Heavy Metal-Induced Carcinogenicity: Depleted Uranium and Heavy-Metal Tungsten Alloy

Chapter

Abstract

Continued development of novel munitions for the battlefield opens the possibility of wounds containing embedded fragments of metals or metal mixtures whose toxicological properties may not as yet be well-understood. This chapter reviews what is currently known about two recent additions to many nations’ arsenals: depleted uranium and heavy-metal tungsten alloy. The toxicological and genotoxic properties of these materials, derived from both in vitro and in vivo studies, will be discussed, as will the health effects of known human exposures. Finally, areas requiring further research will be detailed.

Introduction

Advances in weapon design and the expanding terroristic use of Improvised Explosive Devices have opened the possibility of human exposure to metals or metal mixtures whose toxicological properties and physiological effects are not known. In this chapter, two of the more recent additions to the weapons arena, depleted uranium and heavy-metal tungsten alloy, will be discussed. The known toxicological properties of uranium and tungsten will be addressed with respect to a variety of human exposure scenarios, including inhalation, ingestion, and embedded fragments. The influence of depleted uranium and heavy-metal tungsten alloy on gene expression and signal transduction pathways leading to carcinogenicity will be considered and finally, areas requiring further research will be detailed.

Depleted Uranium (DU)

Uranium was first identified by Klaproth in 1789 and named after the planet Uranus. However, it was not until over 100 years later that the radioactive properties of uranium were described by Becquerel. Uranium is a naturally occurring element widely spread in the environment. It is normally found at low levels (parts per million) in soil, water, plants, and animals, including humans (ATSDR 1999). Average daily uranium intake in humans is approximately 3 µg, primarily through food and drinking water. Uranium, as found in nature, is slightly radioactive and consists predominantly of three isotopes, 234U, 235U, and 238U (Table 10.1). Although all three isotopes are radioactive, 234U and 235U have a much higher specific activity than 238U. Natural uranium consists largely of 238U (99.274%) with smaller amounts of 234U (0.006%) and 235U (0.72%). The processing of uranium for use in nuclear reactors and nuclear weapons involves increasing the percentages of the high specific activity isotopes with respect to 238U. This process is known as “enrichment” and results in the production of two different uranium fractions. The “enriched” fraction consists of approximately 97.010% 238U, 0.03% 234U, and 2.96% 235U. The “depleted” fraction consists of approximately 99.745% 238U, 0.005% 234U, and 0.25% 235U. Although radiologically different, both fractions remain identical chemically.
Table 10.1

Uranium characteristics

Chemical symbol

U

Atomic number

92

Atomic weight

238.029

Category

Actinide

Group/series/block

n.a./7/f

Melting point

1135°C

Common oxidation states

+4, +5, +6

Density

18.95 gm/cm3

Depleted uranium has several commercial applications including shielding for radioactive material and as counterweights in aircraft and ships. However, it is because of its military applications that depleted uranium has received much of its attention. Because it is extremely dense, with a density second only to tungsten, depleted uranium is used for shielding for tanks and vehicles and as kinetic-energy armor-penetrating munitions. The use of DU munitions presents the greatest chance of human exposure. Although the toxicological hazards of uranium have been recognized for over a 100 years, many of these adverse effects were ascribed to radioactivity. Depleted uranium is approximately 40% less radioactive than natural uranium; thus, although radiation may play a role in the induction of cellular damage, the chemical properties of DU are also of paramount concern. In addition, metals such as titanium or molybdenum are often added during production of DU-containing munitions to provide specific metallurgic properties. The original uranium source from which DU was processed can also add minor contaminants to the final product. For example, DU obtained from reprocessed nuclear fuel can have small amounts of fission products and transuranics present, including strontium, cesium, plutonium, and americium. Also, the normal radioactive decay pathways of uranium can introduce additional contaminants (e.g., thorium) in the final product. Therefore, when evaluating the cellular effects of DU exposure, the presence of contaminants introduced as a result of processing, as well as those resulting from normal radioactive decay, cannot be discounted. As noted above, one of the main military uses of DU is in the production of kinetic-energy armor-penetrating munitions. The first widespread use of these munitions was in the 1991 Persian Gulf War. DU munitions were also used by NATO forces in the recent conflicts in Bosnia and Kosovo. Because of concern over the health and environmental effects of the use of DU munitions there has been a movement toward the use of alternative materials and the heavy-metal tungsten alloys are one of these.

Heavy-Metal Tungsten Alloy

Tungsten was first identified in 1758 by the Swedish chemist Cronstedt. The word tungsten is Swedish for “heavy stone” and is a tribute to its density; the highest of any naturally occurring element (Table 10.2). Tungsten is found in varying concentrations in air, water, and soil. In soil, tungsten is found as a mineral mixture, primarily as wolframite or scheelite (van der Voet et al. 2007). Weathering and dissolution of rocks and soil result in tungsten being released into the air or entering the groundwater. Environmental tungsten levels are generally very low, except in areas of tungsten mines or natural deposits (ATSDR 2005). As such, uptake levels are usually insignificant, with occupational exposure being the most likely route of tungsten internalization in humans.
Table 10.2

Tungsten characteristics

Chemical symbol

W

Atomic number

74

Atomic weight

183.85

Category

Transition Metal

Group/series/block

6/6/d

Melting point

3422°C

Common oxidation states

+6

Density

19.25 gm/cm3

Because of its density and high melting point, tungsten is used in a variety of commercial applications including light bulb filaments, counterweights, radiation shields, and thermocouples. It is also found, as tungsten carbide, in cutting blades and drill bits. Tungsten has also been used as replacement for lead in small-caliber ammunition. In 1991, the U.S. Fish and Wildlife Service banned the use of lead shot for the hunting of waterfowl and advocated the use of ammunition formulations that were not toxic when ingested by wildlife. Many of the subsequently approved ammunitions contain varying amounts of tungsten in combination with other metals such as nickel, tin, iron, copper, and bismuth. In addition, a formulation of tungsten with a polymer matrix has also been approved for use. Toxicity testing has shown no adverse health effects of these materials (Kraabel et al. 1996; Kelly et al. 1998; Mitchell et al. 2001a, b, c; Brewer et al. 2003).

Military applications of tungsten also include the use of tungsten-based composites, primarily tungsten/tin and tungsten/Nylon, as replacements for lead in small-caliber ammunition. As noted earlier, widespread public concern over the health and environmental impact of the continued use of depleted uranium has led many countries to replace depleted uranium with various tungsten alloys in their arsenals of armor-penetrating munitions. In many of these formulations, tungsten is combined with two or more of the following transition metals: nickel, cobalt, iron, and copper. Although these materials are referred to as “tungsten alloys”, they are in fact two-phase composites, due to the extremely high melting point of tungsten. During manufacturing, powders of the appropriate metals are mixed and heated. Heating occurs at temperatures below the melting point of tungsten, but above the melting points of the transition metals. The melted transition metals dissolve a small amount of tungsten; however, most of the tungsten powder remains intact. The result is a material consisting of pure tungsten grains surrounded by a “binder matrix” composed of tungsten and the added transition metals. In contrast, additional material added during the processing of depleted uranium results in a true alloy because of the similar melting points of the components.

Routes of Exposure

Depleted uranium and tungsten alloys can be internalized by three primary routes: inhalation, ingestion, or wound contamination via embedded fragments. Regardless of the route of exposure, several factors govern the eventual health effects induced by the internalized metals. Clearly, the amount of material internalized plays a major role in the end-result of any exposure. Equally important however are the chemical and physical properties of the metal. These properties include solubility characteristics (particularly in biological fluids), particle size, speciation, and chemical reactivity (Yokel et al. 2006). For inhalation exposures, the size of the inhaled particle as well as its solubility will determine its ultimate fate. Approximately 25% of inhaled particles are immediately exhaled (International Commission on Radiological Protection 1966). Of the remaining 75%, particles less than 5 mm in diameter can reach the alveolar space while particles greater than 10 mm tend to remain in upper areas of the lung (Morrow et al. 1967). Once deposited in the lung, the solubility of the particle is of importance. Soluble metals are rapidly dissolved and enter the circulatory system. Less soluble metals will eventually be removed through the process of phagocytosis by the alveolar macrophages. Larger inhaled particles, unable to access the alveolar space, will be removed from the lung via mucocilliary clearance. However, many of the particles, once cleared, will be swallowed and thus enter the gastrointestinal tract. In addition to swallowing after mucocilliary clearance, metals can be ingested through contaminated food or liquids. Once ingested, absorption of the metals will depend upon the chemical form and solubility. Both uranium and tungsten are usually poorly absorbed by the gastrointestinal tract (Leggett 1997; Leggett and Pellmar 2003). Wound contamination can occur as a result of metals entering open wounds (e.g., as dust or liquid) or as embedded fragments. As with other routes of exposure, the physicochemical properties of the metal are of prime importance when determining its fate in vivo. Research with intramuscularly injected metallic radionuclides has shown that even those considered insoluble can be solubilized in vivo (Bistline et al. 1972; Lloyd et al. 1974; Dagle et al. 1985). This fact was dramatically shown in studies investigating the health effects of embedded fragments of depleted uranium where solubilization and urinary excretion of the uranium was found within 48 h after implantation of the solid metal into the leg muscles of laboratory rodents (Pellmar et al. 1999).

Regardless of the route of internalization, several different cell types could potentially be affected by exposure to metals. The epithelial cells of the gastrointestinal tract and the mesenteric lymph nodes have been shown to accumulate depleted uranium after chronic ingestion (Dublineau et al. 2006). Inhalation results in the exposure of epithelial cells and alveolar macrophages (Schins and Borm 1999; Monleau et al. 2006). As noted earlier, the alveolar macrophages play a key role in both particle clearance and retention in the lung (Tasat and De Ray 1987). Wound contamination and embedded fragments present a far more complex situation because of the wide variety of cell types and mediators involved in wound healing. Briefly, the process of wound healing can loosely be divided into three phases: inflammation, proliferation, and remodeling (Broughton and Janis 2006; Tsirogianni et al. 2006; Li et al. 2007). Immediately after a wound is suffered, platelets arrive to begin the process of clot formation to maintain hemostasis. Neutrophils and macrophages are the next cell types to arrive at the wound site and are responsible for eliminating foreign organisms and debris, including nonviable tissue. Macrophages are also capable of phagocytizing small metal particulates and can concentrate these metals in the phagolysosomal vesicles before exiting through the lymphatic system (Berry et al. 1997; Lizon and Fritsch 1999). However, the most important role of the macrophage is the secretion of numerous cell mediators that lead to the proliferation phase of wound healing. In the proliferation phase, fibroblasts migrate to the wound site to produce the extracellular matrix and granulation tissue required for proper wound healing. Maturation of the extracellular matrix occurs during the remodeling phase and, depending upon the type of wound, can take up to a year to complete. In many cases, the specific response of the macrophages to the internalized metals will determine the ultimate outcome of the exposure, including the induction of cancer (Sica et al. 2008).

In Vitro Studies

Depleted Uranium

Cell culture systems have been used for many years to model potential adverse health effects from exposure to metals. Macrophage, kidney, lung, and neuronal cell lines have all been utilized to assess metal toxicity, as well as genomic and proteomic changes occurring as a result of exposure. Treatment of Chinese hamster ovary cells with depleted uranium resulted in decreased cell viability and increased chromosomal aberrations (Lin et al. 1993). Cellular damage, evidenced by an increase in the release of lactate dehydrogenase, was observed in LLC-PK1 kidney cells after uranium treatment (Furuya et al. 1997; Mirto et al. 1999). Several studies have also shown that the mouse macrophage cell line, J774, is capable of internalizing extracellular depleted uranium (Kalinich and McClain 2001). Once internalized, the DU can induce cell death, via apoptosis, in a concentration-dependent manner (Kalinich et al. 2002). Not all cultured cell lines appear capable of internalizing DU. When assessed colorimetrically using the method of Kalinich and McClain (2001), Molt-4, a human T-cell leukemia line, and Reh, a human B-cell lymphoma line, did not appear to internalize DU added to the extracellular medium. In addition, these cell lines were far less susceptible to the cytotoxic effects of DU exposure, showing no significant change in viability compared to untreated cells (Fig. 10.1).
Fig. 10.1

Cell viability assessment of Molt-4, Reh, and J774 cells treated with depleted uranium-uranyl chloride for 24 h at 37°C. Viability was determined using the MTT assay and data normalized to values from untreated cells and are the mean of 6 independent experiments. Error bars represent standard error of the mean. The stippled bars represent a uranium concentration of 1 µg/ml, the black bars represent a uranium concentration of 10 µg/ml, and the slanted-line bars represent a uranium concentration of 100 µg/ml

Peritoneal macrophages and splenic T-cells isolated from mice then exposed to varying concentrations of DU also demonstrated that macrophages are much more sensitive to the cytotoxic effects DU than other immune system cells (Wan et al. 2006). Treatment of cultured human osteoblast cells with either soluble or insoluble forms of DU transformed the cells to a neoplastic phenotype. These transformed cells also formed tumors when injected into immunocompromised mice (Miller et al. 1998; McClain and Miller 2007). As noted earlier, although DU is 40% less radioactive than natural uranium, it is still radioactive and that characteristic also has the potential to induce significant cellular damage. The question of whether the chemical or radiological property is primarily responsible for the cellular damage inflicted by DU is still open to debate. At present, it appears that the chemical characteristics of DU are predominantly responsible for the observed cellular damage, with the radiological component playing a smaller role (Miller et al. 2002a, b, 2003).

Along with inducing genotoxic effects in vitro, low-level DU exposure can also alter gene expression patterns in many cell types. In NR8383 cells, a rat alveolar macrophage cell line, DU has been shown to induce secretion of tumor necrosis factor α (TNF-α), as well as activate the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways (Gazin et al. 2004). This work suggests that macrophages exposed to uranium, either through inhalation or wound contamination, can secrete elevated amounts of TNFα, a major proinflammatory cytokine. Because of the central role TNFα plays in regulating the release of secondary inflammation mediators (Cromwell et al. 1992), any perturbation can have far reaching consequences for the organism. DU can also induce a number of stress-related genes in HepG2 cells, a human liver carcinoma cell line. In this assay, HepG2 cells, stably transfected with chloramphenicol acetyltransferase under the transcriptional control of a variety of stress-gene regulatory sequences, were treated with insoluble DU (Miller et al. 2004). Several categories of promoters were affected in a dose-dependent manner including transcription factor binding sites (FOS, NFκBRE, CRE, p53RE, and RARE), cell cycle regulation sites (GADD45, GADD153), transport proteins (GRP78, HSP70), and the promoter for metallothionein IIA (HMTIIA). These data indicate that DU can activate gene expression through a variety of signal transduction pathways, including many that are involved in the carcinogenic process. Using microarray technology, Prat and colleagues (Prat et al. 2005), demonstrated that exposure of cultured HEK292 cells, a human embryonic kidney cell line, to DU resulted in both up- and down-regulation of numerous genes including many involved in signal transduction and trafficking pathways. Microarray technology was also used to assess the effect of DU exposure on gene expression patterns in mouse peritoneal macrophages and CD4+ T cells (Wan et al. 2006). Again, DU was shown to alter gene expression profiles with genes responsible for signal transduction pathways, chemokines, and interleukins affected the greatest. As a result of these findings, Wan et al. (2006) postulated the potential for cancer development as a consequence of DU exposure.

Heavy-Metal Tungsten Alloy

There have been far fewer studies on the toxicological and genotoxic properties of the heavy-metal tungsten alloys. There are several reports investigating the toxicity of tungsten alone. The cytotoxicity of degrading tungsten coils used medically for vascular occlusions was assessed in cultured human smooth muscle, endothelial, vascular, and fibroblast cells. Cytotoxicity was observed only at tungsten concentrations above 50 µg/ml (Peuster et al. 2003). In vitro studies with J774, Molt-4, and Reh cells have shown that, unlike DU, exposure to a heavy-metal tungsten alloy composed of tungsten (92%), nickel (5%), and cobalt (3%) resulted in decreased viability of all three cell lines in a concentration-dependent manner (Fig. 10.2). The same tungsten alloy mixture, as well as one composed of tungsten (92%), nickel (5%), and iron (3%), was found to transform cultured human osteoblast (HOS) cells to a neoplastic phenotype (Miller et al. 2001). The individual metals comprising the alloys were also able to transform the HOS cells, but at a frequency far lower than the mixtures. In fact, when the transformation frequency data from the individual metals are compared with those of the alloys, it appears that there is synergistic effect between two or more of the metals, leading to increased transformation (Miller et al. 2002c). While both tungsten alloys (WNiCo and WNiFe) and all individual component metals could transform HOS cells, only those cells transformed by the tungsten alloys developed tumors when injected into immunocompromised mice (Miller et al. 2001). The tungsten/nickel/cobalt alloy was also capable of inducing the expression of several genes when assayed using chloramphenicol acetyltransferase-transfected HepG2 cells (Miller et al. 2004). As with DU, tungsten alloy had the ability to induce gene promoters in several categories including transcription factor binding sites (FOS, NFκBRE, CRE, and p53RE), transport proteins (HSP70), and the promoter for metallothionein IIA (HMTIIA). Surprisingly, tungsten alloy exposure had no effect on either of the cell cycle regulation promoters tested (GADD45, GADD153) in contrast to DU. When tested individually, the metals comprising the alloy were also able to induce the promoters affected by the alloy, but did so at a much lower level. Again, a synergistic effect of the metals in the alloy on gene induction was observed (Miller et al. 2004).
Fig. 10.2

Cell viability assessment of Molt-4, Reh, and J774 cells treated with heavy-metal tungsten alloy (91% tungsten, 6% nickel, 3% cobalt) for 24 h at 37°C. Viability was determined using the MTT assay and data normalized to values from untreated cells and are the mean of 6 independent experiments. Error bars represent standard error of the mean. The stippled bars represent an alloy concentration of 1 µg/ml, the black bars represent an alloy concentration of 10 µg/ml, and the slanted-line bars represent an alloy concentration of 100 µg/ml

In Vivo Studies

Depleted Uranium

During the Persian Gulf War of 1991, several individuals suffered wounds containing embedded DU fragments. Concern, both chemical and radiological, over the long-term health effects of this unique material led to several studies investigating the toxicological and carcinogenic properties of embedded DU fragments. Development and validation of a rodent model system to study the biological effects of embedded fragments was undertaken at the Armed Forces Radiobiology Research Institute (Castro et al. 1996). This model system involves surgically implanting small pellets of test material into the leg muscles to mimic shrapnel wounds. An x-ray of the location of several 1 ´ 2 mm cylindrical pellets is shown in Fig. 10.3. Sprague Dawley rats, implanted with up to 20 DU pellets (1 ´ 2 mm cylinders) for periods up to 2 years, exhibited no overt adverse health effects. No tumors were observed at the pellet implantation sites for either DU or tantalum, an inert negative control metal (Pellmar et al. 1999).
Fig. 10.3

Radiograph of rat showing location of depleted uranium pellets (1 ´ 2 mm cylinders) surgically implanted in the gastrocnemius muscles of the rear legs

The DU pellets degrade rapidly in vivo, with significant uranium levels measured in the urine as early as 2 days post-implantation. Some of the DU pellet material is not immediately solubilized and can be found at the pellet implantation site upon histopathological examination (Fig. 10.4). Over time, as more of the pellet degrades, DU can be found in a variety of tissues including kidney, liver, brain, testes, and lymph nodes (Pellmar et al. 1999). Similar results were reported by Hahn and colleagues using Wistar rats (Hahn et al. 2002). When DU was implanted as 1 ´ 2 mm cylindrical pellets, no tumors were observed. However, when embedded as squares (2.5 ´ 2.5 ´ 1.5 mm or 5 ´ 5 ´ 1.5 mm), DU induced soft-tissue sarcomas at the implantation sites. Rats implanted with tantalum did not develop tumors indicating that the observed DU effects were not the result of foreign-body carcinogenesis (Brand et al. 1975, 1976). As with the Pellmar study, the DU material began to degrade shortly after implantation. Taken together these studies indicate that once a certain mass is reached, DU fragments are capable of inducing neoplastic changes resulting in soft-tissue sarcomas in laboratory rats.
Fig. 10.4

Histopathological examination of a hematoxylin-and-eosin-stained section of leg muscle from a F344 rat implanted with depleted uranium pellets for 3 months. DU residue is visible at pellet implantation site. Scale bar = 200 µm

Embedded DU fragments also induced gene changes in the muscle tissue surrounding the metal. Using Northern blot analysis, Miller et al. (2000) demonstrated elevated mRNA levels of p53, k-ras, and bcl-2 in muscle tissue adjacent to embedded DU pellets. Utilizing immunohistochemical techniques, Hahn (2007) showed increased p53 protein levels in tissue surrounding the implanted DU. However, MDM2, c-myc, and p21 levels were found to be no different than control.

Heavy-Metal Tungsten Alloy

Thus far only one study has been published in the peer-reviewed literature describing the health effects of embedded tungsten alloy. F344 rats implanted with a tungsten alloy comprised of tungsten (91.1%), nickel (6.0%), and cobalt (2.9%) developed highly aggressive rhabdomyosarcomas at the pellet implantation sites (Kalinich et al. 2005). The tumors grew rapidly and metastasized to the lungs requiring euthanasia of the animals. Tumor incidence was 100%. Rats implanted with nickel, a known carcinogen, also developed tumors, but did so at a slower rate than those rats treated with the tungsten alloy. Rats implanted with tantalum did not develop tumors. As with DU, the metals comprising the tungsten alloy rapidly solubilized and were found in the urine at an order of magnitude higher than control values (Kalinich et al. 2008). However, in contrast to DU, no particulate material was observed at the pellet implantation site upon histopathological examination (Fig. 10.5). Significant hematological changes were observed as early as 1 month after implantation; well before any neoplastic changes had occurred. Whether these changes are attributable to an individual metal in the alloy or a synergistic effect between two or more components is not yet known.
Fig. 10.5

Histopathological examination of a Gomori trichrome-stained section of leg muscle and tumor from a F344 rat implanted with heavy-metal tungsten alloy pellets (91.1% tungsten, 6% nickel, 2.9% cobalt) for 6 months. “P” indicates site of pellet implantation. “T” denotes the tungsten alloy-induced tumor (rhabdomyosarcoma). “MF” shows area of normal muscle fibers. Scale bar = 1.0 mm

Human Exposures

Depleted Uranium

As noted above, the first widespread use of DU was in the 1991 Persian Gulf War. During this conflict several individuals were wounded with DU fragments. United States military personnel with retained DU fragments have been followed clinically by the Veterans Affairs Medical Center in Baltimore, Maryland. After 16 years of follow-up surveillance, there has been no indication of any clinically significant DU-related health effects (McDiarmid et al. 2000, 2001, 2004, 2006, 2007a, b, 2009; Dorsey et al. 2009). However, there is some indication of a weak genotoxic effect as a result of the embedded DU fragments. This was determined by fluorescent in-situ hybridization (FISH) analysis of the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in peripheral blood lymphocytes (McDiarmid et al. 2007a, b). As a result of these findings, the authors have recommended continued surveillance of these individuals.

Heavy-Metal Tungsten Alloy

A variety of tungsten-based munitions have been proposed as replacements for DU in armor-penetrating shells and for lead in small-caliber ammunition. As yet, there have been no reports on whether there are individuals with retained fragments of these materials and, further, that these fragments are resulting in adverse health effects. There are several reports in the literature describing adverse health effects due to exposure to tungsten and tungsten-based materials. As part of an initiation rite, a French artillery soldier drank 250 ml of a beer and wine mixture that had been used to rinse a gun barrel. Shortly after, he suffered seizures and was comatose for 24 h (Marquet et al. 1996). Extremely high levels of tungsten were found in his blood and urine and persisted for 2 weeks (Marquet et al. 1997). Although his malady was blamed on tungsten intoxication, there are some who believe the organic residue left in the gun barrel as a result of the explosive charge of the shell was actually to blame for his condition (Lison et al. 1997). There have also been two reports in the literature of granuloma formation as a result of embedded metal from a lawn mower blade (Saruwatari et al. 2009) and a chain saw blade (Osawa et al. 2006), respectively. In both cases, metal analysis of the excised fragment showed that it was composed primarily of tungsten with smaller amounts of other metals.

Conclusions

Little is known about metal-induced gene expression changes and carcinogenicity especially with respect to militarily-relevant metals. Improvement in weapons design and the terroristic use of Improvised Explosive Devices will continue to increase the possibility of embedded fragment injuries with metals or metal mixtures whose toxicological properties are not fully understood. The metals discussed in this chapter, depleted uranium and heavy-metal tungsten alloy, are only two such examples. In this final section, areas requiring additional research in order to enhance our understanding of heavy metal carcinogenicity will be discussed.

Although in vivo exposure scenarios may differ, the common factor in most is the presence of the macrophage. Macrophages are not only capable of phagocytizing small metal particulates (Berry et al. 1997; Lizon and Fritsch 1999), but have also been shown to interact with and modify the surface composition of metal alloys through the production of reactive chemical species (Thomsen and Gretzer 2001; Lin and Bumgardner 2004). The critical role the macrophage plays in wound repair, as well as its postulated regulatory link between inflammation and cancer induction (Sica et al. 2008), makes this cell type key in understanding heavy metal-induced carcinogenicity.

In vitro studies have demonstrated that macrophage viability is affected by both DU and heavy-metal tungsten alloy. In addition, gene expression patterns are perturbed by both treatments. No consensus has been reached on an exact list of specific up- and down-regulated genes by metal exposure primarily due to experimental design differences between the published studies. However, there is a pattern of up-regulation of those genes involved in transcription regulation and signal transduction, as well as those coding for the interleukins and transport proteins. Areas that require further research include an investigation of gene induction by insoluble as well as soluble metals and alloys. As seen with DU, even though a fragment begins to rapidly degrade once implanted (as determined by urine uranium levels), substantial particulate material is still found at the implantation site. As yet unknown is whether exposure to the insoluble DU will result in a similar pattern of gene induction as for the soluble material. Exposure to heavy-metal tungsten alloy raises similar concerns and may be more difficult to decipher because of the number of metals comprising the alloy and their proposed synergism with respect to biological effects (Miller et al. 2004). Again, a detailed investigation of the alloy, as well as the individual metal components, in both soluble and insoluble forms will greatly enhance our knowledge of metal-induced gene expression.

Although in vitro studies will provide a foundation for our understanding of heavy metal-induced carcinogenicity, in vivo models will be necessary to definitively determine if embedded fragments of these materials have the potential to cause cancer. In addition, recent advances in laser microdissection and microarray techniques will be crucial in elucidating metal-induced gene-expression changes in the tissue immediately adjacent to the embedded fragment. Not only will this information allow correlation of in vitro and in vivo findings, but it will be critical if changes in treatment strategies, either surgically or pharmacologically, are required in order to maintain the health and well-being of wounded individuals.

Notes

Acknowledgements

The views expressed here are strictly those of the author and not those of the Armed Forces Radiobiology Research Institute, the Uniformed Services University, or the United States Department of Defense. Mention of any commercial reagents or devices does not constitute endorsement by the United States Government. Dr. Kalinich has been supported in part by grants from the U.S. Army Medical Research and Materiel Command (Award #: DAMD17–01-1–0821) and the U.S. Army Peer-Reviewed Medical Research Program (Award #: W81XWH-06–2-0025). The author would like to thank Dr Steven Mog, DVM for obtaining histopathological images.

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Armed Forces Radiobiology Research InstituteBethesdaUSA

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