Tungsten or Wolfram: Friend or Foe?

Send Orders for Reprints to [email protected] Current Medicinal Chemistry, 2017, 24, 1-10

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REVIEW ARTICLE

Tungsten or Wolfram: Friend or Foe? Maria Antonietta Zoroddua,*, Serenella Medicia, Massimiliano Peanaa, Valeria Marina Nurchib, Joanna I. Lachowiczb, Freda Laulicht-Glickc and Max Costac,* a

Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy; bDepartment of Chemical and Geological Sciences, University of Cagliari, Cagliari, Italy; cDepartment of Environmental Medicine, New York University School of Medicine, New York, USA

ARTICLE HISTORY Received: January 18, 2017 Revised: February 06, 2017 Accepted: February 08, 2017 DOI: 10.2174/0929867324666170428105603

Abstract: Tungsten or wolfram was regarded for many years as an enemy within the tin smelting and mining industry, because it conferred impurity or dirtiness in tin mining. However, later it was considered an amazing metal for its strength and flexibility, together with its diamond like hardness and its melting point which is the highest of any metal. It was first believed to be relatively inert and an only slightly toxic metal. Since early 2000, the risk exerted by tungsten alloys, its dusts and particulates to induce cancer and several other adverse effects in animals as well as humans has been highlighted from in vitro and in vivo experiments. Thus, it becomes necessary to take a careful look at all the most recent data reported in the scientific literature, covering the years 2001-2016. In fact, the findings indicate that much more attention should be devoted to thoroughly investigate the toxic effects of tungsten and the involved mechanisms of tungsten metal or tungsten metal ions.

Keywords: Tungsten, toxicity, biological role, carcinogenesis, leukemia, environment, pollution, weapon. 1. INTRODUCTION 1.1. Chemistry The name tungsten comes from a Swedish word that means “heavy stone” due to the high density (19.3 g/cm3), one of the highest of any metal. Tungsten’s name is used together with the name Wolfram, believed to be the German word for 'wolf's foam' and used by the mid-European tin miners few centuries ago, to mean that its presence interferes with the smelting process of tin, hence being “devoured” like a wolf can do. Thus, though it was generally considered for years as an impurity in the tin mining, it is now clear that, because of its hardness and high melting point (3350 *Address correspondence to these authors at the Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy; Tel/Fax: ++39-079-229529, +39-079-229559; E-mail: [email protected]; and Department of Environmental Medicine, New York University School of Medicine, New York; Tel /Fax: ++1-845-731-351539, +1-845 351 4510; E-mail: [email protected]

0929-8673/17 $58.00+.00

°C, carbon is the only element with a higher melting point), its use has increased dramatically. The high melting point has reserved to tungsten its place in the history for the use in the filaments of incandescent light bulbs since ‘900. Tungsten has atomic number 74, atomic weight 183.85 and five naturally occurring isotopes: 184W (31%), 186W (29%), 182W (26%), 183W (14%), and 180 W (0.1%). Thirty-three tungsten artificial radioisotopes have been prepared, with mass numbers spanning from 157 to 194. Tungsten, considering its density, can be properly classified a heavy metal. Tungsten is considered a chemical brother of molybdenum which precedes it within the transition metals block, in the VIB group of the periodic table, for their identical atomic radius (1.39A) and similar ionic radius (0.59, 0.60 in the VI oxidation state and six coordination number, respectively); electronegativity (1.8, 1.7, respectively); distance in MO bond (1.76) and similar coordination chemistry. In Table 1, data for the chemical properties are shown.

© 2017 Bentham Science Publishers

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Table 1. Chemical and physical properties of tungsten Name, Symbol

Tungsten, W

Density @ 20 ºC (gm/cc)

19.25

Alternative name

Wolfram

Melting Point ºC

3422

Atomic Number Z

74

Boiling Point ºC

5555

Standerd Atomic Weight

183.84

Molar Volume

9.5501×10-6

Group Number, Block

6, d-block

Atomic Radius

193 pm

Electronic shell

14

[Xe] 4f 5d 6s

Covalent Radius

146 pm

Color

gray

Electronegativity (eV) Pauling

2.36

CAS Registry Number

7440-33-7

Lattice Type

Body Centered Cube

CID Registry Number

23964

Stable Isotopes and Abudances (%)

4

2

180

From these data, tungsten should have similar bacterial essentiality for W-enzymes to the Mo-enzymes, though it is known that molybdenum is an essential element for eukaryotes and its enzymes are ubiquitous, while tungsten has an established biological role only in prokaryotes. A key factor that can give account of the different biological role of these two chemical brother elements can be correlated to the lower reduction potential of tungsten compared to that of molybdenum centers, as reported for some enzymes in which tungsten can replace molybdenum, though forming catalytically inactive molecules [1] as well as in some synthesized tungsten complexes [2]. With respect to molybdenum, tungsten has the 4f orbitals being complete in the 6th period ([Xe] 4f14 5d4 6s2), thus its valence shell electrons should be more available for metal-metal interactions. Compared to chromium(VI), a lighter metal which shares the VIB group, tungsten in its higher oxidation state, VI (d0), is much more stable than chromium towards reduction. W(II) can be stabilized only by the presence of metal-metal bonds in cluster units. The coordination compounds can have high coordination numbers up to 9-13.

W (0.12%), 182W (26.5%), 183W (14.31%), 184W (30.64%), 186W (28.43%)

angle of the octahedron through a halogen ion (X), have been found [3]. 1.2. Tungsten Biological Role It is known that elements which possess an atomic number higher than 35 do not usually have functional biological purposes, with only some rare exceptions. Tungsten is the heaviest metal that has a reported biological role, though only in prokaryotes, being essential in some very primitive organisms such as archea. In addition, a certain number of isolated tungsten-enzymes have also been well characterized. In Table 2 the known tungsten-enzymes are shown. In all these tungsten-containing enzymes, the metal is bound to sulfur either in the active site to two dithiolene groups of two pyranopterin or to one dithiolene group of one pyranopterin, according to the borderlinesoft acid character of the metal (Fig. 1).

The tungstate anion WO42- by decreasing the pH condensates in less soluble para-tungstate anions at pH 6 ([HW6 O21]5-), in pseudo meta-tungstate anions at pH 4 ([HW6 O203-]n) which are a little more soluble and then, at pH 1, it gives less soluble WO3⋅2H2 O oxide.

Actually, the crystal structure of an aldehyde ferredoxin oxidoreductase extracted from Pyrococcus furiosus, a hyperthermophilic archea, has been resolved [4]. The active site has one W atom coordinated with two pterin molecules bridged by one Mg ion. The specific requirements of tungsten in these organisms can be explained by the higher thermal stability of W(VI) species, due to stronger π bonding, and to the much lower redox potential. W-enzyme can function for formateCO2 transformations at redox potential of -430 mV, as an example [65].

Some di-nuclear tungsten species have been prepared which have a W-W bond and a low oxidation number. Several cluster compounds with oxidation number (II), in which an octahedral structure of metal atoms in a (W6 X8)4+ clusters with a bridge on each tri-

Anaerobic bacteria as Clostridium need Wenzymes. Few bacteria can utilize tungsten in an en zyme with the aim to reduce carboxylic acids to aldehydes. Bacteria, which live in hydrothermal vent sites, can use W-enzymes to produce ATP [5-8].

Tungsten or Wolfram: Friend or Foe?

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Table 2. Tungsten enzymes. Enzyme AOR

Function AOR family catalyzes the oxidation of aldehydes to carboxylic acids. Ferredoxin are used as redox partner protein

Microorganism

PDB ID

Pyrococcus furiosus

1AOR

AOR

Pyrococcus endeavori

AOR

Thermococcus paralvinellae

FOR

Thermococcus litoralis

FOR

Pyrococcus furiosus

GAPOR

Pyrococcus furiosus

CAR

Clostridium thermoaceticum

CAR

Clostridium formicoaceticum

ADH

Desulfovibrio gigas

FDH

FDH family catalyzes the oxidation of formate to carbon dioxide

1B25, 1B4N

Clostridium thermoaceticum

FDH

Eubacterium acidaminophilum

FDH

Desulfovibrio gigas

1H0H

Methanobacterium wolfei

5T5I, 5M, 61

FMDH

FMDH family catalyzes the reversible reductive carboxylation of methanofuran with carbon dioxide to N-formylmethanofuran

Methanobacterium thermoautotrophicum

FMDH AH

AH catalizes the hydration of acetilene to acetaldehyde

Pelobacter acetylenicus

2E7Z

AOR = aldehyde ferredoxin oxidoreductase; FOR = formaldehyde ferredoxin oxidoreductase; GAPOR = glyceraldehyde-3-phosphate dehydrogenase; CAR = carboxylic acid reductase; ADH = aldehyde dehydrogenase; FDH = formate dehydrogenase; FMDH = formylmethanofuran dehydrogenase; AH = acetylene hydratase [1, 65].

Fig. (1). Coordination sphere of tungsten complexed with the pyranopterin cofactor present in mononuclear tungsten enzymes. PDB entry 1AOR, Tungsten-containing aldehyde ferredoxin oxidoreductase, organism: Pyrococcus furiosus.

2. DISTRIBUTION IN THE ENVIRONMENT AND CONCERNS Tungsten is not abundant in the earth (54th element in abundance). It has been found in several minerals: scheelite (CaWO4 ), wolframite (Fe, MnWO4), ferberite (FeWO4), as tungstate oxoanion, where it has the most common oxidation number (VI). Although very little amount of tungsten has been detected in the soils, it is

possible to find high levels of W near mineral processing plants; 2000 ppm were present near to an ore processing plant in Russia. In fresh water, the tungsten concentration is very low, usually less than 20 nM, while marine sediments can contain quite high level in the range 10-60 µg/Kg outside the areas rich in W minerals. Tungsten is present in a significant amount, higher than 50 nM, in


ground waters directly associated with W-containing ore deposits. 1 mg/Kg is a typical level in the normal soil, slightly less than the general abundance in the lithosphere. In addition to its legendary role in early metallurgy, tungsten is used to enhance the resistance, hardness, and elasticity of steel. Nowadays it finds its best application in the high-tech field, being used, for its stability and high melting point, in electrodes, heating elements, filaments in light bulbs and cathode ray tubes, in heavy metal alloys, carbide tipped tools, cemented tungsten carbide grinding wheels used in mining, tunneling and construction industries. It is also used as stabilizer in aircrafts and in Formula one cars, in the “superalloys” for obtaining wear-resistant coatings, as well as, in military industry as supersonic bullets and armor penetrating munitions in missiles. It is also used in our everyday life in microwave ovens, television sets and as carbide in the tips of pens or in golf clubs. It is noteworthy that tungsten plays a key role in ammunitions. In fact, since the mid-nineties (1990s), in searching safer materials for bullets in guns and large artillery, tungsten was used in place of depleted uranium (DU) and lead bullets for which concerns have been raised [66], regarding human health effects and environmental contamination. Heavy metal-tungsten alloys (HMTA) constituted by a mixture of 91-93% of tungsten, and nickel, iron and cobalt, from 2 to 5 %, have been used in order to reach potential comparable to DU materials for high-velocity penetrating bombs or anti-tank weaponries. Tungsten weaponry has been referred to as a “magic, green and friend ammunition”, though now known as oddly named. In some other cases, tungsten has been previously considered beneficial, as for example replacing the poisonous lead sinkers used for fishing weights. Actually, the “green” bullet first believed to be insoluble, with no toxicity and to be more environmental friendly than lead or DU, previously used in the Gulf war and in the Balkan war, it is nowadays recognized to dissolve very readily in water, being mobile enough to be readily dispersed into the environment [9]. There are few toxicological studies showing that tungsten may pose a hazard to human and ecological targets, as reported in the 2008 EPA Integrated Risk Services Information Agenda. Firing of tungsten/nylon bullets resulted in the release of tungsten into the surrounding sites. It can, in contact with water, readily oxidize and solubilize as tungstate WO42- ions. The solubilization is accompanied by a lowering of pH that can give very high levels of tungsten, due to the

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probable formation of even more soluble polytungstate ions. The dissolution of tungsten is also enhanced by the presence of iron [10-11]. For example, federal facilities have detected dissolved tungsten in groundwater areas where small munitions ranges were located. High levels of tungsten were found in soil pore-water beneath bullet collection areas to a depth of up to 65 cm with concentrations up to 400 mg/l (corresponding to about 2 mM), and in down gradient monitoring wells up to 560 µg/l (corresponding to about 3 µM) [9, 12]. Usually very low amount of tungsten has been found in eatable plants, less than 0.1ppm. It is known that plants can take up tungsten from the soil in which they grow, up to 100 ppm of metal has been found in some trees. For example, barley removed most radioactive 185W applied to a sample of soil, absorbing it in the form of tungstate [5, 13]. From a study performed by Sardinian Regional Agency for Environmental Protection (ARPAS) in 2011, a high level of tungsten has been determined in lichens in the areas close to military sites [14]. As a result, environmental exposure to tungsten via water, soil and food has become a significant concern. Recent discussion regarding tungsten, alone or in combination, as emerging pollutant of environmental health concern has also been made [15]. In fact, a few in vitro and animal studies have been published and emerging evidence points to potential adverse health risks related to tungsten, even if the mechanisms underlying tungsten toxicity are still unclear. Though environmental outcomes of tungsten are not well studied, it is reported that by mixing tungsten powder to soil (more than 1% on mass basis), death of considerable part of bacterial population and increase in fungal biomass have been detected [16, 17]. Tungsten dust is known to be strong irritant for the eyes and skin, even if it is considered only mildly toxic as a bulky material. Indeed, it is known that nanoparticles as well as fine and ultrafine particles of different chemistry and shape may exert several adverse effects on human health [18]. Due to the increasing exposure to this metal, the National Toxicology Program (NTP) together with the Environmental Protection Agency (EPA) has considered tungsten as an “emerging toxic agent” and thus there is an urgency to investigate its toxicity in greater depth [1-3]. As reported by NTP – Department of Health on Human Services, Co-W carbide particles are “reasonable anticipated to be human carcinogens” on the basis of data detected in humans and studies on molecular mechanisms involved in car-



cinogenesis [19]. For these reasons, numerous research programs have been aiming to understand potential health concern, uptake, transport, and fate together with eventual cleanup strategies for tungsten. Here, we will give an account of the recent literature, covering the years 2001-2016, regarding the most important evidence concerning cancer and non-cancer adverse health effects, with the aim of drawing attention to the potential toxic effects due to the present use of tungsten materials. The values for tungsten in humans and in the environment are reported in Table 3. Table 3. Values for W in environment and in human body. Abundances in Environments

%

Universe

5×10-8

Sun

4×10-7

Meteorites

1.2×10-5

Earth's Crust

1.1×10-4

Oceans

1.2×10-8

Abundances in Human Body*

ppb

Blood

1

Bone

0.2

Tissue

NA

*The total amount of tungsten in an average 70 kg man is 0.02 mg

3. HUMAN AND ANIMAL EXPOSURE 3.1. Human Exposure to Tungsten According to a report from the EPA, tungsten enters environment through ore processing, alloy fabrication, tungsten carbide production and use, as well as during municipal waste combustion [20]. Tungsten production has been increasing; actually, in 2011, there were 72,000 tons of tungsten produced as opposed to 40,000 tons, in 2002 [21]. The risk of exposure for humans, in addition to the natural presence in the environment in rocks and minerals, occurs most commonly in occupational setting (mines, industries), in medical devices and in military activities. Principal health hazards derived from tungsten and related compounds arise from inhalation of aerosols generated by milling and mining operations, wherein it acts as a potent pulmonary irritant; it can irritate eyes, respiratory system, skin and blood. Military personnel are exposed through inhala-

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tion of dusts and particles or shrapnel pieces deriving from explosions or detonated ammunitions. In fact, it has been reported that a significant number of veterans had high levels of tungsten in urine, higher than the upper limit value which was in the range 0.003 – 2.70 µg/(g of creatinine) as established from National Health and Nutrition Examination Survey (NHANES) data [22]. Most of the tungsten that enters the body is rapidly excreted, but some is retained in the liver, spleen, kidney and bone [23]. Studies have found that the retained tungsten is predominantly stored in the bones [16]. There are limits for occupational exposure that take into account the water solubility [24-31]: 1 mg/m3 for tungsten water soluble compounds or 5 mg/m3 for water insoluble for an exposure of 8-10 h, and 3 mg/m3 for soluble or 10 mg/m3 for insoluble for short term exposure, respectively. Different regulations depending upon the country are in effect (at a large range in drinking water in the State of Massachusetts [24] or Russia [25], for an example) [15]. The estimated daily tungsten intake is about 12 µg/day, though, as studied by using radioactive Wtracer, only a small quantity was absorbed in the body. In the human body (70 Kg), about 20 µg can be found distributed between bones (0.2 ppb) and blood (1ppb) [5]. 3.2. Tungsten Toxicity Though limited data are available in the literature regarding the effects on health, especially long term, enough work documenting significant adverse effects on health has been published in these last 15 years (2001-2016). One of the first studies reporting specific genotoxic effects of tungsten and tungsten alloys appeared in 2001 [32]. In this report, HMTAs containing also Ni, Co and Fe, used in military applications, demonstrated the ability to transform human osteoblast-like cell line (HOS) to the tumorigenic phenotype. These HMTA transformants are characterized by anchorage independent growth, tumor formation in nude mice and high level expression of K-ras oncogene. The molecular mechanisms involve direct DNA injury, as an increased breaking of DNA, and several chromosomal aberrations. For comparison, tantalum oxide in the same conditions was not able to induce HOS transformation. The activity of tungsten carbide (WC) and cobalt tungsten carbide (CoWC) particles (1µ diameter) has been tested on human peripheral blood cells after 24 hours of exposure [32]. Whereas cobalt metal particles solubilized, the insoluble WC particles were not


dissolved but were gradually phagocytized by monocytes. Thus, first considered as an inert material in biological systems, it has now been shown that WC insoluble particles are indeed able to induce apoptotic effects in monocytes, which are dependent on the activation of caspase-9 in the monocytes. A few years later, the same authors analyzed the gene expression profile induced in vitro in peripheral blood mononuclear cells after 24 of exposure with WC and CoWC particles [34]. The aim was to better understand the molecular mechanisms involved in the apoptotic and genotoxic effects caused by these particles. They showed that several genes were affected, giving rise to a severe inflammatory and a hypoxia like response. In addition, a higher mutagenic potential of the mixture CoWC, when compared to the single components, was demonstrated suggesting that surface properties of CoWC mixture can be responsible for the enhanced activity of the dusts. Particular attention has been devoted to the tungsten materials used in military applications [35]. Heavy metal tungsten alloys have been examined for their property to induce stress genes in several cell lines from human liver carcinoma cells (HepG2). The pure metal tungsten, as well as W-Ni-Co mixture, activates gene expression that can be involved in carcinogenesis. A study published in 2005 describes the carcinogenic activity of embedded weapons – grade tungsten alloy shrapnel in rats [36]. The experiments were carried out with the aim to mimic shrapnel loads found in personnel from the 1991 Persian Gulf War. Male F344 rats were intramuscularly implanted with W-alloy pellets of weapons, in order to simulate shrapnel wounds. Intramuscular implantation of W-alloy pellets caused the development of metastatic high-grade rhabdomyosarcomas within a very short period, 4-5 months. The yield of tumors was 100% either from low doses (4 pellets) or from high doses (46 pellets) of implanted cylinders of 1x2 mm in dimension, and these tumors quickly metastasized to the lung. Important hematopoietic changes were evident at the high dose within one month of implantation prior to carcinogenesis. It is noteworthy that the implantation of tantalum, as an inert metal for comparison, at high doses, resulted in no rhabdomyosarcomas in rats. This result raises concerns on the use of tungsten munitions. In the year 2007, a review concerning the latest opinions on the toxicity of tungsten was published [37]. This paper highlighted that the internalization of tungsten metal as an embedded shrapnel may be considered as a pathway for long-term exposure. In addition, it

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was emphasized that pellets made from heavy metal tungsten alloys possess carcinogenic potential previously unseen for depleted uranium and/or lead. In patients following surgery to remove tungsten fragments, tungsten could be detected in the urine for two years after surgery, suggesting that there is a secondary storage site for W in humans [38]. Several papers also reported the adverse respiratory health effects related to inhalation of W ballistic aerosol. Higher levels of W particles have been found in particulate from 3.7 to 8.95 mg/m3, in comparison with 0.415 or 0.7411.28 mg/m3 for Co or Ni, respectively [39]. Aerosol particulates with a dimension around 10 nm containing tungsten, as well as nickel, cobalt and iron, were present on filters collected from ballistic penetration after rod projectiles penetrating into steel plates. After 48 hours of exposure of human epithelial A549 lung cells to the particles aerosol, cell death and production of cytokines were assessed [40]. In particular, it is noteworthy that the elements of nanoparticles sizes demonstrated the highest cytotoxic activity, in agreement with several studies reported in the literature on this issue. These results support the potential short-term human toxicity on the respiratory tract of aerosols or ballistic aerosol containing tungsten [39]. Also, the effects of exposure to soluble tungsten compounds, as tungstate WO42- ions, on the cells of the immune system have been published. Exposure of isolated human peripheral blood lymphocytes (PBL) in vitro to soluble tungstate resulted in an increase of apoptosis and in a reduction of interleukin-10 production [41]. Multiple potential exposures to tungsten have drawn attention to several leukemia clusters [42,43], in Fallon in Nevada, and in Elk Grove California, as well as in Sierra Vista in Arizona [44-47]. Although definitive conclusions have not yet been reported regarding the contribution of tungsten to the occurrence of this leukemia [48], one study reported data showing elevated levels of tungsten in residents of Fallon (medium values in urines were 2.3 ppb for children and 0.8 ppb for adults). Tungsten concentration in people living in Fallon was higher in children than in adults and the highest value found in the environment was 934 ppm. [42] Most of the cases of leukemia in the Fallon cluster showed acute preB lymphocytic leukemia [44]. The first evidence that B lymphocytes are targets for Winduced toxicity has been reported in a paper published in 2011 [47]. The in vivo investigation on oral tungsten exposure of C57BL/6 wild type mouse that drank water (15-200 µg/ml of W) resulted in DNA injury in the bone marrow. The highest tungsten concentration used


was 934 ppm (≈ 1g/l of W), the same as “surface dust hot-spot” measured in Fallon [49]. Despite all the raised concerns, tungsten and its compounds still remained relatively poorly investigated with regard to their potential carcinogenic activity. In a paper published in 2015 by Laulicht et al. [50] tungsten's ability to induce carcinogenic related endpoints was demonstrated and various molecular mechanisms involved in the carcinogenesis were studied. Activation of multiple pathways of gene expression related to cancer was studied in tungsten transformed clones, and increased migration was detected. W-treated cells formed more colonies than controls in soft agar and the tungsten-transformed clones were able to form in vivo tumors in nude mice. The Wtransformed clones, when compared to control clones, had altered the expression of many genes associated with cancer, as revealed by RNA-sequencing data. Tungsten ions were able to deregulate genes involved in lung cancer, leukemia, as well as in other general cancer genes. Taken together, all the reported data strongly highlight the carcinogenic potential of tungsten. Sodium tungstate has been previously reported to induce DNA damages after modulating ATM function [51]. A study examining the distribution of radioactive tungsten has been performed in an animal model [52]. It has been reported that, after a single dose, 50% of tungsten was retained after a month in the bone, 70% after two years and 90% after four years, thus suggesting that the bones can be a reservoir that could serve as a source of chronic exposure even if the source of the metal is removed [53]. It was also reported that tungsten accumulates more in older mice compared to younger mice [54]. DISCUSSION AND CONCLUSION Human exposure can arise both from the natural occurrence of tungsten compounds in the environment and from the use of tungsten in many occupational settings. Tungsten production for industrial and military purposes has almost doubled over the past decade and it is continuously increasing. The data reported in the literature regarding tungsten activity point to the adverse outcomes both as a free metal or as metal oxoanion, alone as well as in combination with other metals. From all the reported literature, the effect of tungsten may depend on the route of exposure, on the form of the metal (dust, particle, fragment), on the solubility, on its amount in the environment together with the extent of contact. Several agencies and scientists have


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studied potential impacts of tungsten on human health and the environment. Currently, tungsten is not classified as a human carcinogen by the Department of Health and Human Services or the International Agency for Research on Cancer, or the U.S. EPA [16, 20]. The Armed Forces Radiobiology Research Institute was published in June 2005; the results of its fouryears investigation aimed to understand long-term health effects of fragments from combat operations containing tungsten, nickel and cobalt alloys. The conclusions of the investigation were the following: “The health effects of tungsten/nickel/cobalt alloy pellets warrant further investigation, and it is too early to draw definitive conclusions from the results. Scientific research has shown that some chemicals that cause cancer in rats do not cause cancer in humans. For this reason, one study in rodents is not enough evidence to indicate that people will or will not develop cancer as a result of embedded fragments. More research is needed to see if similar effects might be expected in humans” [9]. The most critical findings were related to aerosol debris or dusts and exposure mainly through the respiratory tract, or internalization as embedded shrapnel. The later may be considered as a new route for longterm exposure resulting from heavy metal tungsten alloys. Carcinogenic potential was noted for Wcontaining shrapnel but similar effects were not seen for DU nor lead, even though in this study, there is no information regarding the role played by tungsten alone. Nevertheless, taking into account that weapon tungsten alloys usually contain more than 90 % of tungsten, it is likely that this metal plays a major role in the carcinogenic response. It is important not to underestimate the co-exposure of several metals (W-Fe-Ni, W-Fe-Co, W-Ni-Co) that are present together with tungsten in different applications. Usually additive effects can be found. For an example, comparing the in vitro apoptogenic effects of cobalt, tungsten carbide or tungsten carbide-cobalt in conditions which can induce genotoxicity, it has been found that when compared with its individual components tungsten, carbide-cobalt displayed an additive apoptotic effect in the DNA fragmentation test [33]. In the same way, a comparison of the pure metals (tungsten, nickel, or cobalt) with the W-Ni-Co alloys demonstrated that each metal exhibited a similar pattern of gene induction, but at a significantly decreased magnitude than that of the W-Ni-Co mixture [35]. Indeed, from the in vitro study of internalized tungsten alloys, the hazard by tungsten exposure on some


type of cells as human osteoblast has been found comparable to that of well known carcinogens, such as insoluble nickel compounds, i.e. nickel subsulfide or nickel oxide, which are included as carcinogenic compounds of class A by EPA and class 1 by IARC [55]. In addition, based on the evidence provided in the recent study about the carcinogenic potential of tungsten alone, in the form of soluble tungstate [50], it should be regarded as potentially carcinogenic. Considering the low mutagenicity of tungsten metallic species, significant attention should be devoted to epigenetic mechanisms in order to fully characterize tungsten’s carcinogenic potential. Future research should consider investigating other epigenetic mechanisms such as histone modifications [56, 57], possible modifications to the DNA methylome that may occur after tungsten exposure. Both global hypomethylation of tumor-suppressor genes and gene-specific hypermethylation emerge after exposure to carcinogenic metals. Like nickel, as insoluble or soluble compounds, or chromium, as chromate anion, tungsten also modifies histone methylation by affecting the epigenetic machinery that removes methyl groups from histones. It may also be possible that the binding of tungsten to a specific biological target can have a role for the carcinogenic activity of the metal, as has been demonstrated for other carcinogenic metals such as nickel [58-61] or Cr(VI-V) [62, 63], for which also redox property of the metal can be involved. Considering the borderline-soft (“b”) character of tungsten, though mostly associated with oxygen in oxygenate anions, it may readily form W-S coordination bonds with biological molecules inside cells. Indeed, several W-enzymes become inactive if tungsten is substituted for example with its brother chemical element because of the lower reduction potential of the tungsten center compared to molybdenum center. In addition, some synthesized W-complexes showed higher oxidation properties than analogues Mo-complexes [64]. Thus, to catalyze a reaction with low potential, tungsten should be preferred over other metals. This view is reinforced by the fact that W-enzymes catalyze reactions at potentials close to or below that of the standard electrode of hydrogen. The suggestion, raised by Kalinich et al. [36], that free radical formation at the interphase of tissue and W-pellet can trigger extremely aggressive tumors within a short period after implantation is plausible. Considering all these data together, several findings point to the serious health concerns regarding the use of tungsten in everyday life or W-based munitions as an alternative for toxic metals. Further studies, espe-

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cially chronic in vivo exposure, are necessary in order to validate tungsten as carcinogenic to humans. CONSENT FOR PUBLICATION Not applicable. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS We gratefully acknowledge financial support from Regione Autonoma Sardegna L.R.7/2007, “Promozione della ricerca scientifica e dell’innovazione tecnologica in Sardegna” program, project CRP 26712 “Nanopolveri e nanoparticelle metalliche: il vero responsabile della sindrome di Quirra?” (“Metallic nanoparticles: the real cause of Quirra syndrome?”). REFERENCES [1] [2] [3] [4]

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