Review of the Toxicologic and Radiologic Risks to Military Personnel from Exposures to Depleted Uranium During and After Combat (2008)

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7 Uranium Carcinogenicity and Genotoxicity Reviews of uranium compounds (ATSDR 1999; IOM 2000; Royal Society 2002) have found that their carcinogenicity in animals depends on exposure conditions and the chemical nature, particularly the solubility, of the uranium compound. Some inhalation-exposure studies of insoluble uranium oxides that remain in the lungs and pulmonary lymph nodes have shown that lung cancers develop after extended periods; other studies have shown damage to lung tissue but no evidence of neoplasia. Powdered or solid implants of metallic uranium placed in muscle tissue have also shown evidence of uranium carcinogenicity. Exposure studies of soluble uranium compounds that clear the lungs quickly, however, have not shown evidence of tumor development in the lungs or other tissues of animals. The carcinogenic potential of uranium compounds has historically been at- tributed to its radioactivity (see Chapter 6 for discussion of radiologic effects), inasmuch as studies of natural uranium with and without coexposure to radon have suggested that carcinogenicity associated with uranium exposure is due to DNA damage resulting from radiation alone. However, more recent genotoxicity findings suggest that the chemical properties of uranium might enhance its car- cinogenic potential. Nonradioactive metals, such as nickel and chromium, are well-established carcinogens in their insoluble or partially soluble forms. Whether uranium is a chemical carcinogen is critical in that potential carcino- genic effects of DU exposure determined in the Capstone Report are based solely on estimates of radiologic doses. This chapter explores the evidence of a chemical contribution to the carcinogenic potential of DU. ANIMAL CARCINOGENICITY STUDIES Natural Uranium An early study of the carcinogenicity of uranium reported that sarcomas developed in rats in which powdered metallic uranium was injected in the femo- 86 

Uranium Carcinogenicity and Genotoxicity 87 ral or pleural cavity (Hueper et al. 1952). Eleven of 33 rats with injection of ura- nium in the right femur developed sarcomas that surrounded or were in the im- mediate vicinity of uranium deposits. Six of the sarcomas produced metastases in the inguinal, abdominal, or mediastinal lymph nodes or the lungs. Sarcomas also developed at the site of intrapleural injections in two of 33 rats. The authors concluded that the results clearly established the carcinogenic nature of uranium; however, a definitive conclusion could not be reached regarding the role of the chemical vs radiologic properties of uranium in the cause of the sarcomas. The latent period and the histologic structure of the sarcomas that formed in response to the uranium injections were noted to be similar to those of sarcomas induced by injection of metallic nickel powder, and this suggested a similar chemical- based mechanism of action. Both nickel and uranium injections produced local necrotizing, hyalinizing, and fibrosing reactions, which were often associated with a local proliferation of periosteal and endosteal cancellous bone. However, the authors noted that the intensity of exposure to alpha radiation in tissue im- mediately adjacent to uranium deposits was much higher than the radiation in- tensity that occurs from uranium stored in bone after systemic exposure to solu- ble uranium. In addition, the latent period for the uranium-induced sarcomas in these experiments was similar to reported latent periods for sarcomas produced in rats by the radioactive elements radium and thorium. Thus, the basis of the powdered metallic uranium’s carcinogenic effects remained uncertain. Since the 1950s, when this study was conducted, research has suggested that local sarco- mas forming around many types of embedded metals may be due to chronic local inflammation (IARC 1999); this mode of action should be considered in determining cancer risks associated with DU exposure. Leach et al. (1970, 1973) conducted 5-y inhalation studies in monkeys, dogs, and rats with natural uranium dioxide dust about 1 µm in mass median diameter. Animals were exposed to uranium at 5 mg/m3 6 h/d 5 d/wk for up to 5 y. Evidence of neoplastic changes (pulmonary glandular neoplasms and atypical epithelial proliferation) was observed 2-6 y after exposure ceased and only in dogs. That finding was important because tumor incidence was 50-100 times higher than in controls. Evidence of pulmonary neoplasia was observed in six of 13 dogs followed for up to 6.5 y after exposure to uranium dioxide. In contrast, no pulmonary tumors or areas of atypical epithelial proliferation were observed in six monkeys followed for up to 7.5-y after exposure (Leach et al. 1973). One monkey had lymphoma that involved some tracheobronchial lymph nodes. The difference in cancer incidence between the dogs and the monkeys, the authors noted, might have been due to the different proportions of the life spans over which the dogs and monkeys were tested and observed. Leach et al. also noted that glandular neoplasms occur infrequently in humans, so quantitative extrapo- lation of the results of this study to humans would be difficult. The authors were concerned, however, that neoplasms developed in the dogs at radiation doses that were about 20-25% lower than those produced by 239Pu, leading them to propose that natural uranium might exert both chemical and radiologic effects. 

88 Risks to Military Personnel from Exposure to Depleted Uranium The carcinogenicity of uranium after chronic inhalation exposure to ura- nium-ore dust was also studied by Cross et al. (1981a) in Syrian golden hamsters exposed to carnotite uranium dust with an activity-median-aerodynamic diame- ter of 1.5-2.1 µm. At a uranium-ore concentration of 19 mg/m3, there was no evidence of neoplasia in alveolar regions of the lung in 99 animals and only one area of bronchiolar epithelial hyperplasia with squamous metaplasia after 16-27 mo of exposure. No areas of bronchial or alveolar metaplasia were observed in control animals. The primary lung lesions observed in animals exposed to ura- nium dust were inflammatory responses (macrophage accumulation) and prolif- erative responses (alveolar-cell hyperplasia and adenomatous alteration of alveo- lar epithelium). Small (statistically nonsignificant) differences in tumor incidences in other tissues were noted in the uranium-dust-exposed animals (one pheochromocytoma, one melanoma, and one adrenal-cell carcinoma) and none in the control animals. Cross et al. (1981a) also exposed hamsters to radon and radon daughters alone or in combination with uranium dust. The animals exposed to only radon and radon daughters showed higher occurrence of both bronchiolar and alveolar epithelial squamous metaplasia than controls (nine in 96 animals vs none in 82 animals, respectively). Combined exposures to uranium ore and high doses of radon and radon daughters increased the occurrence of lung epithelial squamous metaplasia (13 alveolar carcinomas and seven bronchiolar carcinomas in 101 animals). The statistical significance of that increase was not reported. In ani- mals exposed to both uranium dust and radon and radon daughters, there were two osteosarcomas and none in the control animals, four reticulum-cell sarco- mas and three in the controls, and one adrenal-cell sarcoma and zero in the con- trols. Those results suggest that radon in uranium ore is an important factor in the carcinogenicity associated with exposure to natural uranium ore. Cross et al. concluded that the hamster may not be an appropriate animal model for studying the pulmonary carcinogenic potential of uranium ore, in that evidence from other laboratories indicated that the Syrian golden hamster is highly refractory to carcinoma induction by inhaled alpha-emitting radionuclides. Mitchel et al. (1999) used a nose-only inhalation system to expose rats to natural uranium ore at two concentrations. Exposure to uranium-ore dust at 19 mg/m3 or 50 mg/m3 (containing 44% uranium and no significant radon content) took place for 4.2 h/d 5 d/wk for 65 wk. The animals were observed for their remaining lifetime. The frequency of primary malignant and nonmalignant lung tumors was higher in both exposure groups than in controls. Primary malignant tumors were observed in one of 63 control rats, 22 of 126 rats in the 19-mg/m3 group, and 20 of 61 rats in the 50-mg/m3 group. Similarly, primary nonmalig- nant tumors were found in one, 17, and eight rats of the three groups, respec- tively. There was no difference in tumor latency between the two dose groups. Calculations indicated that malignant lung-tumor frequency was more directly related to the radiation dose rate (determined from uranium concentrations in pulmonary tissues at the end of the exposure period) than to absorbed dose to the lung. Nonmalignant lung tumors correlated significantly with lower lung bur- 

Uranium Carcinogenicity and Genotoxicity 89 dens. It should be noted that the strength of evidence that inhalation of uranium dust is associated with lung cancer is weakened by the fact that the uranium- exposed rats in this study lived longer than the nonexposed animals. Taken together, those studies indicate that long-term inhalation exposure to natural uranium-ore dust can cause malignant changes in lung tissue. Results from the Mitchel et al. study suggest that inhalation of uranium ore (without substantial radon and radon-daughter content) can be carcinogenic to lung tissue after an extended period in animals. Leach et al. (1973) used a different strain of rats and did not observe lung tumors after chronic exposure to uranium dust but did observe lung tumors in dogs exposed in a similar manner. The collective findings suggest that both the chemical and radiologic properties of natural ura- nium may play a role in its carcinogenicity. Depleted Uranium Recent studies have been designed specifically to assess the carcinogenic- ity of DU under exposure conditions experienced by Gulf War soldiers who had embedded DU metal fragments. Hahn et al. (2002) conducted a carcinogenicity study of DU metal fragments embedded in the thigh muscles of rats. They used three sizes of DU containing 0.75% titanium: 1.0 × 2.0-mm pellets and 2.5 × 2.5 × 1.5-mm and 5.0 × 5.0 × 1.5-mm fragments). Tantalum metal fragments were used as negative controls, and a colloidal suspension of radioactive thorium di- oxide injected into the thigh muscle was used as a positive control. The study was designed with 50 rats per group and was extended for the lifetime of the animals. More soft-tissue sarcomas were found in the immediate vicinity of the DU metal implants than the tantalum controls; the incidence was greater near the larger implants. No significant increases in the number of benign or malignant tumors were found in any other tissues. Hahn et al. concluded that DU fragments embedded in muscle tissue are carcinogenic if they are large enough; however, the mechanism involved is un- clear. The number of tumors that developed around the DU implants was not proportional to the surface area of the implants; this suggests the absence of a foreign-body response, which is critical because rats are sensitive to foreign- body carcinogenesis (Oppenheimer et al. 1956; O’Gara and Brown 1967; Autian et al. 1975; Brand et al. 1976; Greaves et al. 1985; McGregor et al. 2000). A foreign-body mechanism of tumor formation was considered unlikely because the DU implants did not remain smooth—an important criterion for foreign- body carcinogenesis. Tantalum pellets remained smooth, whereas DU fragments became corroded and roughened. In addition, DU implants were associated with more inflammation and fibrosis than the tantalum fragments. Although it was not possible from the Hahn et al. study to determine whether the mechanism of carcinogenicity of the DU fragments involved only radiation effects, the authors suggested that the observed correlation between the number of tumors and the initial surface alpha radioactivity of the implanted 

90 Risks to Military Personnel from Exposure to Depleted Uranium materials indicated that radioactivity may have played a role. The chronic tissue damage indicated by the inflammation and fibrosis associated with the DU im- plants also may have played a role and could be a response to cytotoxic effects of uranium related to its radiation or chemical toxicity. A second study examining the development of sarcomas near embedded DU pellets in rats also found thick fibrous capsules, black granular material, and mild to moderate inflammation but no evidence of proliferation or preneoplastic changes at the implantation sites (Arfsten et al. 2007). The absence of neoplastic changes may well have been due to the short period that elapsed between im- plantation of the pellets and the end of the study (150 d). GENOTOXICITY OF URANIUM A number of in vivo and in vitro studies have demonstrated that uranium is a genotoxic metal and provided support for a mixed chemical-radiologic mechanism of uranium carcinogenicity. In Vitro Studies Using Chinese hamster ovary (CHO) cells, Lin et al. (1993) demonstrated that uranyl nitrate is genotoxic and cytotoxic. Uranium exposure decreased cell viability with a 50% inhibitory concentration (IC50) of 0.049 mM. Over a con- centration range of 0.01-0.3 mM, uranyl nitrate depressed cell-cycle kinetics and increased micronuclei, sister-chromatid exchange (SCE), and chromosomal ab- errations. More recently, in vitro studies of the carcinogenicity of DU showed that human osteoblasts exposed to DU-uranyl chloride were transformed to a tu- morigenic phenotype characterized by anchorage-independent growth and tumor formation in nude mice (Miller et al. 1998a). Transformation frequency in- creased by a factor of 9.6 (+ 2.8) in DU-exposed cells compared with 7.1 (+ 2.1) in cells exposed to nickel sulfate, a well-established metal carcinogen. DU- transformed cells also expressed high levels of the k-ras oncogene, reduced pro- duction of the Rb tumor-suppressor protein, and had increased frequencies of SCE per cell. In a followup study, Miller et al. (2003) reported genetic instabil- ity in the human osteoblast line after DU exposure manifested as delayed lethal- ity and micronuclei formation. Wise et al. (2007) showed that human bronchial fibroblasts exposed for up to 72 h to soluble uranium (as uranyl acetate) at con- centrations that decreased cell viability did not increase DNA damage above background levels but that exposure to insoluble uranium (as uranium trioxide) caused chromosomal aberrations in 15% of metaphase cells. Knobel et al. (2006) used a variety of assays to study the genotoxicity of uranyl nitrilotriacetate in nontransformed human colon cells and in a human colon adenoma cell line. Uranium caused DNA strand breaks measured by using the comet assay at concentrations that did not arrest cell growth or decrease cell 

Uranium Carcinogenicity and Genotoxicity 91 viability. Evaluation of damage induced in the gene TP53, known to be altered during carcinogenesis, showed a concentration-dependent migration of TP53 into the comet tail. Fluorescence-in-situ-hybridization (FISH) analysis for chro- mosomal aberrations showed a concentration-dependent increase in aberrant metaphases; uranium caused proportionally more deletions and fewer transloca- tions than the positive control ethylmethanesulfonate. To address the question of whether genotoxicity of uranium occurs through chemical rather than radiologic mechanisms, Miller et al. (2002a) con- ducted in vitro experiments with uranyl nitrates of different isotopic composi- tions and specific activities. The frequency of dicentric chromosomes in human osteoblasts increased in a radiation-dose-dependent manner that suggested that alpha radioactivity was responsible for at least this specific type of chromosomal aberration. The mechanism of uranium’s genotoxicity was studied by Yazzie et al. (2003) with a cell-free system. Their work demonstrated that uranyl acetate caused DNA strand breaks in the presence of ascorbate. Addition of catalase inhibited strand breaking, and this indicated that hydrogen peroxide is involved in the DNA damage; however, there was a lack of protection by hydroxyl- radical scavengers. Miller et al. (2002b) and Periyakaruppan et al. (2007) re- ported results indicating the involvement of oxygen radicals in uranium-induced DNA damage. Hamilton et al. (1997) had shown that uranyl ion in the presence of hydrogen peroxide catalyzes hydroxyl-radical–mediated oxidation. Direct evidence of a chemical-based mechanism of uranium genotoxicity was recently reported by Stearns et al. (2005), who studied CHO cells. Uranium- DNA adducts were formed under exposure conditions that increased hypoxan- thine phosphoribosyl transferase (HPRT) mutations and DNA strand breaks. The authors concluded that molecular analysis of the HPRT mutations induced by uranyl acetate exposure showed mutation spectra consistent, at least in part, with a chemical-induced effect. Uranyl acetate exposure induced more major ge- nomic rearrangements (multiexon insertions and deletions) than occurred spon- taneously. Similarities between hydrogen peroxide and uranyl acetate mutation spectra, however, suggested that oxidative DNA damage also played a role in the uranyl acetate mutagenesis (Coryell and Stearns 2006). Hartsock et al. (2007) reported evidence of a chemical mechanism by which uranium might alter DNA transcription and repair. In in vitro assays, uranyl acetate, but not sodium arsenite, inhibited the DNA-binding activity of both zinc-finger (Aart and Sp1) and non-zinc-finger (AP1 and NFκB) DNA- binding proteins. Animal Studies Evidence that DU is mutagenic, as measured by the Ames Salmonella re- version assay (strain TA98 and AmesII™ mixed strains TA7001 to 7006), was reported by Miller et al. (1998b) in a study of rats with embedded DU pellets. 

92 Risks to Military Personnel from Exposure to Depleted Uranium The gastrocnemius muscles of Sprague-Dawley rats had implants of various combinations of 20 DU and tantalum pellets. The high dose consisted of 20 im- planted DU pellets; the medium dose, 10 DU and 10 tantalum pellets; and the low dose, four DU and 16 tantalum pellets. Controls received 20 tantalum pel- lets. Urine and serum samples were collected 6, 12, or 18 mo after implantation. Urine was passed through a chromatograph column of Amberlite XAD-4 resin, and the effluent was collected and passed through a column of Amberlite XAD- 8 resin. The serum was separated by centrifugation, vacuum-dialyzed, and then tested. Both the urine samples and the serum samples were tested in the Ames assay with strain TA98 and strains TA7001-7006. The latter is made up of six individual strains in equal numbers all measuring base pair substitution. The XAD-4 urine fraction produced statistically significant increases at all periods and at all doses in the TA98 strain. For strains TA7001-7006, results were vari- able for the 6-mo period, but the 12- and 18-mo results were statistically signifi- cant for all doses. The XAD-8 fraction produced less mutagenicity, with no sig- nificant increases at 6 mo, and only the medium and high doses produced significant increases at 12 and 18 mo in TA98. In strains TA7001-7006, statisti- cally significant increases were seen at all doses and all periods. Serum did not produce significant increases in TA98 or TA7001-7006. If the standard of “a doubling of the control rate” is used to indicate a positive response, the study established that exposure to urine from rats with implants of DU increases the mutation frequency of S. typhimurium, causing frame-shift and base-pair substi- tution mutations. Monleau et al. (2006) recently reported evidence of DNA damage in bron- choalveolar lavage (BAL) cells from rats exposed by inhalation to an insoluble form of DU. Animals were exposed to uranium dioxide in an acute and repeated regimen or to uranium peroxide in an acute regimen only. The animals were killed at various times after exposure (see Table 7-1), and DNA damage was assessed in BAL cells and in the kidneys. The comet assay was used to deter- mine single- and double-strand breaks in DNA. It was conducted under alkaline and neutral pH conditions. Alkaline conditions allow detection of single- and double-strand breaks, whereas neutral conditions allow detection only of double- strand breaks. The olive tail moment was used to quantify DNA damage; this index measures the amount of DNA damage and the distance of migration of the genetic material in the tail. In BAL cells, the highest single exposure to uranium dioxide, 375 mg/m3 for 3 h, caused a significant increase in DNA breaks at days 1 and 8 after expo- sure, whereas exposures at 375 mg/m3 for 2 h and 190 mg/m3 for 30 min were without effect (alkaline condition). Repeated exposures to uranium dioxide (190 mg/m3 for 30 min 4 d/wk for 3 wk) resulted in significant DNA breaks under alkaline conditions on all days after exposure. Acute exposure to uranium perox- ide did not cause any significant increases. Under neutral pH conditions, cells with the highest acute exposure (375 mg/m3 for 3 h) and repeated exposure to uranium dioxide showed increases in double-strand DNA breaks. The authors 

Uranium Carcinogenicity and Genotoxicity 93 TABLE 7-1 Experimental Protocol for Inhalation Study Inhalation Type Duration Aerosol Concentration Euthanasia after Exposure Acute UO2 30 min 190 mg/m3 ± 41 mg/m3 4 h and 1, 3, 8 d Acute UO2 2h 375 mg/m3 ± 70 mg/m3 1, 3, 8 d 3 3 Acute UO2 3h 375 mg/m ± 70 mg/m 1, 3, 8, 14 d 3 3 Repeated UO2 30 min 4 d/wk 190 mg/m ± 41 mg/m 1, 3, 8, 14 d for 3 wk Acute UO4 30 min 116 mg/m3 ± 60 mg/m3 4 h and 1, 3, 8 d Air 30 min 4 d/wk — 1, 3, 8, 14 d for 3 wk Note: UO2, uranium dioxide; UO4, uranium peroxide. Source: Monleau et al. 2006. Reprinted with permission; copyright 2006, Oxford Univer- sity Press. concluded that uranium dioxide induces both single- and double-strand breaks in BAL cells. In renal cells, only the repeated-exposure group demonstrated a sig- nificant increase in DNA damage under alkaline conditions; the damage oc- curred 3 and 8 d after exposure. The authors also investigated the expression of genes associated with in- flammation, given that the inflammatory process may play a role in the genera- tion of reactive oxygen species and thereby cause genotoxicity. They discovered that gene expression was increased for interleukin-8 and tumor-necrosis factor alpha. They also showed increases in aqueous and lipid hydroperoxides in the lungs. The genotoxicity thus seen was attributed to a secondary result of activa- tion of reactive oxygen species. Therefore, the genotoxicity observed should have a threshold. Human Studies A number of researchers have conducted cytogenetic assessments in ura- nium-exposed populations. Martin et al. (1991) reported increases in SCE and chromosomal aberrations in lymphocytes of uranium-production workers ex- posed to a mix of soluble and insoluble uranium compounds and possibly en- riched uranium. Studies of two populations of uranium miners, however, had mixed results. Lloyd et al. (2001) reported no increased incidence in chromoso- mal aberrations in the white blood cells of Namibian uranium miners, whereas Popp et al. (2000) observed a significantly increased incidence of micronuclei in the lung macrophages of former German miners. Cytogenetic assessments of DU-exposed Gulf War veterans conducted bi- ennially since 1999 have had mixed, generally negative, results with respect to an effect of uranium exposure on SCE and chromosomal-aberration frequencies 

94 Risks to Military Personnel from Exposure to Depleted Uranium (McDiarmid et al. 2001b, 2004b, 2006, 2007). Mutations of the HPRT gene in peripheral blood lymphocytes, however, have been consistently about twice as high in veterans with higher uranium exposure than with lower uranium expo- sure as measured by urinary uranium. The correlation between HPRT mutations and urinary uranium, however, has not always been statistically significant. That may be due in part to the relatively small number of soldiers involved in each of the assessments, which ranged from 32 to 39. Chromosomal abnormalities measured with FISH analysis in 2005 provided additional evidence of a weak genotoxic effect of DU in this Gulf War cohort (McDiarmid et al. 2007). Inter- pretation of the results is difficult because the studies were not designed to de- termine whether the incidence of abnormalities was larger than expected in “normal” people; the studies were designed only to determine a difference be- tween low- and high-exposure groups and thus an association with urinary ura- nium. Consequently, there were no control cultures for comparison. SUMMARY Experimental evidence indicates that insoluble forms of uranium are weakly carcinogenic in animals. Lung cancer is the primary cancer that occurs after inhalation exposure to insoluble uranium compounds. However, it should be noted that many of the animal studies involved exposures to exceptionally high concentrations of uranium particles, so particle load could have been a fac- tor in the responses. Sarcomas have also been observed in the vicinity of in- jected or embedded uranium metal. Although in vitro studies suggest that ura- nium can have genotoxic effects because of both its chemical and radiologic nature, cytogenetic assessments in exposed human populations (uranium- production workers, uranium miners, and Gulf War DU-exposed veterans) are few and have had mixed results, most likely owing to differences in the solubil- ity and size of the uranium compounds involved. Overall, the carcinogenicity and genotoxicity results are consistent with the relatively low lung-cancer risks predicted by the Capstone study for soldiers who inhaled DU in the Gulf War friendly-fire incidents. RECOMMENDATIONS • Assessment of the risk of cancer and organ dysfunction posed by mili- tary exposure to DU is limited by lack of knowledge of the mechanisms by which uranium causes cancer and by the absence of experimental animal data from studies with exposure scenarios similar to those during the Gulf War. Ad- ditional animal studies should be performed that use DU oxide particles that are similar in size and solubility to those created by the penetration of DU armor by DU munitions; that involve a single, short (up to 1-h) inhalation exposure to DU particles at concentrations similar to those predicted in the Capstone Report (up to 10,000 mg/m3); and that extend observations over the entire lifetime of the 

Uranium Carcinogenicity and Genotoxicity 95 animals. Measured outcomes from such studies should include gross and his- tologic pathology of the lungs, pulmonary lymph nodes, and kidneys for evi- dence of fibrosis, proliferative lesions, and tumors and include tumor-frequency data based on dose and tissue uranium concentrations. Biomarkers of cytoge- netic effects (such as SCEs, micronuclei, HPRT mutations, and chromosomal abnormalities) in lymphocytes should be measured immediately after exposure and periodically thereafter. Spermatogonial cells also should be examined for chromosomal aberrations in the testes. The latter would provide information on the possible heritable nature of the aberrations. • Dust collected from inside tanks should be analyzed for other metals and chemicals that could potentially enhance the carcinogenicity of DU through such mechanisms as inhibition of DNA repair. On the basis of such analyses, it may be necessary to factor in the effects of coexposures to other carcinogens in cancer risk assessments. • The committee recommends research into whether there is a chemical mechanism of uranium carcinogenesis.