pb and Mn content in bottom sediments. Cr and Cd accumulated in plants at a higher rate from bottom sediments than from water. The accumulation rate of Fe in plant tissues was higher with an increase in Fe in water than with an increase in Fe in bottom sediments.     

Exposure of human proximal tubule cells to cd2+, zn2+, and Cu2+ induces metallothionein protein accumulation but not metallothionein isoform 2 mRNA.

The organization of the human metallothionein (MT) gene family is more complex than the commonly used mouse and rat models. The human MTs are encoded by a family of genes consisting of 10 functional and 7 nonfunctional MT isoforms. One objective of this study was to determine if the accumulation of MT protein in cultures of human proximal tubule (HPT) cells exposed to metals is similar to that expected from the knowledge base obtained from rodent models. To accomplish this objective, HPT cells were exposed to both lethal and sublethal concentrations of Cd2+, Zn2+, Cu2+, Ag2+, Hg2+, and Pb2+ and MT protein levels were determined. The results were in general agreement with animal model studies, although there were some exceptions, mainly in areas where the animal model database was limited. In clear agreement with animal models, Cd2+, Zn2+, and Cu2+ were demonstrated to be potent inducers of MT protein accumulation. In contrast to the similarity in MT protein expression, we obtained evidence that the human renal MT-2 gene has a unique pattern of regulation compared to both animal models and human-derived cell cultures. In the present study, we determined that MT-2A mRNA was not induced by exposure of HPT cells to Cd2+ or the other metals, a finding in contrast to studies in both animal models and other human cell culture systems in which a high level of MT-2 mRNA induction occurs upon exposure to Cd2+ or Zn2+. While MT protein expression may be similar between humans and animal models, this finding provides initial evidence that regulation of the genes underlying MT protein expression may be divergent between species.     

Cooperation of divalent ions and thiamin diphosphate in regulation of the function of pig heart pyruvate dehydrogenase complex

The role of Mg2+, Ca2+, and Mn2+ in regulation of purified pig heart pyruvate dehydrogenase complex (PDC) containing endogenous thiamin diphosphate (TDP) was studied. It was found that the effects of the cations depended on the presence of exogenous TDP. In the absence of added TDP, the divalent cations led to a shortening of a lag phase of the PDC reaction and a strong reduction of the Km value for pyruvate. The relative efficiency of the three types of ions are presented as follows: Mn2+>Ca2+>Mg2+. The other sources claim that in the presence of exogenous TDP, which alone strongly increased the affinity of PDC for pyruvate, any significant additional effects of the cations were not observed. However, Mg2+, Ca2+, and Mn2+ decreased the Km value for CoA in both cases, the absence and presence of exogenous TDP, in approximately a similar extent (about twofold). The affinity of PDC for NAD+ seems to be not sensitive to the presence of the divalent cations. The data obtained suggest that Mg2+, Ca2+, and Mn2+ can cooperate with TDP as positive regulatory effectors of pig heart PDC on the level of pyruvate dehydrogenase and lipoamide acetyltransferase components of the complex.     

Observations on metal concentrations in tilapia (Oreochromis mossambicus) in reservoirs of south Sri Lanka

Samples of the muscle and liver of tilapia (Oreochromis mossambicus) obtained from the five reservoirs in four catchments in southern Sri Lanka in 1998 were analyzed for 16 elements: As, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Sr, and Zn by inductively coupled plasma emission spectrophotometry, and Hg by atomic absorption spectrophotometry. The concentrations of Cr, Ni, and Pb were below the detection limits of the instrumental techniques employed in all samples. The elements As, Ca, Co, Cu, Fe, Hg, K, Mg, Mn, Na, Sr, and Zn were detected in the muscle and liver tissue, with Cd detected in some liver samples. There were no statistically significant differences between the individual concentrations of any of the metals and the site from which the tilapia were collected (P>0.05). Furthermore, no statistically significant correlations were found between total length of fish and metal concentration. No elements were found at concentrations of toxicological concern. However, a principal component analysis suggests that the populations of tilapia in the reservoirs may be exposed to different regimes of metals, possibly associated with different catchment land-use patterns.