Metal uptake and acute toxicity in zebrafish: common mechanisms across multiple metals

Zebrafish larvae (Danio rerio) were used to examine the mechanisms of action and acute toxicities of metals. Larvae had similar physiological responses and sensitivities to waterborne metals as adults. While cadmium and zinc have previously been shown to reduce Ca(2+) uptake, copper and nickel also decreased Ca(2+) uptake, suggesting that the epithelial transport of all these metals is through Ca(2+) pathways. However, exposure to cadmium, copper or nickel for up to 48 h had little or no effect on total whole body Ca(2+) levels, indicating that the reduction of Ca(2+) uptake is not the acute toxic mechanism of these metals. Instead, mortalities were effectively related to whole body Na(+), which decreased up to 39% after 48 h exposures to different metals around their respective 96 h LC50s. Decreases in whole body K(+) were also observed, although they were not as pronounced or frequent as Na(+) losses. None of the metals tested inhibited Na(+) uptake in zebrafish (Na(+) uptake was in fact increased with exposure) and the observed losses of Na(+), K(+), Ca(2+) and Mg(2+) were proportional to the ionic gradients between the plasma and water, indicating diffusive ion loss with metal exposure. This study has shown that there is a common pathway for metal uptake and a common mechanism of acute toxicity across groups of metals in zebrafish. The disruption of ion uptake accompanying metal exposure does not appear to be responsible for the acute toxicity of metals, as has been previously suggested, but rather the toxicity is instead due to total ion loss (predominantly Na(+)).     

The vascular system as a target of metal toxicity.

Vascular system function involves complex interactions among the vascular endothelium, smooth muscle, the immune system, and the nervous system. The toxic metals cadmium (Cd), arsenic (As), and lead (Pb) can target the vascular system in a variety of ways, ranging from hemorrhagic injury to subtle pathogenic remodeling and metabolic changes. Acute Cd exposure results in hemorrhagic injury to the testis, although some strains of animals are resistant to this effect. A comparison of Cd-sensitive with Cd-resistant mouse strains showed that expression of the Slc39a8 gene, encoding the ZIP8 transporter, in the testis vasculature endothelium is responsible for this difference. Endogenously, ZIP8 is a Mn(2+)/HCO(3)(-)symporter that may also contribute to Cd damage in the kidney. Chronic Cd exposure is associated with various cardiovascular disorders such as hypertension and cardiomyopathy and it is reported to have both carcinogenic and anticarcinogenic activities. At noncytotoxic concentrations of 10-100nM, Cd can inhibit chemotaxis and tube formation of vascular endothelial cells. These angiostatic effects may be mediated through disruption of vascular endothelial cadherin, a Ca(2+)-dependent cell adhesion molecule. With regard to As, ingestion of water containing disease-promoting concentrations of As promotes capillarization of the liver sinusoidal endothelium. Because capillarization is a hallmark precursor for liver fibrosis and contributes to an imbalance of lipid metabolism, this As effect on hepatic endothelial cells may be a pathogenic mechanism underlying As-related vascular diseases. With regard to Pb, perinatal exposure may cause sustained elevations in adult blood pressure, and genetically susceptible animals may show enhanced sensitivity to this effect. Taken together, these data indicate that the vascular system is a critical target of metal toxicity and that actions of metals on the vascular system may play important roles in mediating the pathophysiologic effects of metals in specific target organs.     

Amelioration of Metal-Induced Toxicity in Caenorhabditis elegans: Utility of Chelating Agents in the Bioremediation of Metals

The presence of toxic amounts of transition metals in the environment may originate from a range of human activities and natural processes. One method for the removal of toxic levels of metals is through chelation by small molecules. However, chelation is not synonymous with detoxification and may not affect the bioavailability of the metal. To test the bioavailability of chelated metals in vivo, the effects of several metal/chelator combinations were tested in the environmentally relevant organism Caenorhabditis elegans. The effect of metal exposure on nematode growth was used to determine the toxicity of cadmium, copper, nickel, and zinc. The restoration of growth to levels observed in nonexposed nematodes was used to determine the protective effects of the polydentate chelators: acetohydroxamic acid (AHA), cyclam, cysteine, calcium EDTA, desferrioxamine B, 1,2-dimethyl,3-hydroxy,4-pyridinone, and histidine. Cadmium toxicity was removed only by EDTA; copper toxicity was removed by all of the chelators except AHA; nickel toxicity was removed by cyclam, EDTA, and histidine; and zinc toxicity was removed by only EDTA. These results demonstrate the utility of polydentate chelators in the remediation of metal-contaminated systems. They also demonstrate that although the application of a chelator to metal contaminants may be effective, binding alone cannot be used to predict the level of remediation. Remediation depends on a number of factors, including metal complex speciation in the environment.     

Chemical and biological properties of toxic metals and use of chelating agents for the pharmacological treatment of metal poisoning

Exposure to toxic metals is a well-known problem in industrialized countries. Metals interfere with a number of physiological processes, including central nervous system (CNS), haematopoietic, hepatic and renal functions. In the evaluation of the toxicity of a particular metal it is crucial to consider many parameters: chemical forms (elemental, organic or inorganic), binding capability, presence of specific proteins that selectively bind metals, etc. Medical treatment of acute and chronic metal toxicity is provided by chelating agents, namely organic compounds capable of interacting with metal ions to form structures called chelates. The present review attempts to provide updated information about the mechanisms, the cellular targets and the effects of toxic metals.     

Pathogenic Mechanisms of Heavy Metal Induced-Alzheimer’s Disease

Alzheimer’s disease is an increasing neurodegenerative disease in the aging population. The disease is associated with toxic chemicals of industrial origin. Industrial processes can result in airborne contamination such as fine dust, and water and soil contamination. In the processes, heavy metals are one of the major environmental pollutants. Also, heavy metals are widely used for many appliances. In particular, heavy metals are seriously toxic to the neural system. In several studies, researchers have emphasized the toxicity of heavy metals such as lead, mercury, and cadmium, as a cause of neurofibrillary tangles, aggregation amyloid beta peptides (AβPs) as well as neuronal cell loss. Based on neurotoxic studies showing that heavy metals induce Alzheimer’s disease, this paper discusses molecular mechanisms by which exposure to heavy metals contributes to the pathogenesis of Alzheimer’s disease. Also, we indicate pathway for heavy metal related Alzheimer’s through integrated analysis based on molecular networks. We suggest that the study of signaling networks contributes to our ability to select significant factors for curing heavy metal induced Alzheimer’s disease.     

Independence and additivity of cultured hepatocyte killing by Ca2+ overload and ATP depletion.

In two competing models of toxic cell death, hepatocyte killing by chemical hypoxia (CN/IAA) is attributed to ATP depletion and killing by A23187 is attributed to Ca(2+)-induced damage. The independence of these models can be questioned because CN/IAA elevates Ca2+ before killing 1c1c7 hepatoma cells and because the ATP source fructose prevents hepatocyte killing by Br-A23187. In the present studies, cultured mouse hepatocytes were exposed to CN/IAA, A23187, or treatments in combination. A23187 produced toxicity proportional to Ca(2+)-activated DNA fragmentation. CN/IAA caused comparable toxicity but no fragmentation of DNA. Treatments in combination were more toxic than either treatment alone. Aurintricarboxylic acid, a Ca(2+)-endonuclease inhibitor, decreased DNA fragmentation and the toxicity of A23187 and combination treatment without affecting CN/IAA toxicity. ATP plus oligomycin decreased CN/IAA and combination treatment toxicity but not that of A23187. These findings indicate that cultured mouse hepatocytes are killed through mechanisms that are independent and additive in their toxicities.     

Hg(2+) and Cu(2+) interfere with agonist-mediated Ca(2+) signaling in isolated Mytilus digestive gland cells

The effects of mercury and copper on agonist-mediated Ca-signaling were investigated in isolated cells from the marine mussel, Mytilus galloprovincialis Lam., by single cell fluorescence microscopy. In isolated digestive gland cells, short-term exposure (10 min) to both Hg(2+), a highly toxic metal and Cu(2+), an essential metal, in the nano-low μM range caused a sustained increase in cytosolic [Ca(2+)]. The effect of mercury on resting [Ca(2+)] was stronger than that of copper. The Hg-induced elevation in [Ca(2+)] seemed to be mainly due to an increased influx through Verapamil-sensitive Ca-channels, whereas the effect of Cu(2+) was related to a release from thapsigargin-sensitive intracellular stores. Agonists, such as epidermal growth factor (EGF), bradykinin (BK) and ATP, evoked Ca(2+) transients in isolated digestive gland cells through different mechanisms similar to those observed in mammalian cells, demonstrating the presence of common pathways of Ca-mediated cell signaling in both invertebrates and vertebrates. The agonist-mediated Ca(2+) response was affected by exposure to Hg(2+) and Cu(2+) in a concentration dependent manner: both metals significantly reduced the amplitude of the Ca(2+) spikes elicited by BK and ATP and decreased the percentage of EGF-responsive cells. The effects of Hg(2+) and Cu(2+) were apparently independent of their different type of interaction with the mechanisms involved in Ca(2+) homeostasis. The results clearly demonstrate that, in marine invertebrate cells, short-term exposure to heavy metal concentrations comparable to environmental exposure levels results in alterations of intracellular Ca(2+) homeostasis which compromise the cell response to extracellular stimuli involving Ca-mediated signaling. The mechanisms of heavy metal interference with Ca-homeostasis and signaling are discussed.     

Evidence that E-cadherin may be a target for cadmium toxicity in epithelial cells

E-cadherin is a Ca(2+)-dependent cell adhesion molecule that plays an important role in the development and maintenance of epithelial polarity and barrier function. This commentary describes the results of recent studies showing that the environmental pollutant Cd(2+) can damage the E-cadherin-dependent junctions between many types of epithelial cells and reviews the evidence indicating that this effect results from the direct interaction of Cd(2+) with the E-cadherin molecule. In addition, the implications of these findings with respect to the mechanisms of Cd(2+) toxicity in specific target organs such as lung, kidney, bone, and the vascular endothelium are discussed.     

Toxicity of metal ions used in dental alloys: a study in the yeast Saccharomyces cerevisiae

Metal ions are released from dental alloys into the oral environment, which can cause biological responses over short and extended periods. Since most toxic metal ions are capable of inducing oxidative stress on cells through the mitochondrial respiratory chain, mitochondria may contribute to and be a target of metal toxicity. In this study, we investigated the effect of metal ions on growth of the budding yeast, Saccharomyces cerevisiae, and on the morphology and function of yeast mitochondria. Moreover, we tested whether mitochondrial respiratory activity contributes to metal toxicity. Metal ions affected yeast cell growth. The toxicity of metal ions to yeast cells, ranked in decreasing order are as follows: Hg > Ag > Au > Cu, Ni, Co, Zn. This result mostly correlates with the degree of toxicity of those metal ions to growth of human cells. The MIC90 of Hg, Ag and Au ions in synthetic complete media are 0.325, 5 and 320 microM, respectively. None of the toxic metal ions resulted in loss of mitochondrial respiratory activity. However, respiration-deficient rho0 cells appeared to be resistant to Ag ion, but not to Hg and Au ions. Furthermore, at high concentrations, Ag ion caused morphological changes in mitochondria. These studies indicate that yeast may be used as a model system to screen for toxic effect of metals ions from dental alloys, and that oxidation activity in mitochondria may play a role in acute toxicity of silver ion.     

The effect of divalent cations on neuronal nitric oxide synthase activity.

Neuronal nitric oxide synthase (NOS I) is a Ca(2+)/calmodulin-binding enzyme that generates nitric oxide (NO*) and L-citrulline from the oxidation of L-arginine, and superoxide (O(2)*(-)) from the one-electron reduction of oxygen (O(2)). Nitric oxide in particular has been implicated in many physiological processes, including vasodilator tone, hypertension, and the development and properties of neuronal function. Unlike Ca(2+), which is tightly regulated in the cell, many other divalent cations are unfettered and can compete for the four Ca(2+) binding sites on calmodulin. The results presented in this article survey the effects of various divalent metal ions on NOS I-mediated catalysis. As in the case of Ca(2+), we demonstrate that Ni(2+), Ba(2+), and Mn(2+) can activate NOS I to metabolize L-arginine to L-citrulline and NO*, and afford O(2)*(-) in the absence of L-arginine. In contrast, Cd(2+) did not activate NOS I to produce either NO* or O(2)*(-), and the combination of Ca(2+) and either Cd(2+), Ni(2+), or Mn(2+) inhibited enzyme activity. These interactions may initiate cellular toxicity by negatively affecting NOS I activity through production of NO*, O(2)*(-) and products derived from these free radicals.     

Generation of DNA damage by anti-neoplastic agents.

DNA has been one of the major targets of cancer chemotherapy. A variety of anti-neoplastic agents can cause different types of DNA lesions, including base alterations, single- or double-strand DNA breaks, DNA-DNA cross-links and DNA-protein cross-links. The exact processes by which these DNA lesions lead to cell death remain uncertain. However, pivotal roles of intracellular Ca2+ ion mobilization, activation of Ca(2+)-Mg(2+)-dependent endonuclease and induction of several oncogenes have been proposed. Understanding the mechanism of DNA damage and subsequent cell death will be important to improve the efficacy of cancer chemotherapy.     

Metal-based inhibitors of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are a large family of signaling enzymes playing critical role in signal transduction and regulation of cellular processes. Dysfunction of PTP activity is associated with diabetes, cancer, autoimmune disorders, and neural diseases. PTP inhibitors therefore emerged as promising therapeutic targets. Recent research indicates that besides small organic molecules, metal ions and metal complexes can also strongly inhibit PTPs both in vitro and in vivo, resulting in the increase of phosphorylation of corresponding substrates and the modulation of cellular process. Structure of metal complexes influences the potency and selectivity of PTP inhibition. Detailed studies on this subject are not only expected to yield metal-based drugs targeting individual PTPs, but also to support understanding the function of metals in organisms. This review focuses on recent advancements in this area of research.     

Gene expression influences on metal immunomodulation

Heavy metals in the environment originate from both human activities and natural processes. Exposure to these metals can result in important changes to immune activity. Depending on the metal and dose, these changes can result in enhanced immune function, diminished immune responses, or altered responses that produce autoimmune disease. One of the intriguing aspects of these various phenomena are the multiple points of interaction with cellular machinery at which metals elicit these changes. The individual sections of this review serve to underscore the variety of targets that can be altered by exposure to heavy metals, and provide some comparisons between the effects of specific heavy metals on the immune system. These observations may ultimately lead us to a comprehensive understanding of the mechanisms by which metals alter the immune system, and may enable the development of countermeasures to offset these effects.     

Protein kinase C isoforms as specific targets for modulation of vascular smooth muscle function in hypertension

Vascular contraction is an important determinant of the peripheral vascular resistance and blood pressure. The mechanisms underlying vascular smooth muscle (VSM) contraction and the pathological changes that occur in hypertension have been the subject of numerous studies and interpretations. Activation of VSM by vasoconstrictor stimuli at the cell surface causes an increase in [Ca(2+)](i), Ca(2+)-dependent activation of myosin light chain (MLC) kinase, MLC phosphorylation, actin-myosin interaction and VSM contraction. Additional signaling pathways involving Rho-kinase and protein kinase C (PKC) may increase the myofilament force sensitivity to [Ca(2+)](i) and MLC phosphorylation, and thereby maintain vascular contraction. PKC is a particularly intriguing protein kinase as it comprises a family of Ca(2+)-dependent and Ca(2+)-independent isoforms, which have different tissue and subcellular distribution, and undergo differential translocation during cell activation. PKC translocation to the cell surface may trigger a cascade of protein kinases, such as mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) that ultimately interact with the contractile myofilaments and cause VSM contraction. Also, PKC translocation to the nucleus may promote VSM growth and proliferation. Increased PKC expression and activity have been identified in several forms of hypertension. The subcellular location of PKC may determine the state of VSM activity, and may be useful in the diagnosis/prognosis of hypertension. Vascular PKC isoforms may represent specific targets for modulation of VSM hyperactivity, and isoform-specific PKC inhibitors may be useful in treatment of Ca(2+) antagonist-resistant forms of hypertension.     

Large-conductance Ca2+- activated K+ channels:physiological role and pharmacology

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels differ from most of other K(+) channels in that their activation is under dual control, i.e., activated by either increase in intracellular Ca(2+) or membrane depolarization. These channels, which are widely distributed in a variety of cells, can control Ca(2+) influx as well as a number of Ca(2+)-dependent physiological processes. In neurons or neuroendocrine cells, BK(Ca) channels are believed to play an important role in controlling hormonal secretion by altering the duration and frequency of action potentials. The activity of BK(Ca) channels functionally expressed in vascular endothelial cells can control K(+) efflux and affect intracellular Ca(2+) concentration. Experimental observations have revealed that a variety of compounds can directly modulate BK(Ca) channel activity. Epoxyeicosatrienoic acids, a metabolite of arachidonic acid, and the increase in intracellular cyclic GMP with vinpocetine or YC-1 can stimulate BK(Ca) channel activity. The increased activity of BK(Ca) channels thus serves as a negative feedback mechanism to limit Ca(2+) influx in excitable cells. Clotrimazole, an imidazole P-450 inhibitor used for the management of sickle cell anemia, can directly suppress BK(Ca) channel activity. Riluzole, a drug used for the treatment of amyotrophic lateral sclerosis, can directly enhance channel activity in neuroendocrine cells. This effect may explain its inhibitory action on excitatory neurotransmission. 2-Methoxyestradil, an endogenous metabolite of 17beta-estradiol, suppresses BK(Ca) channel activity, whereas resveratrol, a natural phytoalexin present in grapes and wine, directly stimulates BK(Ca) channel activity in vascular endothelial cells. These effects may be responsible for their actions on functional activities of endothelial cells. The fenamates, a family of nonsteroidal anti-inflammatory drugs, are also openers of BK(Ca) channels. Therefore, the modulation of BK(Ca) channel activity in excitable and non-excitable cells can be important for therapeutic interventions.     

Inhibition of ADP-induced platelet adhesion to immobilised fibrinogen by nitric oxide: evidence for cGMP-independent mechanisms

Nitric oxide (NO) is an established regulator of platelet function, although the processes by which NO modulates platelet adhesion are unclear. We studied the importance of Ca(2+) and phosphoinositol-3-kinase (PI3kinase) as targets for NO signalling, in the physiological context of platelet adhesion using adenosine diphosphate (ADP)-stimulated adhesion to immobilised fibrinogen. DPTA-NONOate induced a time and concentration-dependent inhibition of adhesion, and reduced protein tyrosine phosphorylation. The action of NO was cGMP-independent despite activation of the cGMP-signalling cascade, as evidenced by VASP phosphorylation. Furthermore, the cGMP-independent mechanism did not involve PKA. Platelet activation by ADP requires Ca(2+) and PI3kinase-dependent signalling pathways. We examined the effect of NO on these pathways using two approaches. Firstly, we dissected the signalling pathways using the P2Y(1)-receptor antagonist A3P5P, and secondly, directly inhibited Ca(2+) mobilisation and PI3kinase activity. ADP-induced adhesion was reduced but not abolished by A3P5P, suggesting signalling from P2Y(12) can induce adhesion. NO further reduced adhesion in the presence of A3P5P, indicating that NO inhibited adhesion independently of any effects on Ca(2+) mobilisation. Dimethyl bis-(o-aminophenoxy) ethane-tetraacetic acid (BAPTA) and wortmannin both partially inhibited ADP-induced adhesion, but completely abolished adhesion when used in combination, demonstrating that ADP-induced adhesion requires Ca(2+) and PI3kinase-regulated pathways. Combination of either dimethyl-BAPTA or wortmannin with DPTA-NONOate enhanced inhibition of both the Ca(2+) and PI3kinase-dependent pathways when compared to the levels of inhibition with either agent alone. Thus, we demonstrate that NO inhibits alpha(IIb)beta(3)-mediated adhesion, by targeting both Ca(2+) and PI3kinase pathways in a cGMP-independent manner.     

Cellular accumulation and distribution of uranium and lead in osteoblastic cells as a function of their speciation

Uranium (U) and lead (Pb) are accumulated and fixed for long periods in bone, impairing remodeling processes. Their toxicity to osteoblasts, the cells responsible for bone formation, is poorly documented. It has been previously shown that cytotoxicity and phenotypic effects of both metals on osteoblasts are highly influenced by metal speciation. Differences in sensitivity between cell types have been underlined as well. In this paper, cellular accumulation of U and Pb in cultured and primary osteoblastic cells was assessed by trace element analysis. Distribution of different species at the cell scale was investigated by electron microscopy. Internalization of both metals was shown to be correlated to cytotoxicity and population growth recovery after exposure. For each metal, the amount of metal uptake leading to 50% cell death was shown to be speciation-dependent. Scanning and transmission electron microscopy showed the formation of precipitates with phosphate in lysosomes for both metals, whose role in toxicity or cell defence remains to be clarified. Although a clear link was established between cytotoxicity and accumulation, differences in sensitivity observed in terms of speciation could not be fully explained and other studies seem necessary.     

A review of the effects of heavy metals on freshwater mussels

The widespread recent decline in the species diversity and population density of freshwater mussels in North America may be partly related to chronic, low-level exposure to toxic metals. As benthic filter-feeding organisms, freshwater mussels are exposed to metals that are dissolved in water, associated with suspended particles and deposited in bottom sediments. Thus, freshwater mussels can bioaccumulate certain metals to concentrations that greatly exceed those dissolved in water. In adult mussels, the most common site of metal uptake is the gill, followed by the mantle and the kidney. The toxic effects of metals on freshwater mussels have been examined in a few acute toxicity tests, but the sublethal effects of long-term exposure to low environmental concentrations are little understood. Sublethal exposure to metals can alter growth, filtration efficiency, enzyme activity and behaviour. Sublethal effects are frequently observed at concentrations that are only half the lethal concentrations. However, few toxicity tests have used environmentally realistic exposure concentrations. Total concentrations of Cd, Cu, Hg and Zn in many oxic surface waters are in the ngl^-1 range, yet many toxicity studies have exposed mussels to concentrations in the gl^-1 or even the mgl^-1 range. An understanding of the processes by which metals affect freshwater mussels would provide insights on the ecotoxicological significance of metal contamination to natural mussel populations and aid in the development of water-quality criteria that adequately protect mussels.     

反向分子对接应用于黄曲霉素B1毒性机制及药物Oltipraz靶点的探究

本研究旨在探究黄曲霉素B1的作用靶点,以探讨其致毒机理,并分析药物Oltipraz的减毒机制,为更好地防治黄曲霉素中毒提供依据。本研究采用反向分子对接的方法,使用了3个在线反向分子对接服务平台预测黄曲霉素B1,Oltipraz可能的作用靶点,综合分析所得结果,结合黄曲霉素中毒的临床表征对结果进行分析。黄曲霉素B1的对接结果表明AFB可能通过作用于细胞凋亡、激素代谢、免疫调节等过程中的相关蛋白扰乱了机体的正常功能,以及通过作用于组织特异性的蛋白造成肝、肾、胃等器官毒性。另外,药物Oltipraz的对接结果表明,Oltipraz可能一方面通过抑制AFB产生毒性的生物转化过程,另一方面通过拮抗多个黄曲霉素可能作用的靶点产生减毒作用。本研究用反向分子对接的方法系统而有效地预测了黄曲霉素B1和药物Oltipraz的靶点,为研发更有效的抗黄曲霉毒素的药物提供了一定依据。     

Toxicity of copper and cadmium in combinations to Duckweed analyzed by the biotic ligand model

The biotic ligand model (BLM) of acute toxicity to aquatic organisms is based on the concept that metals binding onto biotic ligand may cause toxic effect on the organism. The BLM can take into incorporation between metal speciation and the protective effects of competing cations account. The demonstrated BLM can provide a good estimation of the amount of single metal effect under various conditions such as pH, coexistence of other non toxic cations. However, toxic metals are often found as mixture in nature. This study estimated combined toxicity of Cu and Cd examined by growth inhibition of Duckweed (Lemna paucicostata) by using single toxicity data as toxic unit (TU) derived by three types of model, BLM and two conventional models, free ion activity model (FIAM), and total metal concentration model. According to our results, single toxicity data derived by the BLM can estimate combined toxicity described as a function of TU. Particularly under the high level of heavy metals stress, BLM clearly predicted toxicity of heavy metals compared with other two models. According to numeric correlation (R(2), root mean square error), the order is BLM (R=0.83, RMSE=13.5)> total metal concentration model (R=0.41, RMSE=24.9)> FIAM (R=0.36, RMSE=26.1).