Tungsten Toxicity and Carcinogenesis

Abstract

Tungsten is an emerging contaminant in the environment. Research has demonstrated that humans are exposed to high levels of tungsten in certain settings, primarily due to increased use of tungsten in industrial applications. However, our understanding of the potential human health risks of tungsten exposure is still limited. An important point we have learned about the toxicity profile of tungsten is that it is complex because tungsten can often augment the effects of other co-exposures or co-stressors, which could result in greater toxicity or more severe disease. This has shaped the tungsten toxicology field and the types of research questions being investigated. This has particularly been true when evaluating the toxicity profile of tungsten metal alloys in combination with cobalt. In this chapter, the current state of the tungsten toxicology field will be discussed focusing on data investigating tungsten carcinogenicity and other major toxicities including pulmonary, cardiometabolic, bone, and immune endpoints, either alone or in combination with other metals. Environmental and human monitoring data will also be discussed to highlight human populations most at risk of exposure to high concentrations of tungsten, the forms of tungsten present in each setting, and exposure levels in each population.

1. Introduction

Tungsten is a rare transition metal that falls in group VI of the periodic table along with molybdenum and chromium. Tungsten is a very unique metal and contains multiple beneficial properties including strength, flexibility, high density and melting point, and good conductive properties. Historically, tungsten was also thought to be biologically inert, having a low toxicity profile, a point which has been disputed over recent years (EPA, 2017; ATSDR, 2005; Keith et al., 2007; Keith et al., 2015). Because of these beneficial properties there has been an increase in the use of tungsten in numerous different types of manufactured goods including light bulb filaments, power tools, jewelry, armor penetrating munitions, electronics, golf clubs, drill bits, and implanted medical devices (EPA, 2017; ATSDR, 2005; Keith et al., 2007; Keith et al., 2015). In fact, tungsten was recently listed as one of the top 35 materials deemed critical through the United States Department of Interior (Department of Interior, 2018), further highlighting its importance in the manufacturing industry. In the 2000s both the Environmental Protection Agency (EPA) and the National Toxicology Program (NTP) classified tungsten as an emerging environmental toxicant warranting further investigation because of increased human exposure to tungsten and limited knowledge of the human health risks (NTP, 2002; EPA, 2008). Great strides have been made in our understanding of environmental exposure levels in human populations and the toxicity profile of tungsten either alone or in combination with other metals found to co-occur with tungsten in the environment. An important point we have learned about tungsten toxicity is that tungsten can augment the effects of other co-exposures or co-stressors, which could result in greater toxicity or more severe disease. This means that in order to understand the complex toxicity profile of tungsten researchers need to consider tungsten alone as well as in combination with other co-exposures and co-stressors in experimental models. Because of these advancements in tungsten toxicology research, regulations are also changing. As of 2022 tungsten is now listed as an emerging contaminant of concern in federal facility sites (EPA, 2022) and on the draft drinking water contaminant candidate list 5 (EPA, 2021) though the EPA. In this chapter, the current state of the tungsten toxicology field will be discussed focusing on data investigating tungsten carcinogenicity and other major toxicities including pulmonary, cardiometabolic, bone, and immune endpoints either alone or in combination with other metals.

Naturally, tungsten exists in the environment as tungstate (WO₄²⁻) in minerals with other elements such as wolframite and scheelite (ATSDR, 2005). In industrial settings tungsten ore is extracted and used in the manufacturing of industrial goods. Tungsten exists industrially in multiple forms including tungsten metal, tungsten carbide (equal parts tungsten metal and carbon) and tungstate. Tungsten metal or tungsten carbide is often found to exist industrially as an alloy with other metals including cobalt, nickel, and iron (Kraus et al., 2001; Roedel et al., 2012; van der Voet et al., 2007). Historically, tungsten was thought to be inert in the environment and relatively insoluble (Hartung et al., 1991; Langard et al., 2001). However, tungsten mobilization in the soil and dissolution into ground water sources is dependent on pH of the soil as well as phosphorous content. Under alkaline soil conditions tungsten is more soluble, making it more likely to contaminate water sources (ATSDR, 2005, ASTSWMO, 2011). High phosphorous content in the soil also increases tungsten solubility into water by decreasing tungsten sorption in soil due to competitive binding (Koutsospyros et al., 2006). In solution tungsten exists as an anion (WO₄²⁻) under alkaline conditions (Baes Jr et al., 1978). Tungsten can polymerize with itself and other oxyanions to form polytungstates and heteropolytungstates at high concentrations or in acidic pH environments (Lassner and Schubert, 1999). Dissolution of tungsten into solution results in a decrease in pH which can further propagate the formation of these polytungstates (Dermatas et al., 2004). Tungstates are more soluble than tungsten metal or tungsten carbide and are the most common form of tungsten found in water (Lassner and Schubert, 1999; Weast, 1973; CDC-NIOSH, 2010; OECD SIDS, 2005; RCC NOTOX BV, 1992). Conversely, tungsten metal and tungsten carbide contamination in soil would more likely remain in the soil and become aerosolized to contaminate air sources.

2. Susceptible Human Populations to High Exposure to Tungsten

Environmental and human monitoring of tungsten concentrations has identified individuals most at risk to exposure to high levels of tungsten and who would be most at risk of adverse health effects following exposure. Human exposure to increased concentrations of tungsten occurs in four main groups of individuals in different settings including occupational, military, medical, and environmental.

2.1. Occupational.

Occupational exposure to tungsten primarily occurs in mining and manufacturing settings where exposure can be to tungsten ore, pure tungsten metal, tungsten carbide, tungsten-containing alloys, or tungstates. It is estimated that upwards of 800,000 workers are exposed to tungsten in the United States alone (McKernan et al., 2009). Workers are most commonly exposed to tungsten through inhalation or dermal contact of particles in contaminated air (ATSDR, 2005). Hard metal is a common tungsten-containing alloy composed of tungsten carbide and cobalt found occupationally. Another common form of tungsten found occupationally is tungsten metal alloys containing cobalt and nickel or iron. Ambient air concentrations of tungsten in hard metal manufacturing plants have been reported to range between 0.003 – 2.13 mg/m³ (Kraus et al., 2001; Stefaniak et al., 2009). Back in the 1970s ambient air concentrations were reported as high as 6.1 mg/m³ (NIOSH, 1977). Human biomonitoring in hard metal manufacturing plants report urinary tungsten concentrations ranging between 0.33 to 168.6 μg/g creatinine (parts per billion (ppb); Kraus et al., 2001), with concentrations as high as 1.1 parts per million (ppm) reported back in the 1970s (NIOSH, 1977). The highest urinary tungsten concentrations occurred in plant workers that manufacture and grind hard metal alloys (Kraus et al., 2001). Importantly, the bioavailability of tungsten compounds is drastically different. Tungstates are the most bioavailable followed by tungsten carbide and then pure tungsten metal (Kraus et al., 2001). Interestingly, co-exposure of tungsten carbide and tungsten metal increased tungsten bioavailability compared to tungsten carbide alone. The bioavailability of the exposure tungsten compound is important information to consider in toxicological studies because ambient air and biological exposure concentrations might not always be correlative so monitoring of both is necessary to get an accurate estimate of exposure and identify at risk populations.

2.2. Military.

Tungsten has replaced lead and uranium in multiple types of projectile munitions including armor penetrating bullets and improvised explosive devices because of its assumed inertness and impressive ballistic properties. The primary form of tungsten found in munitions is tungsten mixed metal alloys contain 98% by weight tungsten metal in combination with cobalt, nickel, iron, or copper (van der Voet et al., 2007). Active military are exposed to tungsten mixed metal alloys through embedded shrapnel pieces or inhalation of airborne particulates following detonation of ammunition or improvised explosive devices containing tungsten. In a recent survey of 579 Veterans enrolled in the Department of Veterans Affairs’ Embedded Fragment Registry, tungsten was the second most commonly detected metal elevated in the urine (Gaitens et al., 2017). A significant percentage of these Veterans (11.6%) had urinary tungsten levels above the upper limit reference value set from data from the National Health and Nutrition Examination Survey (NHANES) database with concentrations as high at 2.70 μg/g creatinine (ppb) reported. Importantly, researchers have shown in rats that embedded metal fragments rapidly solubilize and metal concentrations detected in the urine are significantly higher than control levels as early as one-month post implantation (Hoffman et al., 2021). These data suggest that embedded tungsten metal alloy fragments could be an important source of systemic tungsten exposure in these veteran populations. In addition, analysis of tungsten particulate concentrations following detonation of tungsten mixed metal alloy-containing improvised explosive devices reveal ambient levels ranging between 3.7 - 8.95 mg/m³, of which 1.95 - 3.08 mg/m³ is in the respirable fraction (≤ 10 μm size; Machando et al., 2010). However, researchers don’t have an estimate of human levels of tungsten in the blood or urine following these types of exposures.

2.3. Medical.

Tungsten is a major component of multiple types of implanted medical devices, which have been shown to breakdown in the body resulting in increased systemic exposure to tungsten. A cohort of breast cancer patients were exposed to tungsten through the use of a tungsten-based shield placed internally during intraoperative radiotherapy. The shield broke down and tungsten fragments remained in the breast tissue (Bolt et al., 2015). Tungsten could be detected in the urine of patients even 2 years-post surgery, with concentrations as high as 2.71 ppb reported. Implanted tungsten coils are also used to perform transcatherter embolization of blood vessels and have been reported to corrode and degrade overtime (Bachthaler et al., 2004; Barrett et al., 2000). Urinary tungsten concentration in these patients have been reported as high as 837.7 ppb (Bachthaler et al., 2004). In a second study, elevated tungsten levels were detected in the blood for years after the embolization procedure (Barrett et al., 2000). These data strongly indicate that tungsten-based implantable devices do breakdown and corrode over time, leading to increased concentrations of tungsten in the systemic circulation and chronic exposure. Back in the 2000s tungsten was also proposed as a potential drug for the treatment of diabetes and obesity (Barbera et al., 2001; Claret et al., 2005; Hanzu et al., 2010). In a proof-of concept trail, patients treated with sodium tungstate (200 mg/day) orally for 6 weeks, for the treatment of obesity, had circulating plasma tungsten concentrations that averaged 2,148 μg/L (ppb) (Hanzu et al., 2010). However, no significant changes in weight, food intake, or energy expenditure were observed following treatment.

2.4. Environmental.

Tungsten minerals are found naturally in the rock and soil and contamination can occur into water and air sources through runoff, erosion, or weathering. However, increased tungsten contamination in the environment can also occur near areas of mining, manufacturing, and military activity due to the generation of industrial waste and emissions. Since tungsten is not routinely measured in water and air sources we have limited data on environmental exposure levels. Importantly, elevated levels of tungsten have been reported in certain locations particularly in regions near mining, industrial, and military sites (ATSDR, 2005; Rubin et al., 2007; Clausen et al., 2007; Seiler et al., 2005; Dams et al.,1970; Haddad and Zikobsky, 1985). Around the town of Fallon, Nevada in the Southwestern United States, where there is a pediatric leukemia cluster, concentrations of tungsten in municipal drinking water ranged between 2.98 – 7.30 ppb (Rubin et al., 2007). Groundwater concentrations in the area were reported as high at 742 ppb (Seiler et al., 2005). Individuals living in this area had elevated levels of tungsten in their urine (Adults: mean = 0.81 ppb, Children: mean = 2.31 ppb; Rubin et al., 2005). In Sicily, Italy near the volcano Mount Etna, where there is increased incidence of thyroid cancer, water concentrations of tungsten were 50x higher compared to concentrations reported in non-volcanic areas (mean water tungsten concentration in volcanic area = 0.2 ppb; Malandrino et al., 2016). However, these values were drastically lower than what was measured in the ground water in the area near Fallon, Nevada. Urinary tungsten levels in this region were approximately 2x higher than control values (mean = 0.12 μg/g creatinine; Malandrino et al., 2016). Near the Nahanni River in the Northwest Territories of Canada, an area rich in tungsten-bearing minerals, water concentrations of tungsten range from <0.1 to 224.5 ppb (Hall et al., 1988). In Camp Edwards Massachusetts sampling near three small arms ranges reported ground water tungsten concentrations ranging between <1 – 560 ppb (Clausen et al., 2007). Ambient air tungsten particulate concentrations in the environment are substantially lower than concentrations found in occupational settings with most measurements reported being < 10 ng/m³ (Dams et al., 1970; Haddad and Zikobsky, 1985; Jagielak and Mamont-Cieśla, 1979). The concentrations of tungsten in the air in the urban setting of East Chicago, Indiana were 5.8 ng/m³ compared to samples taken in a more rural setting of Niles, Michigan were 0.4 ng/m³ (Dams et al., 1970). Comparable concentrations were also reported in Montreal, Quebec (5.2 ng/m³; Haddad and Zikobsky, 1985). In a cross-sectional study across 6 urban cities in the United States, urinary tungsten concentrations were positively associated with particulate matter (PM) 2.5 levels (Pang et al., 2016). These data could suggest that exposure to PM containing tungsten particulates, in certain geographic locations, might be driving the increased urinary levels observed, however no ambient air tungsten concentrations were reported. Importantly, multiple studies have correlated urinary tungsten levels, from environmental biomonitoring data, with health outcomes further stressing that environmental exposure to high levels of tungsten could be affecting human health (Table 1).

Table 1.

Tungsten epidemiological studies

Disease	Tungsten endpoint	Route of exposure	Outcomes	Reference
Pulmonary Disease	Cemented tungsten carbide- cobalt	Inhalation, dust	Interstitial lung disease – bronchiolar inflammation and interstitial fibrosis	19, 37, 90, 114
Cardiovascular Disease	Urinary Tungsten	Unknown Route	Increased risk of Hypertension, Peripheral arterial disease, Stroke; Increased mortality from composite cardiovascular and cerebrovascular disease	3, 92, 93, 110, 116
Diabetes	Urinary Tungsten	Unknown Route	Increased fasting blood glucose, increased risk of diabetes	36, 85, 104,
Chronic Kidney Disease	Urinary Tungsten	Unknown Route	Decreased time to develop chronic kidney disease; Increased risk of kidney disease	38, 100
Risk of Fracture	Urinary Tungsten	Unknown Route	Increased risk of facture	100
Cancer
Thyroid Cancer	Urinary/Ground Water Tungsten	Drinking water	Water and urinary tungsten concentrations were elevated in communities with increased incidence of thyroid cancer	82
Lung Cancer	Tungsten carbide-cobalt	Inhalation, dust	Increased mortality to lung cancer	89, 122
Pediatric leukemia	Urinary/Ground Water Tungsten	Drinking water	Water and urinary tungsten concentrations were elevated in communities with increased incidence of pediatric leukemia	106, 109

2.5. Regulatory Status.

Tungsten is not federally regulated in municipal or ground water sources or in the ambient air. It is also not routinely measured in water or air sources, which makes accurate estimates of exposure in the general United States population difficult. Several states have established their own regulatory limits (EPA, 2017). Texas has set soil (820 ppm) and groundwater (7.3 ppm) protective concentrations for sodium tungstate dihydride (TCEQ, 2016). North Carolina has preliminary protection of groundwater goals for tungsten (8.4 ppm; NCDEQ, 2016). Indiana has set soil (88 ppm) and groundwater (0.016 ppm) screening levels for tungsten (IDEM, 2016).

Occupationally, regulatory safety limits have been established through the Occupational Safety and Health Administration (OSHA) and National Institute for Occupational Safety and Health (NIOSH) (EPA, 2014). Tungsten safety limits are set at 5 mg/m³ for insoluble tungsten compounds and 1 mg/m³ for soluble tungsten compounds over an 8- to 10-hour work day. Short-term exposure limits are set at 10 mg/m³ for insoluble tungsten compounds and 3 mg/m³ for soluble tungsten compounds for a 15-minute work period (CDC-NIOSH, 2010; OSHA, 2013).

3. Tungsten Deposition and Retention in the Body

An important aspect of assessing the toxicity profile of a toxicant is understanding tissue deposition and retention in the body following exposure to be able to predict organs/organ systems most at risk. Following both inhalation and oral exposure, absorbed tungsten is rapidly excreted from the body through the kidneys into the urine (Lagarde et al., 2002; Kaye et al., 1968; Aamodt, 1975). However, tissue accumulation and retention in certain organs does occur, especially following chronic exposure. Tissue deposition following continuous sodium tungstate exposure (62.5 – 200 mg/kg/day) in the drinking water for 28 days resulted in tungsten accumulation in all organ analyzed. The bone was the site of greatest tungsten accumulation with almost a third of the oral drinking water concentration deposited in the bone at 28 days, followed by the spleen, colon, kidney, liver, and brain (Guandalini et al., 2011). Longitudinal assessment of tungsten deposition in the bones of mice demonstrates that tungsten rapidly accumulates in the bone within 1-week of exposure and reaches maximal accumulation in the bone by 4 weeks (Kelly et al., 2013). By performing washout experiments in mice, the rates of removal of tungsten from the bone were slower than the rates of accumulation, making the bone a site of long-term storage of tungsten (Kelly et al., 2013). Tungsten deposition in the bone is affected by age. In mice following a 4-week oral exposure to sodium tungstate (15 ppm), tungsten only accumulated in the bones of young mice (5-weeks old), but not in adult mice (9-months old), suggesting that active bone remodeling is necessary for tungsten incorporation into the bone (Bolt et al., 2016). Tungsten deposition in the bone is also influenced by co-exposures with other metals. Mice implanted with tungsten, cobalt, nickel pellets had more tungsten accumulation in the bone compared to mice implanted with tungsten, nickel, iron pellets, suggesting that co-exposure with cobalt enhanced tungsten deposition in the bone (Emond et al., 2015a). This was confirmed in a follow-up study that demonstrated a greater tungsten accumulation in the bone in mice implanted with tungsten, cobalt, tantalum pellets compared to mice implanted with either tungsten, tantalum or tungsten, nickel, tantalum pellets (Emond et al., 2015b). Importantly, speciation of tungsten in the bone following exposure showed that soluble tungstate (WO₄²⁻) forms complexes with phosphorus in the bone to form phosphotungstate species, which are known redox active catalysts (VanderSchee et al., 2018). Following nose-only inhalation of tungsten blue oxide (0.08 – 0.65 mg/L air) for 28 days, followed by a 14-day recovery period, the predominate site of tungsten accumulation was the lungs followed by the bone than the kidney (Rajendran et al., 2012). In a second study, nose-only inhalation of radiolabeled tungsten oxide found that 60% of the inhaled tungsten concentration deposited in the respiratory tract (Aamodt, 1975). At the end of the 165-day study, the highest tungsten concentrations were found in the lungs and kidney and 10-fold lower in the bone. Interestingly, in a recent study using a whole-body inhalation exposure to tungsten metal particles (1.7 mg/m³; <1 μm), tungsten deposition persisted in the lungs for up to 7 days post a single 4-hour exposure (75 ppb; Miller and McVeigh et al., 2021). There was substantial deposition of tungsten in the bone (175 ppb) as well, probably from oral ingestion of particles. So, in addition to soluble tungstates, there is evidence that insoluble tungsten particulates also accumulate in the bone. However, how these tungsten particles incorporate into the bone and what chemical forms of tungsten are present still needs to be defined. Tungsten distribution and retention in humans has been proposed based on kinetics of radiolabeled tungsten from laboratory animal (Leggett et al., 1997). Based on these studies tungsten is rapidly excreted from the body through the kidneys, but is retained in the bone. It is estimated that following a single dose of tungsten, 50% of total body tungsten is retained in the bone at 1-month post exposure and tungsten can still be detected in the bone up to 10 years post exposure, suggesting long-term exposure (Leggett et al., 1997).

4. Tungsten Carcinogenesis

Limited epidemiological data have investigated the effects of tungsten exposure on cancer outcomes (Table 1). Drinking water and urinary tungsten levels were reported elevated in several communities with increased incidence of thyroid cancer and pediatric leukemia, however no causative links have been established (Malandrino et al., 2016; Rubin et al., 2007). These communities did not only have elevated levels of tungsten in the environment, but also multiple carcinogens including arsenic, nickel, cobalt, benzene, and cadmium, so more research is needed to dissect out which toxicant or toxicant mixtures are driving cancer incidence in these populations and how. Workers in the hard metal manufacturing industry, exposed to tungsten carbide-cobalt particles, have a 2x higher risk of mortality from lung cancer after adjusting for cobalt exposure alone and smoking status (Moulin et al., 1998, Wild et al., 2000). However, defining the contribution of tungsten verses cobalt on the carcinogenic effects observed is needed to fully understand the effect of tungsten on lung cancer outcomes in this population. More epidemiological studies are needed to more clearly define how tungsten exposure impacts cancer outcomes in diverse human populations.

One fundamental mechanism of environmental carcinogenesis is the ability of a toxicant to induce deoxyribonucleic acid (DNA) damage, which can lead to an increased mutational burden. There is evidence that tungsten compounds alone or in combination with other metals are genotoxic. Using supercoil plasmid DNA, in vitro treatment of tungsten particles induced single-strand breaks (Mazus et al., 2000). Mutagenic activity following tungsten treatment has been reported using multiple bacterial mutagenesis assays (Ulitzur et al., 1988; Singh et al., 1983; Sora et al., 1986; Miller et al., 1999). Both tungsten, nickel, cobalt and tungsten carbide-cobalt particles induce DNA damage through the generation of reactive oxygen species (ROS) in vitro (Harris et al., 2011; Anard et al., 1997; Miller et al., 2001; Table 2). The DNA damage induced by tungsten carbide-cobalt particles was significantly more than either cobalt or tungsten carbide particles alone, stressing the synergistic ability of tungsten carbide-cobalt to induce DNA damage (Anard et al, 1997). Sodium tungstate has been shown to induce DNA damage in both a preB lymphocyte cell line BU-11 and primary B-cells in vitro, as well as in isolated B-cells and total bone marrow harvested from tungsten-exposed mice (Guilbert et al., 2011; Kelly et al., 2013). However, sodium tungstate did not induce sister chromatid exchanges or chromosome aberrations in Syrian hamster cells in vitro (Larramedy et al., 1981). In addition, the NTP investigated genotoxicity of sodium tungstate as part of their comprehensive toxicity and carcinogenesis studies in rodents and found mixed results. Sodium tungstate was not mutagenic in bacterial gene mutagenesis assays using S. typhimurium and E. coli strains with and without activation with S9 (NTP, 2021). Sodium tungstate exposure in the drinking water for 3-months resulted in no changes in erythrocyte micronuclei numbers. Tungsten exposure did increase DNA damage (comet assay) in the liver (male and female rats, male mice) and ileum (male mice), but not in the kidney or blood leukocytes. Data presented to date indicates that multiple forms of tungsten (tungstate, tungsten metal, and tungsten carbide) can induce DNA damage in certain contexts. Co-treatment with cobalt seems to exaggerate the effects of tungsten alone. Factors including tungsten dose and exposure time may be important factors that help to explain inconsistencies in data across experiments.

Table 2.

In vitro and in vivo evidence of tungsten-meditated carcinogenesis/tumorigenesis

Model/organism	Test Compound	Route of exposure	Outcomes	Reference
PBMCs	Tungsten Carbide-Cobalt Particles	In vitro	DNA damage; ROS; Oxidative stress; Apoptosis	4, 77, 78, 79
TE85 Osteosarcoma cell line	W/Ni/Co Heavy Metal Alloys	In vitro	Genotoxicity – DNA damage	87
L6-C11 Muscle cell line	W/Ni/Co Heavy Metal Alloys	In vitro	DNA damage; ROS; Apoptosis	51
BU-11 and primary B-cells	Sodium Tungstate	In vitro	DNA damage	46, 64
TE85 Osteosarcoma cell line	W/Ni/Co Heavy Metal Alloys	In vitro	Cellular transformation	87
Beas-2B	Sodium Tungstate	In vitro	Anchorage independent growth; Increased cell migration; Tumors in nude mice	73
Primary human thyrocytes and thyrospheres	Sodium Tungstate	In vitro	Enhanced proliferation; Reduced apoptosis; Anchorage independent growth; Clonogenic growth; Increased Migration capacity; DNA damage	41
Rat	Tungsten Carbide-Cobalt Particles	Intratracheal Instillation	Cytotoxic; Genotoxic; Micronuclei formation	21
Mouse/Rat	Sodium Tungstate	Ora, Drinking Water	DNA damage in liver and ileum	91
Mouse/Rat	W/Ni/Co pellets	Implantation	Increased incidence of rhabdomyosarcoma; Increased peripheral white blood cells; Enlarged spleen	28, 59
Mouse – NMU carcinogen model	Sodium Tungstate	Oral, Drinking Water	Increased incidence of mammary tumors	120
Mouse	Sodium Tungstate	Oral, Drinking Water	Bone marrow and isolated preB cells: Increased DNA damage; Halt in B-cell development; Increased number of preB cells; Increased clonogenic capacity of lymphoid progenitors	64
Mouse – Orthotopic mammary cancer model	Sodium Tungstate	Oral, Drinking Water	Enhanced Lung Metastasis; Increased numbers of myeloid-derived suppressor cells and cancer-associated fibroblasts; Increased MMP-9	13
Female Rats	Sodium Tungstate	Oral, Drinking Water	Increased incidence of C-cell adenoma or carcinoma in thyroid gland	91
Male Mice	Sodium Tungstate	Oral, Drinking Water	Increased incidence of renal tubule adenoma or carcinoma	91

Despite evidence of DNA damage in certain settings, in vitro and in vivo studies, investigating the carcinogenic potential of tungsten in multiple cell lines/organs have been mixed (Table 2). Chronic exposure (6-weeks) of human bronchial epithelial cells (Beas-2B) to sodium tungstate (50 – 250 μM) resulted in increased colony formation in soft agar, increased cell migration, and formation of tumors in female nude mice (Laulicht et al., 2015). Gene expression profiling of the transformed clones from soft agar indicated up-regulation of multiple genes involved in lung cancer including NQO1, CRYAB, S100A4, SPK2 and HTATIP2. Chronic exposure of human thyrocytes and thyrospheres to soluble tungsten (nM range) had biological effects on thyroid stem/precursor cells by enhancing growth and reducing apoptosis (Giani et al., 2019). Tungsten-exposed thyrospheres had increased anchorage-independent growth, clonogenic growth, and migration capacity as well as increased expression of DNA damage markers H2AX and 53BP1. Exposure to heavy metal tungsten alloys containing 91 - 93% tungsten, nickel, and either cobalt or iron had the ability to transform the human osteoblast like cell line (TE85), characterized by increased anchorage-independent growth, tumor formation in nude mice, and increased expression of the oncogene K-ras (Miller et al., 2001). However, the contribution of tungsten as part of this metal mixture in the transformation process has yet to be defined. Oral drinking water exposure to sodium tungstate (15 – 1,000 ppm) leads to an accumulation of preB cells in the bone marrow and increased clonogenic capacity of lymphoid progenitors. (Kelly et al., 2013). Importantly, this population of preB cells is the same population that expands in patients with preB acute lymphoblastic leukemia. Sodium tungstate, at concentrations as high as 20 μg/mL, failed to induce morphological transformation of Syrian hamster embryo cells following 9-day of treatment (DiPaolo and Casto, 1979). In a life-span study Long-Evans rats were treated with sodium tungstate (5 ppm) in the drinking water throughout their lifetime, no changes in median life-span or tumor incidence were observed (Schroeder and Mitchener, 1975). In the NTP two-year carcinogenesis studies there was no evidence of carcinogenic activity of sodium tungstate in male Sprague Dawley rats at exposure concentrations of 250, 500, or 1,000 mg/L (NTP, 2021). In female Sprague Dawley rats there was equivocal evidence of carcinogenic activity based on increased incidence of C-cell adenoma or carcinoma in the thyroid gland at all exposure concentrations. In male B6C3F1/N mice at exposure concentrations of 500, 1,000, or 2,000 mg/L there was equivocal evidence of carcinogenic activity based on increased incidence of renal tubule adenoma or carcinoma in the kidney at the 1,000 and 2,000 concentrations. In female B6C3F1/N mice there was no evidence of carcinogenic activity following exposure. In summary, exposure to tungsten species (sodium tungstate and tungsten mixed metal alloys), have been shown to contribute to the carcinogenesis process through the induction of cellular transformation. Exposure to sodium tungstate has also been shown to increase the incidence of thyroid and kidney cancer in animal studies, however only at high concentrations.

Multiple in vivo studies also provide evidence that tungsten exposure can enhance tumor progression (Table 2). Rodents implanted with tungsten-heavy metal alloy pellets containing tungsten, cobalt, nickel, to model embedded shrapnel, developed highly aggressive rhabdomyosarcoma around the implantation site (Kalinich et al., 2005; Emond et al., 2015a). However, only in the rat model did they developed metastatic tumors (Kalinich et al., 2005). Importantly, they also evaluated the individual contribution of each metal on the tumorigenesis process. Mice implanted with either cobalt, tungsten or tungsten/cobalt pellets developed malignant sarcomas, however substantially less than the tumor incidence reported in the tungsten, cobalt, nickel alloy implanted mice (80%; Edmond et al., 2015b). Using a mutagen-induced model of mammary cancer (N-Nitroso-N-methylurea; NMU), exposure to sodium tungstate in the drinking water (150 ppm) significantly increased the incidence of mammary tumors compared to mutagen alone (Wei et al., 1985). Using an orthotopic model of mammary cancer using the cell line 66Cl4, oral tungsten exposure (15 ppm) enhanced mammary cancer lung metastasis, but did not affect primary tumor volume (Bolt et al., 2015). Instead, enhanced lung metastasis was associated with changes in the surrounding metastatic microenvironment, including increased numbers of myeloid-derived suppressor cells and cancer-associated fibroblast that could be promoting metastasis in this model. Interestingly, the implantation of tungsten, nickel, cobalt pellets that lead to aggressive metastatic rhabdomyosarcoma also resulted in changes in immune parameters including increased circulating white blood cells and enlarged spleens (Kalinich et al., 2005), so this could be a common mechanism of tungsten-enhanced tumorigenesis. However, the effects of tungsten on tumorigenesis are not consistent for all carcinogen-induced models investigated. Using an N-nitrososarcosine ethyl ester-induced model of esophageal and stomach cancer, tungsten did not increase tumor incidence or accelerate cancer stage (Luo et al., 1983). Oral sodium tungstate exposure also did not alter lung cancer initiation or mammary tumor incidence using a benzo[a]pyrene model (Gunnison et al., 1988). In summary, exposure to sodium tungstate and tungsten mixed metal alloys enhances tumor progression (incidence and metastasis) in multiple co-carcinogen/mutagen in vivo models, but the data is not consistent for all models’ tests. A common mechanism that should be explored further is tungsten-mediated inflammation as a potential driver of enhanced tumorigenesis.

Importantly, the International Agency for Research on Cancer (IARC) has classified cobalt with tungsten carbide as probably carcinogenic in humans (Group 2A) based on experimental animal and mechanistic evidence and some, but not convincing evidence regarding cancer in humans (IARC, 2006). IARC has just recently classified weapons-grade tungsten with cobalt and nickel alloys as possibly carcinogenic to humans (Group 2B) based on sufficient evidence for cancer in experimental animals, yet limited mechanistic evidence and inadequate evidence regarding cancer in humans (Karagas et al., 2022). The data presented to this point strongly suggests that tungsten could have a synergistic role in the carcinogenic/tumorigenic process especially in the context of co-exposures with other carcinogens such as nickel and cobalt. Understanding the underlying molecular mechanisms of tungsten to induce or promote carcinogenesis/tumorigenesis is a gap in the tungsten toxicology field that should be explored.

5. Tungsten Pulmonary Toxicity

Epidemiological data from the hard metal manufacturing industry indicates that exposure to tungsten carbide-cobalt particulates is associated with interstitial lung disease characterized by bronchiolar inflammation and interstitial fibrosis (Table 1; Coates et a., 1971; Naqvi et al., 2008; Figueroa et al., 1992; Tanaka et al., 2014). An extensive amount of research has been completed to investigate the molecular mechanisms driving pulmonary disease following tungsten particulate exposure. In vitro, in both lung epithelial and alveolar macrophages, treatment with tungsten carbide-cobalt particles led to a generation of ROS and induction of cell death (Armstead et al., 2014; Liu et al, 2015; Lison et al., 1990). In Beas-2B cells tungsten carbide-cobalt nano-particles (0.1 – 1000 μg/mL) were more cytotoxic and generated more ROS than tungsten carbide-cobalt micro-particles, likely due to internalization by the lung epithelial cells (Armstead et al., 2014). In alveolar macrophages, tungsten carbide-cobalt particles were significantly more cytotoxic than either cobalt or tungsten carbide particles alone (Lison et al., 1990). Interestingly, using a co-culture model of Beas-2B lung epithelial cells with THP-1 macrophages, less cytotoxicity was induced by tungsten carbide-cobalt nano-particles (1 – 1,000 μg/mL) compared to Beas-2B cells treated in a monoculture, suggesting that the macrophages were playing a protective role against cytotoxicity (Armstead et al., 2015). Engulfment of tungstate nanowires, but not nanospheres led to a generation of ROS and cytotoxicity in RAW 264.7 murine macrophages (Dunnki et al., 2014). Using human peripheral blood mononuclear cells, in vitro treatment with tungsten carbide-cobalt particles, also led to generation of ROS, oxidative stress, DNA damage, and an induction of apoptosis (Lombaert et al., 2004; Lombaert et al., 2008; Lombaert et al., 2013; Anard et al., 1997). Treatment of the skeletal muscle cell line L6-C11 with tungsten, cobalt, nickel particles also induced cell death, DNA damage, and generation of ROS (Harris et al., 2011). In vitro studies indicate that exposure to tungsten carbide and tungsten metal alloy particulates leads to toxicity of lung epithelial cells and alveolar macrophages through the generation of ROS, induction of DNA damage, and cytotoxicity.

In vivo studies indicate that inhalation exposure to tungsten particles leads to inflammation, fibrosis, and alter function in the lungs. Intratracheal instillation of tungsten carbide particles resulted in a mild infiltration of macrophages into the alveoli, however exposure to tungsten carbide-cobalt particles (1 – 10 mg/100g body wt) produced severe alveolitis and fatal pulmonary edema (Lasfargues et al., 1992). Repeated exposures to tungsten carbide-cobalt particles (4 x 1 mg/100g body wt) resulted in makers of fibrosis in the lungs including increased lung hydroxyproline content and histopathological evidence of fibrosis (Lasfargues et al., 1995). Rats exposed to either tungsten, nickel, cobalt or tungsten, nickel, iron particles through intratracheal instillation (1- 4 mg/ 100g body wt) had pulmonary inflammation characterized by an increase in neutrophils and increased levels of pro-inflammatory cytokines and chemokines TNFα, IL-1β, CINC-1, and CINC-3 (Roedel et al., 2012). Exposure to individual metals did not produce the same inflammatory effect in the lungs. Inhalation of tungsten, cobalt, chromium, iron particulates (2.5 – 5mg/ 100g body wt) also led to pulmonary inflammation as well as airway obstruction and hyperreactivity in the lungs following methacholine stimulation (Rengasamy et al., 1999). Rats expose to tungsten blue oxide particles by nose-only inhalation over the course of 28 days, had increased lung weight and an infiltration of macrophages into lungs post exposure (Rajendran et al., 2012). Inhalation exposure to tungsten (V) oxide nanoparticles (10 mg/m³, 4 hours per day for 4 days) resulted in the generation of ROS and activation of the NLRP3 inflammasome in the lungs (Prajapati et al., 2016). A single 4-hour exposure to tungsten metal particles (1.7 mg/m³), using a whole-body inhalation exposure chamber, led to an infiltration of macrophages in the lungs 24 hours post-exposure in mice (Miller and McVeigh, 2021). At day 7 post-exposure, tungsten increased levels of CXCL1 and IL-1β in the bronchoalveolar lavage fluid and the percentage of alpha smooth muscle actin (α-SMA) positive myofibroblasts in the lungs, a crucial early step in fibrosis development. Interestingly, no tungsten was detected in immune cells isolated from the bronchoalveolar lavage fluid at day 1 or day 7 post-exposure. Importantly, when evaluated, these in vitro and in vivo studies show that exposure to tungsten carbide-cobalt or tungsten, cobalt, nickel particles leads to great toxicity (pulmonary inflammation, fibrosis, altered lung function) than individual metals alone, highlight the synergistic effects of tungsten in combination with these other metals to induce pulmonary toxicity.

6. Tungsten Cardiometabolic Toxicity

Multiple epidemiological studies have found associations between increased urinary tungsten levels and increased risk or mortality of cardiovascular diseases, diabetes, and chronic kidney disease (Table 1). Urinary tungsten levels were significantly associated with increased mortality from composite cardiovascular and cerebrovascular disease in a ~14000-person NHANES study (Odds Ratio = 1.78, 95%CI = 1.28-2.48), as was cobalt (Odds Ratio = 2.09, 95%CI = 1.22-3.60, Agarwal et al., 2011). Urinary tungsten levels have also been associated with increased risk of developing specific cardiovascular diseases including peripheral arterial disease, hypertension, and stroke (Navas-Acien et al., 2005; Shiue and Hristova, 2014; Tyrell et al., 2013). Interestingly, in a paper published in 2018, using data from the Strong Heart Study, urinary tungsten levels were positively associated with cardiovascular disease incidence and mortality at lower urinary molybdenum levels (not significant) and significantly inversely associated at higher molybdenum levels (Nigra et al., 2018). These data suggest that molybdenum levels should also be considered in tungsten epidemiological studies because co-exposure could influence tungsten health outcomes. Urinary tungsten levels have also been correlated with diabetes outcomes. In several cross-sectional studies, urinary tungsten levels have been associated with increased fasting blood glucose, increased insulin resistance, and increased risk of developing diabetes (Feng et al., 2015; Menke et al., 2016). In a 1,659 participant cohort from the San Luis Valley in rural Colorado, urinary tungsten levels were positively associated with greater insulin resistances, greater fasting blood glucose, and higher incident diabetes (Riseberg et al., 2021). In this same population urinary tungsten levels were associated with reduced time to develop chronic kidney disease (Fox et al., 2021). These finding were just recently validated in a publication using continuous NHANES survey data where researchers found an association between urinary tungsten levels and increased risk of kidney disease (Park and An, 2022). Very little experimental data have investigated the underlying molecular mechanisms of tungsten contributing to these disease states. A 3-month oral drinking water exposure to sodium tungstate (100 ppm) led to subtle changes in kidney function including decreased urinary flow rate and decreased creatinine clearance (Grant et al., 2021). These changes were associated with an increase in multiple histological markers of fibrosis in the kidneys. Interestingly, multiple in vitro and in vivo have actually shown the anti-diabetic effects of tungsten, however many of these studies were using animal models of diabetes, not looking at the effects of tungsten on disease etiology (Domingo et al., 2002; Girón et al., 2003; Barbera et al., 2001; Heidari et al., 2008; de Souza et al., 2021). More experimental studies are necessary to identify molecular mechanisms of action leading to cardiometabolic disease pathologies following tungsten exposure.

7. Tungsten Bone Toxicity

The bone is a predominate site of tungsten accumulation and it is also a site of toxicity. Tungsten deposition in the bone alters multiple components of bone biology. Mesenchymal stromal cells (MSCs) are multi-potent stem cells in the bone marrow niche that can differentiate into multiple lineages including osteoblast, adipocytes, and chrondocytes (Hu et al., 2018). In vitro, tungsten exposure skewed MSC differentiation to promote PPARγ-mediated adipogenesis, when cultures were co-treated with the PPAR-γ agonist Rosiglitazone, and inhibit osteoblastogenesis (Bolt et al., 2016). Interestingly, tungsten exposure alone did not promote adipogenesis nor was tungsten a PPARγ agonist, but instead further enhanced PPARγ signaling. Tungsten also induced adipogenesis in the bone marrow niche in young male mice following 4 weeks of oral exposure (15 ppm). However, no change in osteoblast differentiation was observed. Interestingly, the in vivo effects of tungsten on adipogenesis were age and sex dependent. In addition, tungsten also alters osteoclasts differentiation. Following the same exposure paradigm above, a 4-week oral tungsten exposure (15 ppm) resulted in an increase in the number of tartrate-resistant acid phosphatase (TRAP) positive osteoclasts in the bone marrow of young male, but not young female mice (Chou et al., 2021). Mechanistically, tungsten enhanced RANKL-induced osteoclast differentiation through sustained activation of downstream p38 signaling. Despite these dramatic changes in bone biology no changes in bone mineral density or bone fragility have been reported following tungsten exposure in vivo. Interestingly, a recent publication evaluating health outcomes associated with cumulative metals exposure using continuous NHANES survey data found an association between urinary tungsten levels and increased bone fracture risk (Park and An, 2022). Tungsten also accumulates in other bones in the body besides long bones. Tungsten also accumulates in the vertebrae of the spine and the intervertebral disks (IVD) following oral exposure (Grant et al., 2021). Tungsten accumulation resulted in disk degeneration including reduction in IVD height, decreased proteoglycan content, and increased fibrosis. Inflammatory markers TNFα and IL-1β were also elevated in exposed IVD. Interestingly, tungsten accumulation in the bone following inhalation exposure to tungsten metal particulates resulted in transient increases in pro-inflammatory cytokines CXCL10 and TNFα expression in the bone marrow (Miller and McVeigh et al., 2021). To date the bone is a predominate site of tungsten storage and toxicity following exposure leading to alterations in bone biology, however more research is needed to investigate if these changes leads to changes in bone mineral density or increased risk of facture.

8. Tungsten Immunotoxicity

In addition to tungsten’s effects on bone biology, tungsten accumulation in the bone also alters immune cells in the bone marrow niche. As mentioned previously, tungsten blocks B-cell development in the bone marrow. Oral drinking water exposure to sodium tungstate (15 - 1,000 ppm) for 16 weeks resulted in a dose-dependent increase in the percentage of preB-cells in the bone marrow (Kelly et al., 2013). Ribonucleic acid (RNA)-sequencing analysis and validation of this preB population demonstrated that tungsten exposure decreased expression of multiple genes in the IL-7R/Pax5 signaling pathways critical for B-cell development (Wu and Bolt et al., 2019). In vitro, tungsten treatment (50 – 500 μg/mL) induced cell death in the preB-cell line BU-11 and primary bone marrow B-cells (Guilbert et al., 2011). However, tungsten’s effects on immune cells is not confined to the bone niche. As mentioned previously, tungsten exposure stimulates proliferation of thyroid stem/precursor cells and reduces apoptosis (Giani et al., 2019). Oral exposure to sodium tungstate (125 – 2,000 ppm) led to decreased T-cell mediated immunity following challenge with immune stimulating agents in female mice (Frawley et al., 2016). In addition, pups exposed to tungsten in utero had a diminished anti-viral immune response following challenge with respiratory syncytial virus (RSV) (Fastje et al., 2011). Tungsten-exposed pups exhibited significant splenomegaly following RSV challenge. Despite these findings in experimental models, no epidemiological studies have found an association between tungsten exposure and immune endpoints. In summary, exposure to sodium tungstate stimulates immune progenitor cell populations in both the bone niche, leading to an accumulation in preB-cells, and in the thymus, leading to increased proliferation of thyroid stem cells. However, systemically exposure to tungstate leads to immunotoxicity including decreased T-cell mediated immunity, and increased susceptibility to infections.

9. Conclusion

The toxicity profile of tungsten is complex because there are multiple routes of human exposure as well as multiple different tungsten compounds that humans can be exposed to. In addition, humans are often not exposed to tungsten alone but to metal alloys consisting of tungsten in combination with other metals including cobalt and nickel. Since classifying tungsten as an emerging contaminant in the 2000s great advancements have been made in the tungsten toxicology field. Especially, in the identification of at-risk human populations, the forms of tungsten present in each setting, and disease outcomes associated with exposure. In addition, identification of molecular mechanisms of action, target organs/organ systems, and disease endpoints in experimental models have further enhanced our understanding (Figure 1). The data presented to this point indicates that tungsten can augment or enhance to effects of other co-exposures, stressors, or stimulants, often resulting in greater toxicity or more severe disease. This has particularly been evident when evaluating pulmonary and carcinogenicity/tumorigenicity endpoints. As the tungsten toxicology field moves forward consideration of all the relevant forms of tungsten as well as co-exposures that exist in the environment, relevant for each disease state, will be important to shape in vitro and in vivo studies. For example, the form of tungsten found in the bone is phosphotungstate not soluble tungstate (WO4²⁻; VanderSchee et al., 2018). The toxicity profile of phosphotungstate has not been defined and should be added to toxicological studies moving forward, especially when evaluating toxicity endpoint follow systemic tungsten exposure. In addition, tungsten has been reported to co-exist in the environment with other toxicant besides cobalt and nickel. More human biomonitoring data needs to be collected to define what other toxicants tungsten is commonly found in the environment with and relevant exposure concentrations. In addition, analysis of other known genetic and environmental stressors that are risk factors of each disease should be considered and incorporated into future experimental studies to evaluate how tungsten might augment those parameters to cause more severe disease. This could also include using two-hit models of disease pathogenesis to more accurately understand how tungsten contributes to disease outcomes.

Figure 1.

Summary of unique and common mechanisms of action/toxicity of tungsten compounds based on in vitro and in vivo studies.

The list of diseases associated with tungsten exposure, based on epidemiological studies is growing (Table 1). However, this is still an area where more research is needed. More epidemiological studies in diverse, highly exposed populations will help to validate and strength existing tungsten associations as well as further define relevant routes, exposure concentrations, and disease outcomes to model in experimental studies.

The evidence supporting tungsten-mediated carcinogenesis/tumorigenesis is growing particularly in the context of exposure to tungsten carbide-cobalt and tungsten, cobalt, nickel alloys (Table 2). Based on the limited epidemiological data, in addition to in vitro and in vivo studies, tungsten’s role in lung cancer, thyroid cancer, and leukemia etiology is becoming more defined. Potential mechanisms of action of tungsten carcinogenesis include the generation of ROS, induction of DNA damage, and cell transformation. In addition, multiple in vivo studies have suggested that tungsten can enhance tumorigenesis through changes in the microenvironment including inflammation, which could be a promising cellular mechanism of action to focus on (Bolt et al., 2015; Kalinich et al., 2005). Importantly future research should focus on linking all these independent studies together to further identify how different forms of tungsten contribute to the carcinogenesis/tumorigenesis process either alone or in combination with other carcinogens. Focusing on organs and organ systems with the highest tungsten accumulation in the body and cancer endpoints supported from in vitro and in vivo studies. In addition, data from in vitro and in vivo studies should shape future epidemiological studies to identify stronger associations between tungsten exposure and relevant cancer biomarkers.

Multiple epidemiological studies have now found associations between urinary tungsten levels and cardiometabolic outcomes (Agarwal et al., 2011; Navas-Acien et al., 2005; Shiue and Hristova, 2014; Tyrell et al., 2013; Feng et al., 2015; Menke et al., 2016; Riseberg et al., 2021; Fox et al., 2021; Park and An, 2022). Interestingly, these studies have been conducted in human cohorts with relatively low levels of tungsten exposure. In addition, very little research has investigated the molecular mechanisms of how tungsten contributes to the pathogenesis of these diseases. Importantly, these diseases are linked so that having one often increases the risk of developing the other co-morbidities, so common mechanisms of action of tungsten could be driving all these disease states. This is a promising area for more research in the tungsten toxicology field. In particular determining what routes of tungsten exposure are associated with cardiometabolic outcomes in human populations, increasing the number of epidemiological studies evaluating cardiometabolic health outcomes in human populations exposed to high levels of tungsten, and determining other relevant co-exposures to model in experimental studies. In addition, conducting research studies in experimental models to define the molecular mechanisms of tungsten alone or in combination with other co-exposures or risk factors that contribute to disease pathogenesis is needed.

A common mechanism of tungsten toxicity and tumorigenesis that has emerged is tungsten-mediated inflammation as a potential cellular mechanism driving disease. Tungsten accumulates in both the bone and the spleen which are important immune organs (Kelly et al., 2013; Guandalini et al., 2011). Tungsten also alters both developing and mature immune cells, as well as induce inflammation in target organs. Inhalation of tungsten particles leads to pulmonary inflammation (Lasfargues et al., 1992; Roedel et al., 2013; Miller and McVeigh et al., 2021). As mentioned previously, tungsten also modulates the tumor immune response in several cancer models (Bolt et al., 2015; Kalinich et al., 2005). Importantly many of the diseases, found associated with tungsten exposure in human studies, have inflammation as a major driver of disease etiology and pathogenesis. Future studies should expand investigations on how tungsten-mediated inflammation contributes to disease pathogenesis in a diverse set of disease outcomes.

An interesting new angle in the tungsten toxicology field is recent research findings identifying that tungsten is a required cofactor for a family of tungsten-containing oxidoreductase enzymes found to be essential for the normal functioning of certain human gut microbes (Schut et a., 2021). Given how changes in the diversity and function of the gut microbiome contribute to disease pathogenesis of multiple disease, future work will be necessary to investigate how tungsten exposure alters the gut microbiome and how these alterations contribute to toxicity and disease.

List of abbreviations

CINC-1

Cytokine-induced neutrophil chemoattractant 1

CINC-3

Cytokine-induced neutrophil chemoattractant 3

CXCL10

C-X-C motif chemokine ligand 10

CRYAB

Crystallin alpha B

DNA

Deoxyribonucleic acid

EPA

Environmental Protection Agency

H2AX

H2A histone family member X

HTATIP2

HIV-1 tat interactive protein 2

IL-1β

Interleukin-1 beta

IL-7R

Interleukin-7 receptor

IVD

Intervertebral disks

IARC

International Agency for Research on Cancer

K-ras

Kirsten rat sarcoma virus

MSC

Mesenchymal stromal cell

NQO1

NAD(P)H quinone dehydrogenase 1

NHANES

National Health and Nutrition Examination Survey

NTP

National Toxicology Program

NIOSH

National Institute for Occupational Safety and Health

NMU

N-Nitroso-N-methylurea

OSHA

Occupational Safety and Health Administration

53BP1

p53-binding protein 1

Pax5

Paired box protein

PM

Particulate matter

ppm

Parts per million

ppb

Parts per billion

PPAR-γ

Peroxisome proliferator-activated receptor gamma

RANKL

Receptor activator of nuclear factor kappa-B ligand

RNA

Ribonucleic acid

ROS

Reactive oxygen species

RSV

Respiratory syncytial virus

S100A4

S100 calcium binding protein A4

SPK2

Sphingosine kinase 2

TRAP

Tartrate-resistant acid phosphatase

TNFα

Tumor necrosis factor alpha