ARTICLE pubs.acs.org/crt
Tissue Distribution of Tungsten in Mice Following Oral Exposure to Sodium Tungstate Gustavo S. Guandalini,† Lingsu Zhang,† Elisa Fornero,† Jose A. Centeno,*,† Vishwesh P. Mokashi,‡ Pedro A. Ortiz,‡ Michael D. Stockelman,‡ Andrew R. Osterburg,§ and Gail G. Chapman|| †
Division of Biophysical Toxicology, Department of Environmental and Infectious Disease Sciences, Armed Forces Institute of Pathology, Washington, DC ‡ Naval Health Research Center Detachment, Environmental Health Effects Laboratory, Wright-Patterson Air Force Base, Ohio § Shriners Hospitals for Children, Cincinnati, Ohio || U.S. Army Medical Research & Material Command, Military Infectious Disease Research Program, Fort Detrick, Maryland ABSTRACT: Heavy metal tungsten alloys have replaced lead and depleted uranium in many munitions applications, due to public perception of these elements as environmentally unsafe. Tungsten materials left in the environment may become bioaccessible as tungstate, which might lead to population exposure through water and soil contamination. Although tungsten had been considered a relatively inert and toxicologically safe material, recent research findings have raised concerns about possible deleterious health effects after acute and chronic exposure to this metal. This investigation describes tissue distribution of tungsten in mice following oral exposure to sodium tungstate. Twenty-four 6 9 weeks-old C57BL/6 laboratory mice were exposed to different oral doses of sodium tungstate (0, 62.5, 125, and 200 mg/kg/d) for 28 days, and after one day, six organs were harvested for trace element analysis with inductively coupled plasma mass spectrometry (ICP-MS). Kidney, liver, colon, bone, brain, and spleen were analyzed by sector-field high-resolution ICP-MS. The results showed increasing tungsten levels in all organs with increased dose of exposure, with the highest concentration found in the bones and the lowest concentration found in brain tissue. Gender differences were noticed only in the spleen (higher concentration of tungsten in female animals), and increasing tungsten levels in this organ were correlated with increased iron levels, something that was not observed for any other organ or either of the two other metals analyzed (nickel and cobalt). These findings confirmed most of what has been published on tungsten tissue distribution; they also showed that the brain is relatively protected from oral exposure. Further studies are necessary to clarify the findings in splenic tissue, focusing on possible immunological effects of tungsten exposure.
’ INTRODUCTION Tungsten (W), also known as Wolfram, is the elemental metal with the highest melting point and tensile strength at high temperatures; for this reason, it is largely used in industry, from light bulbs to high-performance alloys. It has long been considered an inert material, and, due to its resiliency and biocompatibility, W has also become very popular as a constituent of metal alloys in medical implantable devices, such as prostheses in orthopedic and maxillofacial surgery,1 dental implants,2 intravascular embolization coils,3 and mechanic heart valves.4 Tungsten has even been studied as a possible pharmacologic agent in the treatment of diabetes, as it has been shown to lower blood glucose both in insulin-deficient5 and insulin-resistant animal models,6 without significant evidence of severe toxic effects.7 Furthermore, heavy metal tungsten alloy (HMTA)-based materials have been recently introduced as replacement for lead (Pb) and depleted uranium (DU) in small caliber ammunition and armor-penetrating munitions, respectively.8,9 This is justified by concerns regarding the acute and long-term health and environmental effects of exposure to Pb and DU, which have forced the r 2011 American Chemical Society
military in many countries to explore the possibility of applying toxicologically safer metals with comparable material characteristics. The broad use of W-alloys provided them the status of inert and toxicologically safe compounds. However, HMTA have recently been shown to have possible toxic effects in vivo and in vitro, a problem brought to attention particularly after chronic tungsten exposure has been investigated as the possible causative factor in a cluster of childhood leukemia in Fallon, Nevada since 1997.10 Concerns about the toxicity and potential health effects of W-alloys have been corroborated by several in vitro studies showing genotoxicity and neoplastic transformation of cell lines exposed to W-alloy particles.11,12 Also, the use of HMTA in munitions applications elevates the problem of possible deleterious health effects caused by tissue-embedded W-alloys, especially after the discovery that implanted W-alloy pellets containing nickel (Ni) and cobalt (Co) induce high-grade rhabdomyosarcoma in rats.13 Received: September 2, 2010 Published: March 04, 2011 488
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When considering the possible deleterious environmental effects from HMTA used in munitions, the oral route becomes important because most of the W from these alloys, when left in the environment, will oxidize to tungstate (WO42 ), a thermodynamically stable molecule under most environmental conditions.14 To better understand the in vivo effects of W and W-alloys, the main objective of the present study is to describe W distribution in different organ tissues following oral exposure to sodium tungstate (Na2WO4) in laboratory mice. Such an approach leads to further understanding of its uptake and accumulation in vivo, revealing novel insights about tungsten effects and distribution in tissues after subacute exposure. These objectives are achieved by using state-of-the-art highly sensitive and precise instruments for multiple trace element analysis in biological specimens, i.e., sector-field high-resolution inductively coupled plasma mass spectrometry (HR-ICPMS). In addition to tungsten, iron (Fe), nickel, and cobalt concentrations were determined for each organ analyzed, as these metals are the most commonly found as components in HMTA. This approach is justified by the interest in establishing baseline levels for these elements in biological tissues since future studies might be concerned about their possible effects when present in combination with tungsten.
Figure 1. Boxplot showing the animals' water intake in different exposure groups from a preliminary oral exposure study with females only (n = 37, 10 in the control group and 9 in each treatment group). Tungstate concentrations were adjusted to maintain the doses as the mice gained weight. There was no significant difference in daily water intake due to increased sodium tungstate concentration in drinking water.
Table 1. Operating Parameters of the HR-ICPMS Instrument (Finnigan Element 2)
’ EXPERIMENTAL PROCEDURES
HR-ICPMS operating parameters
Animal Procedures. The tissue distribution of WO42
was evaluated in young (6 9 weeks old) pathogen free C57BL/6 mice kept on low-molybdenum rodent chow (Harlan Teklad, Madison, WI). The mice were housed in groups of 3 animals, in Static Micro Isolator (SMI) cages with Bed-o-cob (The Andersons, Inc., Maumee, OH) bedding. All animal procedures were conducted in compliance with the Animal Welfare Act and in accordance with the principles set forth in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animals Resources, National Research Council, National Academy Press, 1996. They were approved by the University of Cincinnati Institutional Animal Care and Use Committee and by the Wright-Patterson Air Force Base Institutional Animal Care and Use Committee. They were acclimatized in quarantine for at least one week and were given access to food and water ad libitum throughout the study. Exposure Groups. A cohort of 24 mice (12 male and 12 female) was divided into four exposure groups (n = 6/group). The treatment groups were continuously exposed to tungstate for 28 days in drinking water containing sodium tungstate dihydrate (Na2WO4 3 2H2O), and received 0, 62.5, 125, or 200 mg/kg/d of Na2WO4 3 2H2O. Body weights and water consumption were monitored, and Na2WO4 concentrations in drinking water were adjusted weekly to maintain exposure levels. Negative control animals received deionized water only (e.g., vehicle controls). There were no significant differences in weight gain or water consumption due to tungstate concentration in a preliminary 10 day exposure study (Figure 1). Tissue Processing and Trace Element Analysis. Twenty-four hours following the final dose, animals were euthanized, and kidneys, liver, colon, bone (femur), brain, and spleen were harvested for tissue processing. Digestion was performed using 70% HNO3 and 37% H2O2 (2:1 mL) solution under a pressure and temperature controlled microwave digestion system (MarsXpress, CEM Inc., Mathews, NC). The digested tissues were then reconstituted with 2% HNO3 to a final volume of 10 mL. After digestion, each tissue sample was analyzed using a sector-field inductively coupled plasma mass spectrometer (Finnigan Element 2, Thermo Scientific Inc., Bremen, Germany), working under the settings listed in Table 1. In addition to W, Co, Ni, and Fe concentrations were determined due to the interest in establishing baseline concentrations for these elements as constituents of HMTA.
setting
plasma gas
argon
plasma power
1,150 W
gas flows nebulizer gas flow rate
0.97 L min
1
auxiliary gas flow
0.90 L min
1
plasma gas flow
16.50 L min
1
lenses [V] extraction focus
2000 1050
X-deflection
9.56
Y-deflection interface cones
3.02 platinum
Calibration was performed employing a 5-point standard external calibration curve, with W, Co, Ni, and Fe standard concentrations ranging from 0.02 μg/kg to 20 μg/kg. Indium (115In) was used as the internal standard. Tungsten concentrations in tissues are reported per wet tissue weight basis. Statistical Analysis. Tungsten accumulation in organ tissues was statistically evaluated for each dose group using the SPSS 14.0 for Windows. Analysis of variance (ANOVA) for mean tissue concentration was carried out to evaluate the data. Dunnett’s posthoc test was used to compare different dose groups, using 0 mg/kg/d as the control group. Mann Whitney U test was applied for gender comparison. Linear regression was used to assess correlation between tungsten and other trace elements in organ tissues. Differences were considered significant when p < 0.05.
’ RESULTS Organ Accumulation under Different Doses. Following 28 days of oral exposure at drinking water concentrations that provided 0, 62.5, 125, or 200 mg/kg/day of Na2WO4 3 2H2O, W tissue concentration, as measured by sector field ICP-MS, was very low for control animals and dramatically increased when animals received increasing daily doses of WO42 (Figure 2). 489
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Figure 2. Increased tungsten levels in all organs as a result of exposure to increasing doses of W (males and females combined, n = 6/group). Gray bars show the median, lower quartile, and upper quartile; lines show the lowest and highest nonoutlier observations; and outliers are shown as circles (mild outliers) or asterisks (extreme outliers). Except for the colon (p = 0.094), p < 0.001 for all organs.
This increase was statistically significant for every organ, except for the colon. Among the six organs analyzed, W accumulation was higher in bone tissue, followed by the spleen, colon, kidney, liver, and brain (Figure 3A). Interestingly, despite inhomogeneous total W accumulation, relative distribution was fairly uniform when expressed as mean concentration proportions among different exposure groups for every organ analyzed (Figure 3B). Gender Discrepancies in Organ Accumulation. Tungsten distribution showed no overall difference between genders. However, females were found to have higher W concentration in their spleen when compared to that of their male matches (Table 2 and Figure 4); no other organ showed similar disparity between males and females. Correlation between Tungsten and Other Trace Elements. The multielement nature of the ICPMS instrument used for quantification of trace metals allowed plotting tungsten concentration results against other metals, such as iron, nickel, and cobalt. No organ accumulation was observed for any of these three metals. Interestingly, tungsten concentrations did correlate to iron levels in spleen (r = 0.760, p < 0.001) (Figure 4), something that was not observed for any other organ or element analyzed (data not shown).
those from a recent phase II clinical trial, in which obese individuals weighing approximately 100 kg received 200 mg/d of Na2WO4 3 2H2O per os (roughly 2 mg/kg/d).16 Interestingly, the first case report of acute intoxication following oral exposure to W estimates the total amount ingested to be as low as 385 mg, as part of a French artillery regiment initiation rite in which the barrel of a 155 mm gun was rinsed with beer and wine after several shots.17 In this specific case, the patient presented with seizures 15 min after ingesting the beverage, followed by coma and acute renal failure;18 nevertheless, the chemical form of tungsten to which this soldier was exposed was not determined, suggesting that WO42 is a much less toxic form than the one responsible for these deleterious effects. The background W concentrations in the kidney and bone in nonexposed animals were determined to be in the same range as those previously reported in the literature (Table 3).19,20 Although the reviewed publications quantified W in several intestine segments instead of the colon only, our results found that W distribution in colon tissue were comparable with those found in the literature for intestine. In addition, the liver concentrations reported here were higher than those previously reported in the literature.19 21 This discrepancy might be explained by the use of a quadrupole ICPMS (PerkinElmer Elan 6000) on these previous studies, an instrument with an expected higher limit of detection than the double focusing magnetic sector ICPMS used in this investigation. The feature gained by using the magnetic sector high-resolution ICPMS was its excellent sensitivity and signal-to-noise ratio, leading to a limit of detection as low as 15 ng/kg for tungsten. The increase in tungsten concentrations among exposed groups shown on Figure 2 was not statistically significant only
’ DISCUSSION The daily amount of Na2WO4 3 2H2O to which the mice were submitted in this study (up to 200 mg/kg/d) resembles the daily exposures in experimental models that used this substance as an antiobesity agent in leptin-receptor deficient rats (225 mg/kg/d) and leptin-deficient mice (180 mg/kg/d).15 Such a regimen uses doses that are significantly higher than 490
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Figure 3. Analysis of tungsten tissue distribution among six organs, with different doses (0 mg/kg/d in black, 62.5 mg/kg/d in dark gray, 125 mg/kg/d in light gray, and 200 mg/kg/d in white). (A) Tungsten accumulation is not uniform and is shown to be the highest in bone and lowest in brain tissue. (B) There is no significant difference among the relative distribution ratios.
Table 2. Tungsten concentration in tissues from nonexposed and orally exposed groups (average ( standard deviation, in mg/kg; N=3/group) oral exposure (mg/kg/d) organ gender
0
62.5
125
200
kidney female 0.03 ( 0.04
1.88 ( 0.82
3.59 ( 0.74
6.96 ( 0.89
0.05 ( 0.04
1.11 ( 0.49
4.51 ( 1.85
4.51 ( 0.35
male liver
female 0.06 ( 0.03
1.86 ( 0.83
1.64 ( 0.08
3.39 ( 1.69
colon
male 0.03 ( 0.03 female 0.06 ( 0.07
1.19 ( 0.64 1.06 ( 1.11
1.87 ( 0.40 4.74 ( 2.42
1.69 ( 0.55 4.94 ( 3.95
0.00 ( 0.01
4.05 ( 6.42
7.21 ( 8.17
13.69 ( 14.25
male bone
female 0.10 ( 0.07 17.54 ( 2.88 male
56.86 ( 24.61 60.79 ( 16.78
female 0.00 ( 0.00
0.01 ( 0.02
0.07 ( 0.02
0.11 ( 0.05
0.00 ( 0.00
0.03 ( 0.03
0.04 ( 0.03
0.11 ( 0.10
spleen female 0.03 ( 0.04
6.34 ( 2.89
10.43 ( 1.13
15.97 ( 3.08
0.04 ( 0.06
3.13 ( 1.30
4.96 ( 1.02
6.38 ( 1.40
brain
male male
Figure 4. Correlation between iron and tungsten levels in the spleen (r = 0.760, p < 0.001). Males are shown as squares (lower concentration) and females as triangles (higher concentration). Such correlation is not seen in any other organ (data not shown).
0.05 ( 0.03 30.86 ( 13.90 75.90 ( 26.20 63.07 ( 18.30
the highest W concentration among all tissues analyzed. As a transition metal, W can be found in different oxidation states, a property that confers to it the ability to replace other endogenous metals. It has been reported, for instance, that W can replace molybdenum (Mo) in Mo-containing enzymes such as xanthine oxidase and sulfite oxidase in kidneys and intestine.20 Moreover, it has been suggested that WO42 could replace phosphate (PO43 ) in bone, a mimetic property which may explain the much higher concentration found in bone tissue when compared to those of other organs.19,20 We were able to compare gender differences regarding tungsten distribution, an observation that other researchers have not been able to do because only one animal gender had been used.19,20 Additionally, only one study analyzed spleen W concentration,22 the only organ in which we found some discrepancy between genders. The higher splenic W concentration in females when compared to that of their male matches might be the explanation for the decrease in some lymphocyte subpopulations in female animals, a finding from another branch of this same study that will be published elsewhere (Osterburg et al., unpublished results). Iron, nickel, and cobalt levels were determined in addition to tungsten to establish baseline levels, due to the use of these metals in HMTA, and their potential deleterious health effects reported in literature.13,23 Iron and tungsten were unexpectedly
for the colon (p = 0.094). This observation might raise the possibility that the colon is not an accumulation organ for W and that the increasing concentrations detected in these tissues could be due to direct deposition to the colonic mucosa (i.e., oral exposure). Nevertheless, the published literature shows that the intestine is an organ in which tungsten accumulates even after IV administration of Na2WO4 in the same range as that observed in the kidney, the organ in which W showed its highest accumulation.19 These findings might implicate the small number of animals in each exposure group for the lack of statistical power. The low W levels found in brain tissue even with increasing daily dose (0.00 to 0.23 mg/kg) could be a result of the choroid plexus restriction to the diffusion of several hydrophilic molecules through the blood brain barrier. Although we found a statistically significant increase in W concentration with increasing oral exposure, even the highest brain tissue concentrations were in the same range as the average baseline concentration for the other organs. Interestingly, none of the reviewed literature on W toxicokinetics studies described the tissue distribution in the brain tissue after oral or parenteral exposure to sodium tungstate.19 22 Another remarkable aspect of the tissue distribution after 4 weeks daily exposure to Na2WO4 is the fact that the bone showed 491
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Table 3. Comparison with Published Data for Endogenous Tungsten Levels for Different Organs in Rodents (Average ( Standard Deviation, in mg/kg) species
instrument
model number gender
present study Na2WO4 3 2H2O Sector-field ICPMS mouse McDonald et al. 2007 Na2WO4 3 2H2O Quadrupole ICPMS mouse rat a
kidney
liver
intestine
femur
brain
spleen
0.04 ( 0.04 0.05 ( 0.03 0.03 ( 0.06 0.08 ( 0.06 0.00 ( 0.00 0.04 ( 0.04 a
6
both
6 6
female 0.06 ( 0.09 ND female 0.03 ( 0.05 ND
0.01 ( 0.01b 0.15 ( 0.13 N/A 0.03 ( 0.04b 0.01 ( 0.02 N/A
N/A N/A
Colon only. b Small intestine.
correlated in spleen; even though the mechanisms leading to this correlation are not fully understood, a review of the literature suggests that W injection in the spleen leads to lymphocytopenia in rats.24 Such a finding might lead to further understanding of possible toxic effects from W to the immune system in general, as this increase in iron levels could be a marker of inflammation and tungsten-induced toxicity in splenic tissue. In conclusion, our findings showed that, following oral exposure to sodium tungstate, tungsten mainly accumulates in the bone and spleen, but retention is also observed in the colon, kidney, liver, and brain (from highest to lowest concentration). The correlation between tungsten retention and increased iron concentration in splenic tissue is yet to be further explained, possibly implicating the immunological effects reported in the literature.
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’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Funding Sources
This work has been funded by a grant from the Office of Naval Research work unit 60862 and by an Interservice Support Agreement (ISSA) from the Naval Health Research Center Detachment Environmental Health Effects Laboratory (N4181709MP9T018).
’ DISCLOSURE This manuscript has been reviewed in accordance with the policy and guidelines of the Armed Forces Institute of Pathology, the Environmental Health Effects Laboratory Naval Health Research Center, and the Department of Defense, and approved for publication. The views expressed are those of the authors and do not reflect the official policy or position of the Department of the Navy, the Department of the Army, the Department of Defense or the United States Government, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. ’ ACKNOWLEDGMENT We thank Dr. Dean Wagner (LT, USN) and Dr. Ayodele Olabisi (LT, USN) for their insightful comments and assistance. ’ REFERENCES (1) Niinomi, M. (2008) Mechanical biocompatibilities of titanium alloys for biomedical applications. J. Mech. Behav. Biomed. Mater. 1, 30–42. (2) Hart, C. N., and Wilson, P. R. (2006) Evaluation of welded titanium joints used with cantilevered implant-supported prostheses. J. Prosthet. Dent. 96, 25–32. (3) Bachthaler, M., Lenhart, M., Paetzel, C., Feuerbach, S., Link, J., and Manke, C. (2004) Corrosion of tungsten coils after peripheral vascular embolization therapy: influence on outcome and tungsten load. Catheter Cardiovasc. Interv. 62, 380–384. 492
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(20) Waylon, M. W., Marr, R., Kracko, D., Gao, Z., McDonald, J. D., and Chearnaigh, K. U. (2008) Disposition of tungsten in rodents after repeat oral and drinking water exposures. Toxicol. Environ. Chem. 90, 445–455. (21) Le Lamer, S., Poucheret, P., Cros, G., de Richter, R. K., Bonnet, P. A., and Bressolle, F. (2000) Pharmacokinetics of sodium tungstate in rat and dog: a population approach. J. Pharmacol. Exp. Ther. 294, 714–721. (22) Wide, M., Danielsson, B. R., and Dencker, L. (1986) Distribution of tungstate in pregnant mice and effects on embryonic cells in vitro. Environ. Res. 40, 487–498. (23) van der Voet, G. B., Todorov, T. I., Centeno, J. A., Jonas, W., Ives, J., and Mullick, F. G. (2007) Metals and health: a clinical toxicological perspective on tungsten and review of the literature. Mil. Med. 172, 1002–1005. (24) Roser, B., and Ford, W. L. (1972) Prolonged lymphocytopenia in the rat. The immunological consequences of lymphocyte depletion following injection of 185 W tungsten trioxide into the spleen of lymph nodes. Aust. J. Exp. Biol. Med. Sci. 50, 185–198.
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