High Urinary Tungsten Concentration Is Associated with Stroke in the National Health and Nutrition Examination Survey 1999–2010

Background

In recent years there has been an exponential increase in tungsten demand, potentially increasing human exposure to the metal. Currently, the toxicology of tungsten is poorly understood, but mounting evidence suggests that both the elemental metal and its alloys have cytotoxic effects. Here, we investigate the association between tungsten and cardiovascular disease (CVD) or stroke using six waves of the National Health and Nutrition Examination Survey (NHANES).

Methods

We investigated associations using crude and adjusted logistic regression models in a cohort of 8614 adults (18–74 years) with 193 reported stroke diagnoses and 428 reported diagnoses of CVD. We also stratified our data to characterize associations in a subset of younger individuals (18–50 years).

Results

Elevated tungsten concentrations were strongly associated with an increase in the prevalence of stroke, independent of typical risk factors (Odds Ratio (OR): 1.66, 95% Confidence Interval (95% CI): 1.17, 2.34). The association between tungsten and stroke in the young age category was still evident (OR: 2.17, 95% CI: 1.33, 3.53).

Conclusion

This study represents the most comprehensive analysis of the human health effects of tungsten to date. Individuals with higher urinary tungsten concentrations have double the odds of reported stroke. We hypothesize that the pathological pathway resulting from tungsten exposure may involve oxidative stress.     

The nitrate reductase inhibitor,tungsten, disrupts actin microfilaments in Zea mays L.

Tungsten is a widely used inhibitor of nitrate reductase, applied to diminish the nitric oxide levels in plants. It was recently shown that tungsten also has heavy metal attributes. Since information about the toxic effects of tungsten on actin is limited, and considering that actin microfilaments are involved in the entry of tungsten inside plant cells, the effects of tungsten on them were studied in Zea mays seedlings. Treatments with sodium tungstate for 3, 6, 12 or 24 h were performed on intact seedlings and seedlings with truncated roots. Afterwards, actin microfilaments in meristematic root and leaf tissues were stained with fluorescent phalloidin, and the specimens were examined by confocal laser scanning microscopy. While the actin microfilament network was well organized in untreated seedlings, in tungstate-treated ones it was disrupted in a time-dependent manner. In protodermal root cells, the effects of tungsten were stronger as cortical microfilaments were almost completely depolymerized and the intracellular ones appeared highly bundled. Fluorescence intensity measurements confirmed the above results. In the meristematic leaf tissue of intact seedlings, no depolymerization of actin microfilaments was noticed. However, when root tips were severed prior to tungstate application, both cortical and endoplasmic actin networks of leaf cells were disrupted and bundled after 24 h of treatment. The differential response of root and leaf tissues to tungsten toxicity may be due to differential penetration and absorption, while the effects on actin microfilaments could not be attributed to the nitric oxide depletion by tungsten.     

Abscisic acid relations of plants grown on tungsten enriched substrates

The effect of tungsten as a potential inhibitor of ABA biosynthesis in plants was tested both under controlled conditions in the greenhouse or laboratory and in the field at a site naturally enriched with tungsten. Although as predicted in all cases the activity of aldehyde oxidase was strongly inhibited by tungsten, in well watered, greenhouse-grown maize plants increased concentrations of tungsten did not reduce ABA concentration in tissues. Depending on the time of incubation, mostly an accumulation due to an increased net ABA synthesis could be observed. Under drought stress, however, tungsten may have an inhibitory effect on ABA accumulation. Field experiments with drought-stressed clover and dandelion plants from a habitat close to a former tungsten mine confirm those of the greenhouse and laboratory. The ineffectiveness of tungsten in decreasing ABA levels in well-watered plants and its minimal effects in inhibiting ABA accumulation of droughted plants indicate that it is unsuitable as an inhibitor of ABA biosynthesis.     

Measurement of the electric conductivity of tungsten in a continuous liquid-to-gas transition

A method is developed that makes it possible to investigate the transition of a metal from a condensed to a gaseous phase while maintaining almost uniform temperature and pressure distributions in the sample. The method consists in the pulsed Joule heating of a sample in the form of a thin foil strip placed between two relatively thick glass plates. This method is used to measure the conductivity of tungsten in a process during which the pressure in the sample is maintained at a level of 40–60 kbar and the density of the sample decreases from the normal solid density to a density 20 to 30 times lower. It is found that, along the 40-kbar isobar, the density dependence of the conductivity of tungsten changes radically at a certain density value, at which it has a pronounced kink. At the kink, the density of tungsten is approximately ten times lower than its characteristic solid density, and the internal energy is about two times the sublimation energy. The method makes it possible to carry out experiments with the almost isobaric heating of tungsten in the parameter range in which the effect in question takes place. No such effect is detected in nonisobaric processes.     

The tungsten metallome of Pyrococcus furiosus

The tungsten metallome of the hyperthermophilic archaeon Pyrococcus furiosus has been investigated using electroanalytical metal analysis and native-native 2D-PAGE with the radioactive tungsten isotope (187)W (t(1/2) = 23.9 h). P. furiosus cells have an intracellular tungsten concentration of 29 μM, of which ca. 30% appears to be free tungsten, probably in the form of tungstate or polytungstates. The remaining 70% is bound by five different tungsten enzymes: formaldehyde ferredoxin oxidoreductase, aldehyde ferredoxin oxidoreductase, glyceraldehyde-3-phosphate ferredoxin oxidoreductase and the tungsten-containing oxidoreductases WOR4 and WOR5. The membrane proteome of P. furiosus is devoid of tungsten. The differential expression, as measured by the tungsten level, of the five soluble tungsten enzymes when the cells are subjected to a cold-shock shows a strong correlation with previously published DNA microarray analyses.     

Hematological effects and metal residue concentrations following chronic dosing with tungsten-iron and tungsten-polymer shot in adult game-farm mallards

The U.S. Fish and Wildlife Service required a chronic dosing study that assessed the health and reproductive effects of tungsten-iron and tungsten-polymer shot in adult game-farm mallards (Anas platyrhynchos) prior to granting permanent approval of the shot for waterfowl hunting. Herein, we present the effects of tungsten-iron and tungsten-polymer shot on various hematologic parameters and metal residue concentrations in the femur, liver, kidneys, and gonads. Thirty-two-bird groups (sexes equal) of adult mallards were dosed orally with eight #4 steel shot (control), eight #4 tungsten-iron shot, or eight #4 tungsten-polymer shot on days 0, 30, 60, 90, and 120 of a 150 day trial (26 January 1998 to 25 June 1998). An additional 12 mallards (sexes equal) received eight #4 lead shot (positive control) on day 0 of the study. Lead-dosed mallards had significantly decreased hematocrit, hemoglobin concentration, and whole-blood delta aminolevulinic acid dehydratase activity on day 7, as well as significant changes in a number of plasma chemistry parameters compared to ducks in the control, tungsten-iron, or tungsten-polymer groups. Mallards dosed with tungsten-iron or tungsten-polymer shot had occasional significant differences in hematocrit and plasma chemistry values when compared to control mallards over the 150 day period, but these changes were not considered to be indicative of deleterious effects. Low concentrations of tungsten were detected in gonad and kidney samples from males and females and in liver samples from females dosed with tungsten-polymer shot. Tungsten was also detected in femur samples from tungsten-polymer-dosed mallards. Higher concentrations of tungsten were detected in femur, liver, kidney, and gonad samples from tungsten-iron-dosed ducks. Tungsten-iron or tungsten-polymer shot repeatedly administered to adult mallards did not cause adverse hematological effects during the 150 day trial. Concentrations of tungsten in the femur, liver, kidneys, and gonads were generally higher in tungsten-iron-dosed ducks when compared to tungsten-polymer-dosed ducks.     

Generation of DNA double-strand breaks and inhibition of somatic embryogenesis by tungsten microparticles in wheat

Particles of metallic tungsten, known also as tungsten microprojectiles, are routinely used to deliver foreign DNA into target cells and tissues. Some side effects of biolistic transformation have been observed but never studied in detail. Here we present evidence that intact tungsten particles can promote a breakage of phosphodiester bonds in native DNA, at a limited number of sites. A single, double-strand break appeared within almost each of the circular pUC119 molecules after a short incubation of plasmid DNA with a suspension of tungsten particles. No further DNA cutting could be induced even if the reaction rate was accelerated by increasing the concentration of tungsten in the incubation mixture. Indirect evidence indicates that similar lesions may be generated in cellular DNA of bombarded tissues. These lesions are rapidly repaired, as evidenced by increasing incorporation of labelled DNA precursors in bombarded wheat embryos. The rate of repair is, however, not high enough to restore all the genome functions. Neither germination of mature embryos nor initiation of callus tissues from immature embryos was inhibited by biolistic bombardment. Nevertheless, the frequency of formation of somatic embryos in calli derived from bombarded embryos was markedly lower than in calli derived from control embryos. Both immediate (generation of a limited number of double-strand breaks) and remote (selective inhibition of somatic embryogenesis) side effects of the biolistic process strongly suggest that biological activity of tungsten deserves special attention. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molybdenum and tungsten in Campylobacter jejuni: their physiological role and identification of separate transporters regulated by a single ModE-like protein

Campylobacter jejuni is an important human pathogen that causes millions of cases of food-borne enteritis each year. The C. jejuni respiratory chain is highly branched and contains at least four enzymes predicted to contain a m etal binding pt erin (MPT), with the metal being either molybdenum or tungsten. Also predicted are two separate transport systems, one for molybdenum encoded by modABC and a second for tungsten encoded by tupABC . Both transport systems were mutated and the activities of the four predicted MPT-containing enzymes were assayed in the presence of molybdenum and tungsten in wild-type and mod and tup backgrounds. Results indicate that mod is primarily a molybdenum transporter that can also transport tungsten, while tup is a tungsten-specific transporter. The MPT-containing enzymes nitrate reductase, sulphite oxidase, and SN oxide reductase are strict molybdoenzymes while formate dehydrogenase prefers tungsten. A ModE-like protein regulates both transporters, repressing mod in the presence of both molybdenum and tungsten and tup only in the presence of tungsten. Like other ModE proteins, the C. jejuni ModE binds DNA through a helix–turn–helix DNA binding domain, but unlike other members of the ModE family it does not have a metal binding domain.     

Electroanalytical determination of tungsten and molybdenum in proteins

Recent crystal structure determinations accelerated the progress in the biochemistry of tungsten-containing enzymes. In order to characterize these enzymes, a sensitive determination of this metal in protein-containing samples is necessary. An electroanalytical tungsten determination has successfully been adapted to determine the tungsten and molybdenum content in enzymes. The tungsten and molybdenum content can be measured simultaneously from 1 to 10 microg of purified protein with little or no sample handling. More crude protein samples require precipitation of interfering surface active material with 10% perchloric acid. This method affords the isolation of novel molybdenum- and tungsten-containing proteins via molybdenum and tungsten monitoring of column fractions, without using radioactive isotopes. A screening of soluble proteins from Pyrococcus furiosus for tungsten, using anion-exchange column chromatography to separate the proteins, has been performed. The three known tungsten-containing enzymes from P. furiosus were recovered with this screening.     

Stimulatory Effects of Sodium Tungstate on the Growth of the Blue-green Alga, Anabaena doliolum

Sodium tungstate increases the growth rate and final growthyields of molybdenum-starved cultures. Also, concentrationsof tungstate much higher than of molybdenum are non-toxic andexert growth promotory effect. like molybdenum, tungsten effectsare stimulatory on inorganic and molecular nitrogen, whereason amino acids as nitrogen source, the effects are toxic. Thechelator 8-hydroxyquinoline nullifies the growth promotory effectof both tungsten and molybdenum. Effects of both the elementson heterocyst size and frequency are also comparable. Implicationsof these findings are discussed in relation to the possiblerole of tungsten.     

Differential effects of sodium tungstate and vanadyl sulfate on vascular responsiveness to vasoactive agents and insulin sensitivity in fructose-fed rats

High fructose feeding induces insulin resistance, impaired glucose tolerance, and hypertension in rats and mimics most of the features of the metabolic syndrome X. The effects of a 6-week treatment with the transition metals administered in drinking water, vanadium (VOSO4.5H2O, 0.75 mg/mL) or tungsten (Na2O4W, 2 g/mL), were investigated on the reactivity to norepinephrine (NEPI) or acetylcholine (ACh) of thoracic aorta rings isolated from fructose (60%) or standard chow fed rats. Maximal effect (Emax) and pD2 (-log EC50) values were determined in each case in the presence or absence of endothelium, while the degree of insulin resistance was determined using the euglycemic hyper insulinemic glucose clamp technique. Aortic segments isolated from 6-week fructose-fed animals were characterized by NEPI hyperresponsiveness (increase in Emax) and endothelium-dependent NEPI supersensitivity (increase in pD2) without any change in the reactivity to ACh. Vanadium or tungsten administered in fructose-fed animals prevented both hypertension and NEPI hyperresponsiveness, while vanadium, but not tungsten, reduced NEPI supersensitivity. Vanadium, but not tungsten, increased the relaxing activity of ACh, both in control and fructose-fed animals. Insulin resistance associated with high fructose feeding was reversed by vanadium but not by tungsten treatment. The differential effects of the two transition metals on vascular responsiveness to NEPI or ACh may be explained by their differential effects on insulin sensitivity.     

Vanadium and tungsten derivatives as antidiabetic agents: a review of their toxic effects

Tungstate is an oxyanion that has biological similarities to vanadate. In recent years, a number of studies have shown the antidiabetic effects of oral tungstate in animal models of diabetes. However, because of the tissue accumulation and potential toxicity derived from chronic administration of vanadium and tungsten compounds, the pharmacological use of vanadate or tungstate in the treatment of diabetes is not necessarily exempt from concern. In the context of a potential use in the treatment of human diabetes mellitus, the most relevant toxic effects of vanadium derivatives are reviewed and compared with those reported for tungsten. Hematological and biochemical alterations, loss of body weight, nephrotoxicity, immunotoxicity, reproductive and developmental toxicity, and behavioral toxicity have been reported to occur following exposure to vanadium compounds. Moreover, vanadium also has a mitogenic activity affecting the distribution of chromosomes during mitosis and inducing aneuploidyrelated end points. In contrast to vanadate, studies about the toxic effects of tungstate are very scant. Early investigations in cats, rabbits, dogs, mice, and rats showed that tungstate was less toxic than vanadate when given intravenously. Although in vitro investigations showed a direct effect of tungstate on the embryo and fetus of mice at concentrations similar to those causing effects in vivo, information on the potential cellular toxicity of tungstate is particularly scarce. Taking into account the recent interest of tungstate as a new potential oral antidiabetic agent, an exhaustive evaluation of its toxicity in mammals is clearly necessary.     

In vitro reconstitution of demolybdosulfite oxidase by molybdate.

Reconstitution of purified demolybdosulfite oxidase from rat liver has been achieved using inorganic molybdate as the source of molybdenum. The activation process has a pH optimum of 7.4 and is dependent on concentrations of molybdate and demolybdoenzyme. The reaction is inhibited by high concentrations of anions and by reduction of the demolybdoenzyme and requires incubation temperatures higher than 30 degrees. A reconstitution mechanism involving loss of tungsten and concomitant replacement with molybdenum in those demolybdo molecules which contain tungsten is supported by the following observations: (a) the extent of activation achieved by molybdate corresponds to the proportion of molecules in the preparation which contain tungsten. (b) Incubation of the demolybdoenzyme preparation at 37 degrees in the absence of molybdate results in progressive and concentration-dependent loss of ability to be reconstituted by molybdate and a corresponding but more rapid loss of tungsten from the enzyme. The reconstituted enzyme displays the molybdenum EPR signal characteristic of native enzyme and is inactivated by incubation at 42 degrees in a manner identical to native sulfite oxidase.     

A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei.

We have recently reported that the thermophilic archaeon Methanobacterium wolfei contains two formylmethanofuran dehydrogenases, I and II. Formylmethanofuran dehydrogenase II, which is preferentially expressed in tungsten-grown cells, has been purified and shown to be a tungsten-iron-sulfur protein. We have now purified and characterized formylmethanofuran dehydrogenase I from molybdenum-grown cells and shown that it is a molybdenum-iron-sulfur protein. The purified enzyme, with a specific activity of 27 U/mg protein, was found to be composed of three subunits of apparent molecular mass 64 kDa, 51 kDa, and 31 kDa and to contain per mol 146-kDa molecule approximately 0.23 mol molybdenum, 0.46 mol molybdopterin guanine dinucleotide, and 6.6 mol non-heme iron but no tungsten (< 0.01 mol). The molybdenum enzyme differed from the tungsten enzyme (8 U/mg) in that it catalyzed the oxidation of N-furfurylformamide and formate and was inactivated by cyanide. The two enzymes also differed significantly in the pH optimum, in the apparent Km for the electron acceptor, and in the chromatographic behaviour. The molybdenum enzyme and the tungsten enzyme were similar, however, in that the N-terminal amino acid sequences determined for the alpha and beta subunits were identical up to residue 23, indicating that the two proteins are isoenzymes. The molybdenum enzyme, as isolated, was found to display an EPR signal derived from molybdenum as evidenced by isotope substitution.     

“CLASPing” tungsten's effects on microtubules with “PINs”

Tungsten, supplied as sodium tungstate, inhibits root elongation in Arabidopsis thaliana, which has been attributed to a diminishing of PIN2 and PIN3 auxin efflux carriers. In this work, we sought to analyze the effect of tungsten on cortical microtubules and CLASP (Cytoplasmic Linker Associated Protein), which are also involved in the anisotropic cell expansion of root cells. Seedlings grown in a tungsten-free substrate for 4 d and then transplanted into a tungsten-containing substrate exhibited randomly oriented microtubules in a time-dependent manner. While tungsten had no effect on roots treated for 3 h, microtubule alignment was obviously affected in the transition and elongation zones after a 6, 12, 24, 48 h tungsten treatment, at prolonged tungsten administrations and in seedlings grown directly in the presence of tungsten. This change in microtubule orientation may be associated with the reduction of CLASP protein expression induced by tungsten, as evidenced in experiments with plants expressing the CLASP-GFP protein. A possible mechanism, by which the coordinated functions of CLASP, PIN2 and microtubules are affected, as revealed by inhibited root growth, is discussed.     

What is a healthy ecosystem?

Rapid deterioration of the world's major ecosystems has intensified the need for effective environmental monitoring and the development of operational indicators of ecosystem health. Ecosystem health represents a desired endpoint of environmental management, but it requires adaptive, ongoing definition and assessment. We propose that a healthy ecosystem is one that is sustainable – that is, it has the ability to maintain its structure (organization) and function (vigor) over time in the face of external stress (resilience). Various methods to quantify these three ecosystem attributes (vigor, organization, and resilience) are discussed. These attributes are then folded into a comprehensive assessment of ecosystem health. A network analysis based ecosystem health assessment is developed and tested using trophic exchange networks representing several different aquatic ecosystems. Results indicate the potential of such an ecosystem health assessment for evaluating the relative health of similar ecosystems, and quantifying the effects of natural or anthropogenic stress on the health of a particular ecosystem over time.     

The particle inflow gun can be used to co-transform Paramecium using Tungsten particles

A particle inflow gun (PIG) was constructed and tested for its utility to transform Paramecium using tungsten or gold as the DNA carrier particle. In the first set of experiments we transformed Paramecium with a plasmid containing the neomycin-resistance gene, obtaining a transformation efficiency of 0.31+/-0.14% (mean+/-SD) for tungsten particles and 1.30+/-0.29% for gold particles. Plasmid DNA precipitated upon tungsten was shown to be stable for transformation purposes for up to 1 h prior to use and had no detectable effects on transformation efficiency. In addition, we demonstrated that at high frequency (71+/-20%) a Paramecium mutant strain could be phenotypically rescued by co-transformation with a second plasmid containing the selectable neomycin-resistance gene. The PIG coupled with tungsten particles as the carrier offers a low-cost alternative for biolistic transformation of Paramecium.     

The detection of high-risk groups among workers in contact with heavy metals based on an analysis of chromosome aberrations and sister chromatid exchanges

Heavy metal salts, the workers from molybdenum, tungsten and cobalt plants to make in contact with, reveal their mutagenic activity. Individual sensitivity to heavy metal salts has been analyzed through the example of molybdenum. Chromosome aberrations and sister chromatid exchanges have been studied for the regularities of their formation in lymphocytes of workers depending on the length of service. Sensitivity of tests of chromosome aberrations and sister chromatid exchanges has been compared to reveal genetic consequences of these types of the effects.     

Issues in Human Particulate Exposure Assessment: Relationship between Outdoor,Indoor, and Personal Exposures

The recent review of the National Ambient Air Quality Standard for particuslate matter and the resultant new health-based PM_2.5 standard was in part motivated by findings from epidemiological studies. These studies reported significant associations between adverse health effects and concentrations of ambient particulate matter at levels below the previously existing PM₁₀ standard. Interpretation of these results has been hindered by our relatively poor understanding of the relationship between personal exposures and concentrations in the indoor and outdoor environments. Individuals spend the majority of their time in indoor environments. Therefore, it is important to understand where and how they may be exposed to the contaminants which may be causing the health effects, and which activities place them at a higher risk of exposure to these agents. In addition, since particulate matter is a complex mixture of contaminants, further research is required to examine its formation process, sources, composition, and health effects. Without an improved scientific understanding of these issues, it is difficult to assess whether the new PM_2.5 standard will be implemented, and if so, whether it can be adequately protective of public health.     

Quality assurance issues and PACS

Quality assessment and assurance is a growing concern in all areas of health care. The concern is fueled by a body of evidence that indicates that quality of care is not optimal, and in many instances, is unacceptably low. Although different standards for quality have been proposed, health outcome is the ultimate standard, since improving health is the goal of the health care system. The effects of PACS on health outcome are not known. A PAC system has the potential to improve quality, especially if it makes the diagnostic process more efficient, but evidence that such improvement actually occurs is so far lacking.