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3 Toxicology T his chapter presents information about the toxicology of uranium. Stud- ies of laboratory animals and other nonhuman systems are essential for understanding mechanisms of action, biologic plausibility, and possible health effects when experimental research in humans is not ethically or practi- cally possible (Cohrssen and Covello, 1989; NRC, 1991). Such studies permit a potentially toxic agent to be introduced under conditionsâsuch as dose, duration, and route of exposureâcontrolled by the researcher to probe health effects on many body systems. Nonhuman studies are also a valuable complement to human studies of genetic susceptibility. Although nonhuman studies often focus on one agent at a time, they enable investigation of chemical mixtures and their potential interactions more easily. Research on health effects of toxic substances includes animal studies that characterize absorption, distribution, metabolism, and elimination. Animal stud- ies may examine acute (short-term) exposures or chronic (long-term) exposures. Animal research may focus on the mechanism of action (how a toxicant exerts its deleterious effects at the cellular and molecular levels). Mechanism-of-action (or mechanistic) studies encompass an array of laboratory approaches with whole animals and with in vitro systems that use tissues or cells from humans or animals. Structure-activity relationships, in which the molecular structure and chemical and physical properties of a potential toxicant are compared with those of a known toxicant, are an important source of hypotheses about mechanism of action. Some sections of this chapter have been adapted from Gulf War and Health, Volume 1: Depleted Uranium, Pyridostigmine Bromide, Sarin, Vaccines (IOM, 2000). 23
24 updated literature review of depleted uranium In carrying out its charge, the committee used animal and other nonhu- man studies in several ways, particularly to look for markers of health effects that might be important for humans. If animal studies showed absorption and deposition in specific tissues or organs, the committee looked especially closely for possible abnormalities at these sites in human studies, as it did for uranium deposition in bone and kidney. One of the problems with animal studies, however, is the difficulty of finding animal models to study symptoms that are related to uniquely human attributes, such as cognition, purposive behavior, and the per- ception of pain. The toxic effects of uranium also have been reviewed in a recent National Research Council report, Review of Toxicologic and Radiologic Risks to Military Personnel from Exposure to Depleted Uranium During and After Combat (NRC, 2008), which assessed the US Armyâs âCapstone reportâ (USACHPPM, 2004) on toxicologic and radiologic risks to soldiers posed by exposure to depleted uranium. This chapter begins with a summary of the findings presented in Gulf War and Health, Volume 1: Depleted Uranium, Pyridostigmine Bromide, Sarin, Vac- cines (IOM, 2000), hereafter referred to as Volume 1. It next addresses experi- mental data from toxicokinetic (also called pharmacokinetic), animal, and in vitro studies published since Volume 1. The chapter ends with a discussion of how the committee applied the experimental data. Tables 3-1 to 3-10 are included at the end of this chapter. Summary of Previous Report Chapter 4 of Volume 1 includes a review of studies of the toxicology of ura- nium. It covers toxicokinetics and animal and in vitro studies. Uranium is both a heavy metal and a low-specific-activity radioactive ele- ment. Studies on the toxicity of uranium have examined both its chemical and its radiologic effects. The primary routes of human exposure to uranium are ingestion and inhalation; the effects of dermal exposure and embedded fragments have also been studied. The amount of uranium that the body absorbs depends largely on the route of exposure and the solubility of the uranium compounds to which a person is exposed. Insoluble uranium compounds may remain in the pulmonary tissues, especially the pulmonary lymph nodes, for a long time and thus pose a localized radiologic hazard. As a general rule, uranium absorption from the intestinal tract is lower than that from the respiratory tract and results in lower doses per unit intake. Renal dysfunction and lung injury are the best-characterized consequences of exposure to uranium compounds. The chemical and radiologic properties of uranium could act cooperatively to cause tissue damage, so it cannot be assumed that excess cancers would be due solely to the radiologic effects of uranium or that organ damage is due exclusively to its heavy-metal properties.
toxicology 25 Toxicokinetics The toxicokinetics of a substance has to do with the routes and rates of absorption, distribution, metabolism, and excretion. Toxicokinetics can be used to determine the amount of a substance that reaches particular organs or cells and might therefore lead to a toxic effect. For a review on biokinetic models of exposure to different forms of uranium with emphasis on depleted uranium, the reader may consult Leggett (2006). Absorption Where uranium particles are deposited in the respiratory tract is the result of a combination of physical forces that govern particle behavior in an air stream and the anatomy of the respiratory tract (Gordon and Amdur, 1991). The site of deposition affects the degree of uranium absorption, the clearance mechanisms that are available to remove uranium particles, and the severity of the conse- quences of damage of tissue of the respiratory system. Which of the various regions of the respiratory tract and lung (extrathoracic, tracheobronchial, and deep pulmonary or alveolar) inhaled uranium-dust particles are deposited in depends on the particlesâ aerodynamic diameter and inspiratory flow rate. An aerodynamic diameter that incorporates both the density and the diameter of particles and their aerodynamic drag is typically assigned to nonspheri- cal particles. It represents a particle as having the diameter of a unit-density sphere that has the same terminal velocity as the particle, whatever its size, shape, or den- sity (Gordon and Amdur, 1991). Larger particles are deposited in the tracheobron- chial region; mucociliary action transports the particles to the pharynx, where they are swallowed. Smaller particles reach the terminal bronchioles and the alveoli. The International Commission on Radiological Protection has developed extensive models of the dosimetry of inhaled radioactive materials (ICRP, 2002). At the alveolar level, the more soluble uranium compounds (categorized as type F for fast dissolution) are taken up by the systemic circulation within days. The less soluble uranium compounds (type M for medium dissolution) are likely to remain in the pulmonary tissue and associated lymph nodes for weeks. The relatively insoluble compounds (categorized as type S for slow dis- solution) are least likely to enter the systemic circulation and may remain in the lung and tracheobronchial lymph nodes for several years (ATSDR, 1999). (See Table 3-1 for examples of uranium compounds of each type.) The lungs and the tracheobronchial lymph nodes are the two major sites of accumulation for type S uranium compounds (administered as uranium dioxide) in dogs, monkeys, and rats, accounting for greater than 90% of the total body burden of uranium after inhalation of the compounds (Leach et al., 1970). Given their high density, most inhaled uranium-particleâcontaining dusts have an aerodynamic diameter that does not permit them to be carried to the
26 updated literature review of depleted uranium peripheral part of the lungs (Berlin and Rudell, 1986; Morris et al., 1992). Esti- mates based on measurements in uranium-processing plants suggest that only 1-5% of uranium-particleâcontaining dusts will enter the lungs (Davies, 1961). The rest will deposit in the upper respiratory tract and eventually be swallowed and go through the gastrointestinal tract. Inhalation studies of depleted-uranium particles in animals suggest that pat- terns of exposure may be important in the bioaccumulation of uranium (Monleau et al., 2006a,b). For example, repeated pre-exposure to insoluble depleted-Âuranium dioxide by inhalation has been shown to increase later uranium peroxide bioac- cumulation in the kidneys and femurs and decrease it in the gastrointestinal tract and excreta concurrently with enhanced genotoxic effects in several of these tis- sues (Monleau et al., 2006b). No change in uranium peroxide bioaccumulation was found in the lungs. Gastrointestinal absorption of uranium has been studied after single oral administration of soluble compounds to rats, swine, dogs, hamsters, and baboons (for review, see Thorne, 2003). The absorption of uranium in the gastrointestinal tract generally increases with increasing solubility of the compound, but only a small fraction of even the soluble uranium compounds is absorbed through the gastrointestinal epithelium. Gastrointestinal absorption was estimated at up to 5% and was highly variable between experiments. Uranium absorption occurs predominantly in the small intestine, and there is no absorption from the buccal cavity, stomach, or large intestine (Dublineau et al., 2005). The apparent uranium permeability measured with ex vivo techniques was similar in the various parts of the small intestine (Dublineau et al., 2005) and probably occurs through a transcellular pathway. The relationship between uranium speciation and gastroin- testinal absorption was investigated in rats after ingestion of five samples of water contaminated with different forms of uranium. The average fractional absorption was about 0.4% for each of the samples, so it was concluded that the chemical form of uranium in the water did not influence its absorption into the systemic circulation (Frelon et al., 2005). Peyerâs patches, the aggregated structures of gut-associated lymphoid tissue, have specific electrophysiologic characteristics of ion conductance and secretory capacity (Brayden and Baird, 1994) and have recognized sites of transport of nanoparticles, microparticles, and macromolecules (Pappo and Ermak, 1989; Powell et al., 1996). A recent quantitative analysis of uranium deposition by inductively coupled plasma-mass spectrometry (ICP-MS) after chronic exposure of rats to depleted uranium during 3 or 9 months demonstrated preferential accu- mulation of uranium in Peyerâs patches compared with epithelium (Dublineau et al., 2006a). However, the apparent uranium permeability of the rat intestine was higher (by a factor of 10) in the mucosa than in Peyerâs patches, and this suggests that the small intestinal epithelium was the preferential pathway for the transmucosal passage of uranium.
toxicology 27 As described in Volume 1, skin absorption is an effective route for the entry of soluble uranium compounds into the systemic circulation. Substantial diffusion of soluble uranium through human or mammalian intact skin has been described (de Rey et al., 1983; Lopez et al., 2000; Tymen et al., 2000; Petitot et al., 2004). Absorption of uranium via deep wounds has also been shown to depend on the solubility of the metal. Pellmar et al. (1999a) assessed distribution of uranium that was implanted in the gastrocnemius (lower leg) muscle of rats in the form of depleted-uranium pellets. A dose-dependent increase in uranium concentra- tion was noted 1 day after implantation, reaching 82.0 Â± 9.7 and 31.3 Â± 6.5 ng of uranium per gram in the kidneys and tibias, respectively, of high-dose animals. Those concentrations were about 58 and 26 times higher than seen in the same tissues of the control group. A recent study evaluated the influence of wounds on the short-term distribu- tion and excretion of uranium in rats (Petitot et al., 2007). The authors reported substantial uptake of a uranyl nitrate solution through intact rat skin within the first 6 hours of exposure. Skin excoriation increased percutaneous absorption of uranyl nitrate, and this suggested that percutaneous diffusion of uranyl nitrate depends heavily on compromised skin-barrier integrity (Petitot et al., 2007). Similar studies with other forms of uranium were not found. However, in vitro studies corroborated greater diffusion of uranium through excoriated skin than intact skin; substantial uptake of uranium through excoriated skin occurred as early as 30 minutes after exposure (Petitot et al., 2004). Transport and Biotransformation Once absorbed, uranium forms soluble complexes with bicarbonate, citrate, or proteins in the plasma (Dounce, 1949; Stevens et al., 1980; Cooper et al., 1982). Little is known about the cellular and molecular mechanisms underlying the uptake of uranium in tissues. In the kidneys, a cytotoxic fraction of uranium was found to be a phosphate complex of uranyl whose uptake is mediated by a sodium-dependent phosphate cotransporter system (Muller et al., 2006). No other information on the mechanisms of transport could be found. The role of nonspe- cific metal transporters, such as divalent metal transporter-1, in the transport of uranium has yet to be defined. Distribution The percentages of uranium absorbed into blood, transferred to tissues, and excreted in urine are independent of the deposition of soluble uranium compounds, such as uranium peroxide or uranium tetrafluoride dust, in the lungs (Houpert et al., 1999). The ratio of K to (K + U), where K equals the percentage of uranium retained in the kidneys and U equals the percentage excreted in urine 24 hours after
28 updated literature review of depleted uranium instillation, may be used to characterize kidney clearance of uranium. The ratio was constant when the concentration of uranium, in the form of the two compounds mentioned, in the kidneys increased from 0.02 to 12.5 Âµg/g. Inhaled uranium accumulates readily in the central nervous system (Monleau et al., 2005). Repeated exposure (4 days/week for 3 weeks) to depleted-uranium dioxide at a high air concentration (197 mg/m3) has been associated with ura- nium accumulation in the following rank order: olfactory bulb > hippocampus > frontal cortex > cerebellum. Accumulation in brain regions appears to be route- d Â ependent: injection of uranium results in homogeneous distribution in various brain regions, whereas inhalation and ingestion result in heterogeneous and specific accumulation (Houpert et al., 2007c). Those differences were thought to reflect differential mechanisms of delivery of uranium to the brain; however, the nature of the transporters remains unknown. Lemercier and colleagues (2003) demonstrated transfer of uranium across the blood-brain barrier in an in situ rat brain perfusion study in which brain uranium was measured with ICP-MS. In chronic exposure to uranium, tissue deposition does not appear linear (Paquet et al., 2006). For example, Tracy et al. (1992) showed that uranium concentrations in rat femurs were 6.3 times higher after 28 days of ingestion of uranyl nitrate in drinking water than after 91 days of ingestion. In the case for the kidneys, uranium concentration increased by a factor of 1.8 from 28 to 91 days. Similarly, in Sprague Dawley rats that had implanted depleted-uranium pellets, Pellmar et al. (1999a) showed that uranium concentrations in kidneys peaked 6 months after exposure began and then decreased by a factor of 1.4-1.6 until 18 months of exposure. That pattern of accumulation was also observed in urine, in which a peak uranium concentration was noted at 12 months of exposure. Similarly, rats exposed to uranyl nitrate via drinking water during their entire adult life at a constant concentration of 40 mg/L showed fluctuations in uranium concentration in almost every tissue (Paquet et al., 2006); this suggested that accumulation of uranium in tissues may vary over the course of chronic expo- sure. Thus, chronic exposure may be associated with physiologic phenomena that modify the pharmacokinetics of uranium over time. Excretion and Retention No new studies were identified on systemic clearance or excretion and retention of uranium after inhalation or oral exposure. As described in Volume 1, Pellmar and colleagues (1999a) reported that bone and kidneys were the primary reservoirs of uranium that had dissolved from embedded depleted-uranium frag- ments. Dissolved uranium also localized in various nuclei of the brain, lymph nodes, testes, and spleen, and low serum concentrations of uranium were noted at all times of measurement whereas the size of the pellets diminished with time (Pellmar et al., 1999a; Fitsanakis et al., 2006). Adult male and female rats with surgically implanted depleted uranium were shown to excrete uranium in urine in
toxicology 29 a dose-dependent manner, but tissue concentrations of uranium were not reported (Arfsten et al., 2005). In mice, 60 days after implantation of depleted uranium, the highest concentration of uranium was noted in the kidneys, but bone marrow, hind limbs, and spleen also had substantial increases in uranium (Miller et al., 2005). Those studies establish that depleted uranium from implanted fragments will readily distribute to various tissues over the life span of an animal. Additional details from multiple studies on the accumulation of uranium in the brain are presented below in the section on âNonmalignant Neurologic Effects.â Toxicity Studies This section reviews key animal and in vitro studies of the toxic effects of uranium published since Volume 1. Studies of cancer and noncancer health end points have used inhalation and oral and dermal exposure. There are also studies of the effects of injected uranium and embedded depleted-uranium fragments. Kathren and Burklin (2008) have proposed a median lethal dose for acute oral intake of uranium in humans of 5.0 g and for acute inhalation of soluble uranium compounds of 1.0 g. Carcinogenic Effects Four studies of the carcinogenic effects of uranium were described in Volume 1; two reported positive findings (Leach et al. 1973; Filippova et al., 1978). Since the publication of Volume 1, three studies were found that examined cancer in depleted-uraniumâexposed animals. The experimental details are presented in Table 3-2. In the first study, rats received thigh-muscle implants of depleted-uranium pellets or fragments and were held for their life span (Hahn et al., 2002). At death, necropsies and histopathologic examinations were conducted. A statistically sig- nificant increase in the incidence of soft-tissue sarcoma at the implantation site was reported in rats that received 5.0 Ã 5.0-mm squares of depleted uranium and a slight increase in rats that received 2.5 Ã 2.5-mm squares. No tumors were observed in the rats that received 2.0 Ã 1.0-mm depleted-uranium pellets. The second study used an in vivo leukemogenesis mouse model (Miller et al., 2005). Mice received implants two to eight depleted-uranium pellets in the gastrocnemius muscle, and 60 days later received intravenous injections of murine multicolony-stimulation-factorâdependent hematopoietic cells. Leukemia developed in 68-75% of the mice that received depleted-uranium implants and 12% of the mice that had no implants. In the third study, Mitchel et al. (1999) exposed rats by nose-only inhalation 4.2 hours/day 5 days per week for 65 weeks to natural uranium-ore dust aerosol (44% uranium) in the absence of substantial radon content at 50 or 19 mg/m3. Lung uranium burdens, determined at the time of death, decreased exponentially
30 updated literature review of depleted uranium after cessation of exposure independently of the initial burden. The frequency of primary malignant lung tumors was dose-dependent: 0.016, 0.175, and 0.328 in the control, low-aerosol, and high-aerosol groups, respectively. The groups were indistinguishable with respect to tumor latency. The average radiation doses received at the low and high dust-aerosol concentrations were 0.87 and 1.64 Gy, respectively, and resulted in an average risk of malignant lung tumors of about 0.20 tumor per animal per gray in both exposure groups. The tumor frequency was not directly proportional to the dose; but when malignant lung-tumor frequency was calculated as a function of dose rate (measured on the basis of lung burden at the end of dust inhalation), a direct linear relationship was noted and suggested that radiation dose rate may be a more important determinant of lung-cancer risk than absolute chemical exposure. The authors concluded that chronic inhalation of natural uranium-ore dust alone in rats creates a risk of primary malignant lung- tumor formation. The study also provided details on urinalysis, which was carried out once per month throughout the animalsâ lifetime. The results demonstrated a constant urinary concentration of uranium throughout weeks 13-55 of exposure, with average concentrations of 0.117 and 0.274 mg/L in the low-uranium and high-uranium groups, respectively. Mitchel et al. (1999) also assessed the effects of inhaled uranium on the incidence of nonmalignant lung tumors in rats. They exposed rats to one of two concentrations of natural uranium-ore dust aerosol by nose-only inhalation for 4.2 hours/day 5 days per week for 65 weeks. The propor- tion of animals with nonmalignant lung tumors was 0.016, 0.135, and 0.131 in the control, low-aerosol, and high-aerosol groups, respectively. Genotoxic Effects Volume 1 describes two genotoxicity studies (Miller et al., 1998a,b), both of which reported positive findings. A number of studies have been conducted since Volume 1 on genotoxic effects of depleted uranium in humans. In a 10-year postwar followup assessment, 13 Gulf War veterans with high concentrations of depleted uranium from embedded fragments had a statistically significantly higher incidence of chromosomal aberrations in their peripheral blood lymphocytes than 26 in the low-exposure group (McDiarmid et al., 2004). However, no significant difference in chromosomal aberrations was reported between high-exposure and low-exposure groups in the 8-, 12-, and 14-year postwar followup assessments (McDiarmid et al., 2001; McDiarmid, June 28, 2007, presentation to the com- mittee). Hypoxanthine-guanine phosphoribosyl transferase mutation frequencies were nonsignificantly higher in the high-exposure group than in the low-exposure group (McDiarmid, June 28, 2007, presentation to the committee). An increase in chromosomal aberrations in peripheral blood lymphocytes was reported in a cohort of 69 people in southern Serbia and Montenegro who were exposed to depleted uranium during air strikes in 1999 (Milacic et al., 2004). Statistically significant increases in micronuclei frequencies in peripheral blood lymphocytes
toxicology 31 were found in 30 people who lived near Sarajevo (Krunic et al., 2005). Urinalysis to determine body burden of uranium was not conducted on that cohort, but water from two local wells contained traces of depleted uranium. As detailed in Table 3-3, uranium-induced genotoxicity was demonstrated in a number of studies of rats; human, hamster, and rat cells; and calf thymus DNA. Respiratory Effects Six toxicologic studies of respiratory effects in several animal species are described in Volume 1; their results were inconsistent. Several in vitro studies of the effects of uranium on lung epithelial cells and macrophages have been published recently; details are presented in Table 3-4. In rat lung epithelial cells, treatment with uranyl (VI) acetate was associated with increased oxidative stress and decreased cell proliferation (Periyakaruppan et al., 2007). The authors attributed the decrease in cell proliferation to loss of total cellular redox potential due mainly to depletion of the tripeptide glutathione and superoxide dismutase. In light of uraniumâs ability to induce pulmonary fibrosis, which is often associated with inflammation, Gazin et al. (2004) evaluated the effects of uranium on cytokine secretionâtumor-necrosis factor-alpha (TNF-alpha), interleukin- 1beta (IL-1beta), and IL-10âin alveolar macrophages. TNF-alpha secretion was increased by exposure to uranium but not by exposure to the metallic ele- ment gadolinium. Uranium-treated and control cells were indistinguishable with respect to IL-1beta and IL-10 secretions. In another study, a 48-hour exposure of a human type II epithelial cell line (A549) to 0.5 mM uranyl bicarbonate solution triggered differential expression of cytokeratin 8 (CK8) and CK8 fragments; this suggested dysfunction of the ubiquitin-proteasome system or a regulator pathway involving CK ubiquitinyl- ation (Malard et al., 2005). Renal Effects It is well established that uranium causes low-level metallotoxic effects on the renal system in animals (IOM, 2000). In general, renal injury occurs within days of exposure and is manifested as a change in the proximal convoluted tubules, which results in increased urinary enzyme excretion (excretion of alka- line phosphatase, lactate dehydrogenase, and leucine aminopeptidase). Hyaline casts (casts containing necrotic cells shed from the tubular epithelium) are present at all levels of the tubular system (Berlin and Rudell, 1986). Glomerular changes occur in parallel with tubular damage, principally in the basement membranes of glomerular capillaries. The corresponding functional changes in the kidney are proteinuria, impairment of p-aminohippurate clearance, increase in clearance of amino acids and glucose, and decrease in sodium reabsorption. After severe
32 updated literature review of depleted uranium damage, renal inulin and creatinine clearance decreases (Stopps and Todd, 1982). Generally, if the uranium dose is sublethal, regeneration of the damaged epithe- lium commences within 2-3 days of the end of exposure (Stopps and Todd, 1982; Berlin and Rudell, 1986; Gilman et al., 1998a; ATSDR, 1999). Renal injury was not observed in dogs and monkeys exposed for 5 years to inhaled dust that con- tained insoluble uranium dioxide at a uranium concentration of 5 mg/m3 (Leach et al., 1970). After a 91-day exposure to uranyl nitrate hexahydrate in drinking water at 0.96, 4.8, 24, 120, or 600 mg/L, histopathologic lesions were observed in the kidneys of male and female New Zealand white rabbits in all groups, including the lowest-exposure groups (Gilman et al., 1998b). Pathologic changes included lesions of tubular epithelial cells (apical nuclear displacement and vesiculation, cytoplasmic vacuolation, and dilation), glomeruli (capsular sclerosis), and renal interstitium (reticulin sclerosis and lymphoid cuffing). Studies of dermal and ocular absorption of uranium trioxide in rabbits indicated that uranium was suf- ficiently well absorbed to cause renal damage and even death from renal failure (Voegtlin and Hodge, 1949). Several mechanisms may account for uranium-induced renal damage. A mech- anism involving bicarbonate activity in the kidney has been postulated. Uranium combines with bicarbonate, citrate, or plasma proteins in blood. At low pH, the bicarbonate-uranyl and citrate-uranyl complexes split (Bassett et al., 1948), and the resulting uranyl ion may combine with proteins on the tubular wall and cause renal damage. A second possibility is that uranium compounds inhibit Â mitochondrial oxidative phosphorylation and sodium-dependent and sodium-Âindependent adenosine triphosphate (ATP) use in renal tubules (Brady et al., 1989). Few animal and in vitro studies of renal effects of depleted uranium have been conducted since Volume 1 (see Table 3-5). In one study, concentrations of N-acetyl-Î²-D-glucosaminidase and creatinine in rats given depleted uranium nitrate by a single intramuscular injection peaked 3 days later, and there was a high correlation between injected dose and those concentrations (Fukuda et al., 2006). Depleted-uranium concentrations in urine decreased rapidly for the first 3 days after exposure. Another study reported a decrease in glucose transport in brush-border membrane vesicles of rats given depleted uranium in the form of uranyl acetate (Goldman et al., 2006). Donnadieu-Claraz et al. (2007) exposed rats to uranium nitrate at 40 mg/L of water for up to 18 months and observed that the proximal tubular cells had more vesicles with dense granular inclusions. The granules were found to be iron oxides; uranium was not associated with them. The authors suggested that the mechanisms of iron homeostasis in the kidneys could be affected by chronic uranium exposure. Neurologic Effects The earlier animal studies considered in Volume 1 indicated that uranium crosses the blood-brain barrier and deposits within the brain parenchyma. In this
toxicology 33 volume, several relevant in vitro studies will be discussed in addition to animal studies on neurological effects published since Volume 1. Study details can be found in Table 3-6. In Vitro Models to Assess Neurologic Effects Lemercier and colleagues (2003) demonstrated transfer of uranium across the blood-brain barrier in an in situ rat brain perfusion study in which brain uranium was measured with ICP-MS. They found that substantial uranium accu- mulated in the brain and that the transport was efficient and rapid, with uranium localized to the brain parenchyma as early as 2 minutes after the initial perfusion. The nature of the uranium transporter is unknown. No functional end points were assessed in the study. One of the earliest studies of the specific effects of uranium on functional end points in the nervous system focused on the presynaptic action in phrenic nerve preparations from mice. This in vitro study demonstrated that uranyl nitrate at very high concentrations (0.2-0.8 mM) facilitated the release of acetylcholine from the nerve terminals and potentiated muscle contraction (Lin et al., 1988). The cytotoxicity of depleted uranium was investigated in an in vitro model of the blood-brain barrier with rat brain endothelial cells (RBE4 cells). The cells were derived from rat brain microvascular endothelial cells immortalized with the plasmid pE1A-neo that contained the E1A region of adenovirus 2 and a neomycin-resistance gene. Cytotoxicity was evaluated with assays for cell- volume increase, heat-shock protein 90 expression, 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) reduction, and lactate dehydrogenase (LDH) activity. The results of the assays showed that uptake of the triuranium octaoxide uranyl chloride form of depleted uranium into RBE4 cells is efficient, but no overt cytotoxicity was detected by common biomarkers (Dobson et al., 2006). In vitro studies in rat cortical neuron cultures exposed to uranyl acetate showed little cytotoxicity at concentrations below 100 ÂµM (Jiang et al., 2007). There were no statistically significant changes in F2-isoprostanes, biomarkers of oxidative stress, or thiol metabolites. The lack of cytotoxicity was corroborated with the MTT-reduction and LDH-activity assays and the finding of only mini- mal changes in total adenosine nucleotides. Additional studies in Caenorhabditis elegans using green fluorescent protein reporter worm strains corroborated the primary neuron culture observations, showing no statistically significant neuronal degeneration after uranium exposure (Jiang et al., 2007). Although in vitro models may not recapitulate human health and disease fully, those focused studies indicate that cultured neurons can tolerate high con- centrations of uranyl acetate without important oxidative injury or death (Dobson et al., 2006; Jiang et al., 2007). The studies have examined neuronal lethality after a relatively acute exposure, but impairment of neuronal function may occur in the absence of acute lethality, and effects might arise after longer exposure. Other
34 updated literature review of depleted uranium types of brain cells, such as astrocytes, may be more sensitive to uranium, but this seems unlikely, given their greater redox potential and sensitivity to oxidative stressors compared with neurons and endothelial cells used in the test systems discussed above (Dringen and Hirrlinger, 2003). In Vivo Studies to Assess Neurologic Effects Abou-Donia et al. (2002) investigated the effects of uranyl acetate on senso- rimotor behavior, generation of nitric oxide, and the central cholinergic system of rats. Intramuscular injection of uranyl acetate at 0.1 and 1.0 mg/kg for 7 days daily was followed by a 30-day observation period. On cessation of treatment, the sensorimotor functions of the animals were evaluated with a battery of tests that included measurements of postural reflexes, limb placing, orientation to vibrissa touch, grip time, beam walking, and inclined-plane performance. Treatment with uranyl acetate was associated with dose-dependent deficits in inclined-plane per- formance, beam-walk score, and beam-walk time. Changes in nitric oxide were inconsistent, increasing in cortex and midbrain and decreasing in brainstem and cerebellum at both doses. Acetylcholinesterase (AChE) activity in the cortex, but not other brain regions, of the animals given the high dosage was statistically significantly increased. Ligand-binding densities for the M2 muscarinic receptor did not show any change. The acute and chronic consequences of exposure to depleted-uranium frag- ments in animal models have been addressed by a number of groups (Gilman et al., 1998a,c; Pellmar et al., 1999a,b; Barber et al., 2005; Houpert et al., 2005; Lestaevel et al., 2005b; Monleau et al., 2005; Fitsanakis et al., 2006). Volume 1 described studies conducted by Pellmar et al. (1999a) to establish an animal model providing insight into the injuries sustained by Gulf War veterans from embedded depleted-uranium fragments and to evaluate the biologic effects of intramuscularly embedded depleted-uranium fragments. Pellmar (1999a) sug- gested that uranium can accumulate in the rat central nervous system. An earlier study by Pellmar (1997) suggested that retained depleted-uranium fragments in the leg muscle are associated with increased brain uranium concentrations. Studies by Fitsanakis et al. (2006) corroborated the earlier observations by Pellmar et al. (1999a). A similar model of surgical intramuscular implantation randomly assigned rats to five groups: nonsurgical control (NS control); no depleted-uranium pellets and 20 tantalum (Ta) pellets (sham); four depleted- uranium pellets and 16 Ta pellets (low); 10 depleted-uranium pellets and 10 Ta pellets (medium); and 20 depleted-uranium pellets and no Ta pellets (high). Uranium content was measured in digested samples as 238U with high-resolution ICP-MS. Three months after implantation, depleted uranium had accumulated substantially in the high group in all brain regions except the hippocampus. By 6 months, however, substantial accumulation was measured only in the cortex, midbrain, and cerebellum in the medium and high groups.
toxicology 35 Barber et al. (2005) studied rats treated with single intraperitoneal injections of uranium as uranyl acetate at 1 mg/kg to determine the temporal and regional distribution of depleted uranium in the brain, as measured with ICP-MS. In agree- ment with studies of Pellmar et al. (1999a,b) and Fitsanakis et al. (2006), uranium readily accumulated in the brain. At 24 hours after exposure, it was concentrated in the hippocampus, striatum, cerebellum, and cortex. Prior exposure to stress (five daily episodes of forced swimming) statistically significantly reduced hip- pocampal and cerebellar uranium then and tended to reduce uranium in all brain regions 7 days after exposure. The authors did not discuss the reason for the increased clearance or allude to the effects of uranium on functional end points in this acute-exposure model. Functional consequences of exposure of rats to depleted uranium in drinking water were addressed by Briner and Murray (2005), who found that depleted- uranium exposure was associated with short-term and long-term differences in brain lipid oxidation and open-field behavior. After 2 weeks of exposure to depleted uranium, brain lipid oxidation, measured with the thiobarbituric acid assay, was increased and correlated with increases in line-crossing and rearing behavior. Although the open-field behavior differences were sustained after 6 months of exposure, brain lipid oxidation did not correlate with the behavioral changes at that time. Male rats appeared to be more sensitive than female rats to the behavioral effects of depleted uranium. Monleau et al. (2005) exposed rats to depleted-uranium dioxide by inhalation (depleted uranium at 197 mg/m3) for 30 minutes a day 4 days a week for 3 weeks. They observed greater spontaneous locomotion activity in treated rats than in controls on the first day after the end of the exposure period and attenuated spatial working memory on the sixth day after exposure. Houpert et al. (2007b) noted decrements in performance on open-field, Y-maze, and elevated plus-maze tests in 2-, 5-, and 9-month-old rats exposed to enriched uranium nitrate (40 mg/L) during gestation and lactation. The effects were consistent with decreased spatial working memory and mimicked the effects in adult rats (Monleau et al., 2005). Additional effects included delayed hyperactivity in the uranium-exposed rats. Exposure to 4% enriched uranium or depleted uranium in drinking water for 1.5 months resulted in substantial accumulation of uranium in the hippocampus, hypothalamus, and striatum of rats, and the uranium concentrations were consis- tently 1.5-2 times higher in the hippocampus, hypothalamus, and adrenal of rats exposed to enriched uranium than of rats exposed to depleted uranium or control rats (Houpert et al., 2005). Increased brain uranium concentrations were associ- ated with a statistically significant increase in the amount of paradoxic sleep, a reduction in spatial working memory capacities, and an increase in anxiety. Sleep-wake cycle disturbances characterized by an increase in rapid eye move- ment (REM) sleep and theta-band power during the light period were also noted in the rats as early as 30 days after exposure to depleted uranium in drinking water at 40 mg/L (Lestaevel et al., 2005a). A large variety of neuronal structures
36 updated literature review of depleted uranium and substances, including neurotransmitters and peptides, are known to modulate REM sleep; the underlying mechanisms for these changes remain unknown. Using a similar exposure paradigm (1-month exposure to depleted ura- nium at 40 mg/L in drinking water), the same group of investigators examined dopamine and serotonin brain metabolism in the rat (Houpert et al., 2004). No statistically significant differences were found in dopamine, serotonin, and their catabolite levels in the striatum, hippocampus, cerebral cortex, thalamus, or cerebellum between depleted uranium-exposed and control rats. Thus, it appears that depleted uranium-induced changes in the sleep-wake cycle are not medi- ated by dopamine and serotonin. Other neurotransmitters might be involved, or, given the role of the hypothalamic-pituitary axis in sleep regulation, these effects may be modulated by glucocorticoids. Bussy et al. (2006) investigated effects of exposure to depleted uranium (as uranyl nitrate) in drinking water at 40 mg/L for up to 9 months on dopaminergic and serotoninergic metabolism in rats and found subtle and transient perturbations of monoamine concentrations and AChE activity in discrete brain areas. Biologic Plausibility Collectively, the results of those studies indicate that depleted uranium is a toxicant that can cross the blood-brain barrier and might produce some acute and prolonged behavioral changes. The mechanisms associated with the effects are difficult to reconcile, given the differences in exposure models and apparent contradictions in the results: some studies established a treatment effect and oth- ers failed to. Although at high concentrations different forms of uranium might be associated with some subtle neurologic dysfunction, the significance of the observations is unknown. Gastrointestinal Effects Absorption of ingested uranium occurs mainly in the small intestine (ICRP, 1979). The chemical form of uranium in ingested water does not appear to influ- ence absorption from the gastrointestinal tract in rats (Frelon et al., 2005). No animal or in vitro studies on the effects of uranium-induced pathology in the gastrointestinal tract could be found since those described in Volume 1. As sum- marized in Volume 1, studies have suggested that up to 2-year exposure of various animal species to high doses of uranium nitrate is not associated with gastrointes- tinal effects. Studies that used other forms of uranium could not be found. Hepatotoxicity Uranium-induced hepatotoxicity has not been a prominent finding in most animal studies (ATSDR, 1999). A few studies of hepatotoxicity have been pub- lished since Volume 1; the experimental details are summarized in Table 3-7.
toxicology 37 In adult male zebrafish exposed to exceedingly high depleted-uranium con- centrations in water, increased hepatic oxidative stress was observed (Barillet et al., 2007) in association with decreases in superoxide dismutase and catalase activity and in total glutathione content. Exposure of rats to high concentrations of depleted uranium (11.5 mg/kg) by subcutaneous administration altered hepatic metabolism of bile acids and xeno- biotics through modulation of cytochrome P450 (CYP) isoenzymes (Gueguen et al., 2006). Subtle effects have been reported in rats exposed for 9 months to depleted uranium in drinking water and then treated with acetaminophen (N-acetyl-p- aminophenol, APAP) (Gueguen et al., 2007). Plasma concentration of APAP was higher and hepatic CYP activities were lower in the group exposed to depleted uranium than in controls. Furthermore, APAP treatment in the depleted-uranium rats was associated with a more rapid increase in plasma alanine aminotransferase and aspartate aminotransferase. Using a similar exposure paradigm, the same group (Souidi et al., 2005) addressed the effects of 9 months of exposure to depleted uranium in drinking water on drug-metabolizing enzymes. Hepatic CYP3A1 and CYP3A2 mRNA expression was statistically significantly higher in rats exposed to depleted ura- nium than in controls, but CYP1A1 mRNA expression was not different. Nuclear pregnane X receptor (PXR) mRNA increased, but 9-cis-retinoic acid receptor mRNA was unchanged in the course of the study. Hepatic activity of CYP2C, CYP3A, CYP2A, or CYP2B remained indistinguishable in the depleted-uranium group compared with the controls. The authors hypothesized that uranium may affect the expression of drug-metabolizing CYP enzymes through the PXR and constitutive androstane receptor and thus potentially interfere with the metabo- lism of xenobiotics. Reproductive and Developmental Effects Volume 1 reported that modest testing of mice orally exposed to uranium did not establish association with reproductive or developmental problems but that no animal studies had used dermal or inhalation exposure. Developmental effects of depleted uranium are of particular interest because developing animals are known to be sensitive to the effects of metals (Briner, 2007). There is some recent evidence that exposure of animals to uranium compounds, including depleted uranium, during development can lead to a variety of adverse effects (recently reviewed in McClain and Miller, 2007, and Briner, 2007). Several stud- ies published since Volume 1 are summarized in Table 3-8. Subcutaneous injection of uranyl acetate dehydrate at 0.415 or 0.830 mg/kg per day in rats on gestation days 6-15 led to reduced fetal body weight and an increase in the total number of skeletally affected fetuses (Albina et al., 2003). Arfsten et al. (2005, 2006) reported some results of a large multigeneration reproductive study in which 12 pellets composed of depleted uranium, tantalum,
38 updated literature review of depleted uranium or steel in various combinations were implanted in the gastrocnemius muscles of nine groups of 21 male and 21 female rats. The parental generation was mated 30 or 120 days after implantation. The depleted uranium did not adversely affect reproduction in the parental generation with respect to effects on male reproduc- tive success, sperm concentration, and sperm velocity (Arfsten et al., 2006), and no increased incidence of developmental effects (birth weight, survival, litter size, gross physical abnormalities, neurodevelopmental effects, and immune function effects) was identified in the offspring of the mating at 30 days after implantation (Arfsten et al., 2005). Briner and Byrd (2000) reported that offspring of female mice exposed to uranium acetate at up to 75 mg/L in drinking water for 2 weeks before mating and during gestation and lactation showed more rapid development on some behavioral tests whereas their ratio of brain weight to body weight was statisti- cally significantly lower than in controls. However, the quickened development of uranium-exposed offspring may adversely affect the development of neural systems (Briner, 2007). In contrast, decrements in performance on open-field, Y-maze, and elevated plus-maze tests were noted in 2-, 5-, and 9-month-old rats whose mothers had been exposed to enriched uranium nitrate at 40 mg/L in drinking water for 3 months before mating and during gestation and lactation (Houpert et al., 2007b). Raymond-Whish et al. (2007) recently addressed the effects of uranium hexahydrate in intact, ovariectomized, or pregnant mice at 0.5 Âµg/L (0.001 ÂµM) to 28 mg/L (120 ÂµM) in drinking water. The study was conducted to assess whether uranium added to the drinking water causes responses in the female mouse reproductive tract that might be accounted for by inherent estrogenic effects of uranium. Increased uterine weight and uterine luminal epithelial cell growth, selective reduction in ovarian primary follicles but increase in growing follicles, accelerated vaginal opening, and persistent presence of cornified vaginal cells were noted in mice that drank uranium, and these effects could be attenuated by coadministration of an antiestrogenic compound (ICI 182,780). Transplacental exposure to uranium was associated with fewer primordial follicles in developing pup ovaries. The estrogenic responses noted above occurred at or below the US Environmental Protection Agency safe drinking-water concentration of 30 Âµg/L (0.126 mM) (EPA, 2006). Uranium concentrations in blood and urine were not reported in the study, so it is difficult to extrapolate the results to humans. Immune System Effects Immunologic effects were not addressed in Volume 1, but several experimen- tal studies of toxic effects of uranium on the immune system have been published. They are presented below and in Table 3-9. In vitro studies have investigated the effects of depleted uranium on cells of the immune system. One addressed the effects of depleted ura-
toxicology 39 nium (as uranyl nitrate) on viability and immune function and on cytokine gene expression in murine peritoneal macrophages and splenic CD4+ T cells (Wan et al., 2006). Depleted uranium was shown to affect signal- transduction pathways (c-jun and NF-kappa Bp65), neurotrophic factors (Mdk), chemokine and chemokine receptors (TECK/CCL25), and IL-10 and IL-5 concentrations (Wan et al., 2006). Depleted uranium also led to apoptosis in mac- rophages and CD4+ T cells (Kalinich et al., 2002; Wan et al., 2006). However, those effects were manifested at uranium concentrations exceeding 100 ÂµM, so the relevance of the findings to in vivo scenarios is uncertain. Increased spleen and lymphoid tissue uranium concentrations have been noted after chronic inhalation of natural uranium in adult rats (Leach et al., 1970, 1973), and depleted uranium has also been shown to accumulate in rat immune organs (Pellmar et al., 1999a), but there is little and contradictory information on the in vivo effects of uranium in general and depleted uranium in particular on immune function in animals. Results of the multigeneration reproductive study discussed above show that in offspring of adult rats that had implanted depleted-uranium pellets there are no statistically significant effects on immune function (Arfsten et al., 2005). Mean thymus and spleen weights and the mean total number of thymocytes per spleen were indistinguishable from the values in a control group. No effects on the intestinal localization and density of neutrophils, helper T lymphocytes, and cytotoxic T lymphocytes were observed 1-3 days after gavage treatment of rats with depleted uranium at 204 mg/kg despite modulation of cytokine (interferon-gamma [IFN-gamma]) and chemokine (MCP-1) expres- sion (Dublineau et al., 2006b). In contrast, chronic exposure to depleted ura- nium in drinking water at 40 mg/L for 3, 6, or 9 months was associated with decreased intestinal mast-cell number, increased IL-1beta and IL-10 concentra- tions, decreased mRNA CCL-2 concentrations, decreased intestinal macrophage density, and increased numbers of neutrophils (Dublineau et al., 2007). The same group (Dublineau et al., 2006a) found no change in cytokine expression patterns (IL-10, transforming growth factor-beta, IFN-gamma, TNF-alpha, and monocyte chemoattractant protein-1) in the Peyerâs patches from intestines of rats similarly exposed to depleted uranium in drinking water. Increases in inflammatory cytokine expression and production of hydroperox- ides in lung tissue from rats indicated that the genotoxic damage may be a result of the inflammatory processes and oxidative stress (Monleau et al., 2006c). Cardiovascular Effects No additional animal or in vitro studies of the cardiovascular effects of ura- nium have been identified since Volume 1. As summarized in Volume 1, studies of high doses of uranium in several animal models suggested that the cardiovascular system is not a sensitive target for this metal.
40 updated literature review of depleted uranium Dermal Effects No additional animal or in vitro studies of dermal effects of uranium have been identified since Volume 1. In several studies reviewed in Volume 1, dermal application of uranium compounds was associated with mild skin irritation, severe dermal ulcers, or superficial coagulation necrosis and inflammation of the epidermis in rabbits and swollen and vacuolated epidermal cells and damage to hair follicles and sebaceous glands in rats. Inhalation and oral exposures to uranium compounds have not led to dermal effects in animals. Ocular Effects No additional animal or in vitro studies of ocular effects of uranium have been identified since Volume 1. Only two studies were reviewed in Volume 1; they reported encrusted eyes and conjunctivitis in animals after direct contact of the eye with uranium aerosol or vapor. Musculoskeletal Effects The effects of inhaled uranium on the musculoskeletal system of animals have not been examined. Studies with intravenously administered uranium in the late 1940s were the first to establish uraniumâs high affinity for bone. About 20-30% of intravenously administered uranium could be found in bone within 2.5 hours of administration, and 90% of the uranium retained in the body 40 days after injection was in bone (Neuman et al., 1948a). Greater amounts of uranium were incorporated in bones of young rats and calcium-deficient mature rats than normal mature rats (Neuman et al., 1948b). Uranium was specifically incorporated in areas of active calcification; the areas of uranium deposition became refractory to resorption as new calcification covered them (Neuman and Neuman, 1948). There were no histopathologic findings in rat or rabbit muscles after expo- sure to orally administered uranyl nitrate in drinking water at uranium concentra- tions up to 40 mg/kg per day for 28 day or up to 53 mg/kg per day for 91 days in Sprague-Dawley rats or up to 53 mg/kg per day for 91 days in rabbits (Gilman et al., 1998a,b). However, acute uranium intoxication in suckling rats given [238U]uranyl nitrate at a uranium concentration of 2 mg/kg of body weight intra- peritoneally has been shown to inhibit bone formation and mandibular growth; this effect is believed to be due to the direct action of uranium on bone-forming cells or their precursors (Guglielmotti et al., 1985; Ubios et al., 1998). Inhibi- tion of bone formation by uranium has been shown in endochondral ossification (Guglielmotti et al., 1984), alveolar bone healing (Guglielmotti et al., 1985, 1987), and alveolar bone modeling and remodeling (Ubios et al., 1990). Bone biochemical markersâsuch as osteocalcin, tartrate-resistance acid phosphatase,
toxicology 41 pyridinoline, and rat parathyroid hormoneâwere increased 28 days after rats received single intramuscular injections of depleted uranium and indicated bone damage (Fukuda et al., 2006). That study and other recent studies of musculosk- eletal effects of uranium exposure are detailed in Table 3-10. Pujadas Bigi and Ubios (2007) noted that after a single injection of uranyl nitrate in 1-day-old rats there was statistically significant but transitory inhibition of tooth eruption, dental development, and mandibular growth retardation; the delay in dental growth was attributed to damage to the odontoblast and cemento- blast cell lineage. Other studies in which rats were orally exposed to depleted uranium found statistically significant decreases in expression of CYP27A1, CYP2R1, CYP27B1, and CYP24A1 enzymes involved in vitamin D metabolism and two vitamin D(3)- target genes (ECaC1 and CaBP-D9K) (Tissandie et al., 2006, 2007). Although depleted-uraniumâinduced changes in the concentrations of the active form of vitamin D and its receptor expression could potentially be associated with the modulation of the expression of vitamin D-target genes and calcium homeostasis and thus effects on bone deposition and remodeling, it is difficult to ascribe physi- ologic significance to these findings, given the high doses of depleted uranium to which the animals were exposed. Hematologic Effects No additional animal or in vitro studies of hematologic effects of uranium have been identified since Volume 1. Results of studies of hematologic effects summarized in Volume 1 are inconsistent. Application of the Toxicologic Data As discussed in this chapter, animal toxicity studies have been conducted primarily in rats, mice, and dogs and to a smaller extent in monkeys. The stud- ies exposed groups of laboratory-bred animals to different concentrations of uranium compounds for various portions of the animalsâ life span by differ- ent routes (for example, inhalation, ingestion via drinking water, and surgical implantation).Â Such experiments provide information that is necessary to deter- mine short-term or longer-term effects on the body, specific organ systems, and biochemical processes. The advantage of the studies is that the conditions of dose and exposure are carefully monitored to maintain control over many of the experimental characteristics and to minimize confounders and so allow scrutiny of the specific effects of the uranium or depleted uranium. The disadvantage is that the animals are not the primary species of interest for human toxicity. Toxic- ity in animals is not always predictive of effects in humans. Toxicologic studies (animal and in vitro studies) typically involve adminis- tration of high doses of a test substance, in this case uranium, that are generally
42 updated literature review of depleted uranium greater than those received by humans. For example, in the animal cancer study by Mitchel et al. (1999) discussed above, average urinary uranium concentrations were 0.117-0.274 mg/L compared with a study of uranium mill workers in which the mean urinary uranium concentrations were 0.0652 mg/L in 1975 and 0.0072 mg/L in 1981 (Thun et al., 1985) and a study of uranium in drinking water in which the mean concentration in residents was 0.000424 mg/L (Kurttio et al., 2002). In addition, the urinary uranium concentration in the 50th percentile of the US population is 6.32 ng/L (Ting et al., 1999). The large difference makes it difficult to extrapolate from effects observed in animals to health outcomes in humans. Toxicologic studies conducted using lower doses (similar to doses received by humans) may provide more relevant information about the relation- ship between effects in animals and human health outcomes. For the reasons discussed above, the committee considered toxicologic stud- ies to be secondary information sources. Information from such studies was used to determine mechanism of action but not to determine human health outcomes. TABLE 3-1 Uranium Compounds, by Dissolution Type Type F (Fast) Type M (Medium) Type S (Slow) Uranium hexafluoride (UF6) Uranium tetrafluoride (UF4) Uranium dioxide (UO2) Uranium tetrachloride (UCl4) Uranium trioxide (UO3) Triuranium octaoxide (U3O8) Uranyl fluoride (UO2F2) Uranyl acetate (UO2(CH3CO2)2 Uranium peroxide (UO4) Uranyl nitrate hexahydrate [UO2(NO3)2Â·6H20]
TABLE 3-2â Carcinogenic Effects Frequency and/or Species Route of Exposure and Dose Duration Outcome(s) Reference Rat 90% enriched 235U as tetravalent Not known Statistically significantly increased Filippova et al., (uranium, 0.57-18.7 mg U/kg of body incidence of osteosarcoma, lung 1978 weight) or hexavalent (uranium, 0.55- and kidney carcinoma, lung 5.32 mg U/kg of body weight) U reticulolymphosarcoma, leukemia in treated animals compared with controls Intratracheal injection Rat, male Natural uranium-ore dust aerosol, 50 4.2 hours/day, 5 days/ Frequency of primary malignant lung Mitchel et al., 1999 Sprague mg/m3 or 19 mg/m3 week for 65 weeks tumors 0.016, 0.175, and 0.328 and Dawley frequency of primary nonmalignant Nose-only inhalation lung tumors 0.016, 0.135, and 0.131 in control, low-exposure, and high-exposure groups, respectively Rat, male DU pellets: 2.0 mm Ã 1.0 mm in Observed for lifetime Significant increase in incidence of soft- Hahn et al., 2002 Wistar diameter, 6.0 Bqa tissue sarcoma in 5.0 Ã 5.0 Ã 1.5-mm group; slight increase in 2.5 Ã 2.5 Ã 1.5- DU fragments: 2.5 Ã 2.5 Ã 1.5 mm, 20 mm group; no increase in 2.0 Ã 1.0-mm Bqa; 5.0 Ã 5.0 Ã 1.5 mm, 59 Bqa group 4 implants/rat, intramuscular Continued 43
44 TABLE 3-2â Continued Frequency and/or Species Route of Exposure and Dose Duration Outcome(s) Reference Mouse, male DU pellets: size and radioactivity not 76% of treated mice developed Miller et al., 2005 DBA/2 specified leukemias compared with 12% of controls 2, 6, 8 implants/mouse, intramuscular 106 FDC-P1 hematopietic cells administered 60 days after pellet implantation Hamster, Uranium-ore dust, 19 mg/m3 16 months No increase in number of tumors in Cross et al., 1981 golden Syrian treated animals compared with controls Monkey and Uranium dioxide aerosol, 5 mg/m3 Up to 5 years Frank neoplasms and foci of atypical Leach et al., 1973 dog epithelial proliferation in 31% and 46%, Inhalation respectively, of surviving dogs kept 75 months after termination of 5-year exposure; pulmonary tumors and atypical epithelial changes not found in any exposed monkeys aEffective alpha-particle radioactivity emanating from the surface of the DU. NOTE: DU = depleted uranium.
TABLE 3-3â Genotoxic Effects Frequency and/or System Studied Route of Exposure/Dose Duration Outcome(s) Reference Human 34 Gulf War veterans High-DU group (n = 10), >0.1 12- and 14-year postwar CA assay: no significant differences McDiarmid, 2007 (BVAMC DU Followup Âµg/g creatinine; low-DU group followup between groups Program) (n = 24), <0.1 Âµg/g creatinine HPRT mutation frequency: no significant differences between groups SCE assay: no association between SCE and urinary DU concentration (assayed at 12-year followup only) 39 Gulf War veterans Urinalysis results: 0.001-78.125 10-year postwar followup CA assay: high-DU group had higher McDiarmid et al., (BVAMC DU Followup Âµg/g creatinine CA frequency per cell 2004 Program) High-DU group (n = 13), >0.1 SCE assay: no association between Âµg/g creatinine; low-DU group SCE and urinary DU concentration (n = 26), <0.1 Âµg/g creatinine HPRT mutation frequency: statistically significant positive association between HPRT mutation frequency and urinary DU concentration 50 Gulf War veterans Urinalysis results: 0.002-31.8 8-year postwar followup CA assay: no association between CA McDiarmid et al., (BVAMC DU Followup Âµg/g creatinine and urinary DU concentration 2001 Program) High-DU group (n = 13), >0.1 SCE assay: statistically significant Âµg/g creatinine; low-DU group increase in baseline SCE in high- (n = 37), <0.1 Âµg/g creatinine exposure group compared with low- exposure group (P = 0.03) Continued 45
46 TABLE 3-3â Continued Frequency and/or System Studied Route of Exposure/Dose Duration Outcome(s) Reference 69 people in south of Urinalysis results: 1999-2002 CA assay: non-statistically significant Milacic et al., 2004 Serbia and Montenegro alpha-spectrometry (range): increase in CA in exposed group (where DU ammunition 1-30.4 mBq/L in exposed group (residents of Vranje and Bujanovac) was used during 1999 air versus âbelow detectionâ in compared with controls; increase strikes) controls was below incidence of CA in people occupationally exposed to ionizing radiation 30 people in HadÅ¾iÄi (near Urinalysis not conducted Blood samples taken in Micronucleus cytochalasin-B test: Krunic et al., 2005 Sarajevo) environmentally 2002, 2003 statistically significant increase in exposed to DU Water from two local wells had micronucleus frequencies in exposed traces of DU (0.38Âµg/L [14% of group compared with controls total uranium was DU] and 0.55 Âµg/L [73.4% of total uranium was DU]) Whole animal exposure Sprague Dawley, male Inhalation exposure: UO4: 30 minutes Comet assay: UO4 exposure alone Monleau et al., 2006b had no effect on DNA damage; UO4: 116 Â± 60 mg/m3 UO2 + UO4: 3 hours + 30 repeated UO2 pre-exposure followed minutes by UO4 exposure increased DNA UO2 + UO4: 375 Â± 70 mg/m3 + damage compared with controls 116 Â± 60 mg/m3 UO2 + UO4: 3 hours 4 days/week for 3 weeks + UO2 + UO4: 190 Â± 41 mg/m3 + 30 minutes 116 Â± 60 mg/m3
Sprague Dawley, male Nose-only inhalation exposure: UO2: 30 minutes (190 mg/ Comet assay: DNA damage occurred Monleau et al., 2006c m3), acute and repeated in groups exposed to UO2 at single UO2: 190 Â± 41 mg/m3 or 375 Â± exposure; 2 hours (375 375-mg/m3 dose for 3 hours and 70 mg/m3 mg/m3); or 3 hours (375 repeated 190-mg/m3 dose; no DNA mg/m3) damage in other groups UO4: 116 Â± 60 mg/m3 UO4: 30 minutes Sprague Dawley, male Pellets 1 mm in diameter Ã 2 mm Urine, serum samples Ames Salmonella reversion assay: Miller et al., 1998b long; implanted in gastrocnemius collected 6, 12, 18 months increased urinary uranium content led muscle; low-, medium-, and high- after pellet implantation to increased mutagenicity dose groups In vitro studies Human bronchial Uranyl acetate: 100, 200, 400, Cells incubated for 24, 48, Both compounds led to time- and Wise et al., 2007 fibroblast cell line that 800 ÂµM 72 hours concentration-dependent cytotoxicity; ectopically expresses uranium trioxide led to increased human telomerase, Uranium trioxide: 0.5, 1, 5, 10 chromosomal damage, but uranyl WTHBF-6 Âµg/cm3 acetate did not Human liver carcinoma DU-UO2 at 0-50 Âµg/mL Cells incubated for 48 DU exposure led to dose-dependent Miller et al., 2004 cells (HepG2) hours induction of nine of 13 promoters assayed Human osteoblast cells Induction assay: 0-50 ÂµM DU- Cells incubated for 24 Induction assay: DU exposure led to Miller et al., 2002b (HOS) uranyl nitrate hours dose-dependent increase in yield of dicentrics Transformation assay: 50 ÂµM DU-uranyl nitrate (46 cGy alpha- Transformation assay: DU exposure particle equivalent dose), 238U- led to specific activity-dependent uranyl nitrate (35 cGy), 235U- increase in neoplastic transformation uranyl nitrate (227.5 cGy) frequency Continued 47
48 TABLE 3-3â Continued Frequency and/or System Studied Route of Exposure/Dose Duration Outcome(s) Reference Human osteoblast cells DU-UO2 at 5 or 10 mg/mL Cells incubated for 24 Transformation assay: DU exposure Miller et al., 2002c (HOS) hours at 10 mg/mL led to 25.5-fold increase in transformation frequency compared with untreated HOS cells Genotoxicity assays (micronuclei induction, SCE concentration, DNA single-strand breaks, dicentric formation): DU exposure at 5 mg/mL led to significant increases compared with untreated cells Human osteoblast cells DU-UO2Cl2 at 10 Âµg/mL DU-UO2 Cells incubated for 24 DU exposure led to increase in Miller et al., 2001 (HOS) or 10 ÂµM hours transformation to tumorigenic phenotype; cells not transformed after exposure to DU and phenyl acetate (RAS protein target) Human osteoblast cells DU-UO2Cl2 at 10 ÂµM Cells incubated for 24 SCE assay: DU exposure led to about Miller et al., 1998a (HOS) hours 2-fold increase in SCE induction Transformation assay: DU exposure led to 9.6-fold increase in transformation frequency compared with controls
Chinese hamster lung DU-uranyl nitrate at 10-50 Âµg/mL Cells incubated for 24 Dose-dependent increase in Miller et al., 2007 fibroblast V79 cells hours mutagenic response after exposure; at equal uranium concentration, higher specific activity led to increase in hprt mutant frequency suggesting that radiation is involved in DU- induced biological effects in vitro Chinese hamster ovary 200 ÂµM UA Cells incubated for 24 UA-exposed cells had statistically Coryell and Stearns, (CHO) EM9 cells hours significantly more genomic mutations 2006 than controls Chinese hamster ovary 0-300 ÂµM UA Cells incubated for 40 Cytotoxicity assay: UA exposure led Stearns et al., 2005 (CHO) EM9 cells minutes (comet assay), to more cytotoxicity in EM9 cells 24 hours (cytotoxicity, (which are DNA-repair-deficient) mutagenicity, comet compared with controls (3.1-fold assays; measurement of increase in cell death at 200 ÂµM) uranium-DNA-P binding), 48 hours (measurement of Mutagenicity assay: UA exposure led uranium-DNA-P binding) to higher induced mutant frequency (about 5-fold) in EM9 cells compared with controls Comet assay: no differences in tail moments between EM9 cells and controls Uranium/DNA-P binding: No significant difference in uranium- DNA adduct between EM9 and control cells Continued 49
50 TABLE 3-3â Continued Frequency and/or System Studied Route of Exposure/Dose Duration Outcome(s) Reference Normal rat renal proximal Up to 700 ÂµM uranium Cells incubated for 24 DNA damage occurred in time- and ThiÃ©bault et al., 2007 cells biocarbonate hours concentration-dependent manner; exposure at 300 ÂµM or higher led to genotoxicity; DNA damage may be reversible at low concentrations and irreversible at higher concentrations pBluescript SK DNA+ 0.1-1.0 mM UA 30 minutes at 37Â°C UA + ascorbic acid exposure led to Yazzie et al., 2003 plasmid DNA single-strand breaks, demonstrating chemical genotoxicity Calf thymus DNA 1-1,000 ÂµM DU-uranyl nitrate 30 minutes at 37Â°C DU exposure led to oxidative DNA Miller et al., 2002a damage without significant alpha- particle decay NOTE: BVAMC = Baltimore Veterans Affairs Medical Center, CA = chromosomal aberration, DU = depleted uranium, hprt = hypoxanthine-guanine phospho- ribosyl transferase, SCE = sister chromatid exchange, UA = uranyl acetate, UO 2 = uranium dioxide, UO2Cl2 = uranyl chloride, UO4 = uranium peroxide.
TABLE 3-4 Respiratory Effects Cell Line Dose Duration Outcomes Reference Human type II epithelial 0.5 mM uranyl acetate 48-hour incubation Exposure triggered differential Malard et al., 2005 cell line expression of 18 spots, of which 14 corresponded to fragments of cytokeratin 8 (CK8) and cytokeratin 18 and one to peroxiredoxin 1; CK cleavage did not result from caspase or calpain activity Rat lung epithelial cell 0.25, 0.5, 1 mM uranyl 3-hour incubation Induction of oxidative stress at 0.5 Periyakaruppan et al., line acetate and 1 mM; response correlated with 2007 dose and time Rat pulmonary alveolar 10-300 ÂµM uranyl acetate 24-hour incubation Dose-dependent increase in TNFa Gazin et al., 2004 macrophage cell line, production; no secretion on IL-1Î², NR8383 IL-10 detected NOTE: IL = interleukin, TNF = tumor necrosis factor. 51
52 TABLE 3-5â Renal Effects Species Route of Exposure and Dose Frequency or Duration Outcomes Reference Sprague Dawley rat, male Ingestion via drinking water 6, 9, 12, 18 months Exposed rats had increased Donnadieu-Claraz et number of vesicles containing al., 2007 Uranium nitrate at 40 mg/L dense granular inclusions in (14.5 Bq) proximal tubular cells; inclusions composed of small granule clusters and increased in number with exposure duration; granules composed of iron oxides Wistar rat, male Intramuscular injection Animals euthanized 28 DU concentrations in urine Fukuda et al., 2006 days after exposure decreased within 3-7 days after DU nitrate at 0.2, 1.0, 2.0 mg/kg exposure; NAG-creatinine concentrations peaked at day 3 with high correlation to injected DU doses BBMV from unspecified Uranyl acetate at 0.25, 0.5, or 1.0 30 min Uranyl acetate exposure led to Goldman et al., 2006 rat species mg/mg of protein of BBMV decrease in glucose transport New Zealand White Uranyl nitrate hexahydrate at 0.96, 91 days Histopathological changes Gilman et al., 1998b rabbit, males and females 4.8, 24, 120, 600 mg/L (0.49-43.02 observed in kidneys of animals mg/kg body weight for females; 0.05- exposed at all doses 28.7 mg/kg body weight for males) Dogs and monkeys Inhalation 5.4 hours/day, 5 days/ Injury to kidneys not observed as Leach et al., 1970 week, up to 5 years result of exposure Uranium dioxide at 5.8 mg/m3 NOTE: BBMV = brush border membrane vesicles, DU = depleted uranium, NAG = N-acetyl-Î²-D-glucosaminidase.
TABLE 3-6â Neurologic Effects Species or Cell Line Route of Exposure and Dose Frequency or Duration Outcomes Reference Rat Sprague Dawley, male Inhalation: uranium dioxide (1.3 Ã Inhalation: 30 minutes/day, 4 Overall, amount of uranium entering Houpert et al., 104 Bq/g, 190 mg/m3) days/week for 3 weeks brain was low; location of accumulation 2007c in brain depended on route of Ingestion (drinking water): Ingestion: 1 mg/day for 42 exposure: in injection group, uranium enriched uranium (6.63 Ã 104 Bq/g) days distributed among different cerebral or DU (1.47 Ã 104 Bq/g) areas; in ingestion group, triata had Injection: single injection highest uranium concentration; in Intraperitoneal injection: enriched inhalation group, olfactory bulbs had uranium (16.5 Bq) + 233U (50 Bq) highest uranium concentration; high accumulation found in hippocampus by each route of exposure Sprague Dawley, male Ingestion (drinking water) 3, 6, 9 months Exposure to enriched uranium for 3, 9 Houpert et al., months significantly reduced spontaneous 2007a Enriched uranium nitrate at 40 mg/L alternation measured in Y-maze; no differences between exposed and control rats in the open-field, object-recognition, forced-swimming tests Sprague Dawley, Cell culture Cells treated 3 weeks after Exposure did not lead to significant Jiang et al., primary rat cortical isolation for 24 hours changes in cellular energy metabolism, 2007 neurons 1, 10, or 100 ÂµM uranyl acetate thiol metabolite oxidation, lipid metabolism Continued 53
54 TABLE 3-6â Continued Species or Cell Line Route of Exposure and Dose Frequency or Duration Outcomes Reference Sprague Dawley, male Ingestion (drinking water) 1.5, 6, 9 months Exposure to uranyl nitrate for up to 9 Bussy et al., months did not affect AChE activity in 2006 Uranyl nitrate at 40 mg/L (daily striatum, hippocampus, frontal cortex; intake about 4-1.5 mg/kg of body exposure for 6 months altered AChE weight) activity in cerebellum; exposure for 6 months led to small change in DAergic turnover ratio in frontal cortex; after 9 months, significant decrease in 5HIAA concentration and 5HTergic turnover ratio in frontal cortex, decrease in the DOPAC concentration and DAergic turnover ratio in striatum Rat brain endothelial 10, 50, 100 ÂµM U3O8 uranyl chloride 15, 30 minutes No overt cytotoxicity observed with Dobson et al., cells (RBE4) assays for cell-volume increase, 2006 heat-shock protein 90 expression, 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide reduction, lactate dehydrogenase activity Sprague Dawley, male Implanted in gastrocnemius muscle 3, 6 months At 3 months, DU accumulated in all Fitsanakis et brain regions except the hippocampus of al., 2006 DU pellets: 1 mm in diameter Ã 2 highest-dose animals; at 6 months, DU mm long accumulation found in cortex, midbrain, cerebellum Sprague Dawley, male Intraperitoneal injection Uranyl acetate administered Stress increased clearance of uranium Barber et al., after forced swimming in the brain compared with clearance of 2005 Uranyl acetate dehydrate at 1mg/kg to induce stress; animals brain uranium in unstressed rats euthanized 8 hours, 24 hours, 7 days, 30 days after exposure
Long Evans, male and Ingestion (drinking water) 2 weeks, 6 months Exposure for 2 weeks led to behavioral Briner and female changes (line-crossing, rearing) in males Murray, 2005 DU acetate dehydrate at 75, 150 and changes in brain lipids in both sexes mg/L (25, 50 mg/kg per day) compared with controls; exposure for 6 months produced additional behavioral changes Sprague Dawley, sex Ingestion (drinking water) 1.5 months Exposure to enriched uranium led to Houpert et al., not specified increases in amount of paradoxical sleep 2005 Enriched or depleted uranium and anxiety and reduction in spatial incorporated as nitrate: 1 mg/day working-memory capacities compared per rat with controls; exposure to DU did not lead to these effects Sprague Dawley, male Intraperitoneal injection Animals euthanized 3 days Exposure at higher concentration led to Lestaevel et after exposure shorter paradoxical sleep compared with al., 2005b Uranyl nitrate at 70, 144 Âµg/kg controls; no effect at lower concentration Sprague Dawley, male Ingestion (drinking water) 3 months By 30 days, exposure to uranium led to Lestaevel, increase in rapid-eye-movement sleep 2005a Uranyl nitrate at 40 mg/L and theta-band power during light period Sprague Dawley, male Nose-only inhalation 30 minutes/day, 4 days/week Exposure led to behavioral changes Monleau et for 3 weeks (spontaneous locomotion activity, al., 2005 Uranium dioxide at 197 mg/m3 spatial working memory) compared with controls Sprague Dawley, male Ingestion (drinking water) 30 days Exposure to DU did not lead to alteration Houpert et al., of dopamine, serotonin, catabolite 2004 DU at 40 mg/L concentrations in striatum, hippocampus, cerebral cortex, thalamus, cerebellum Continued 55
56 TABLE 3-6â Continued Species or Cell Line Route of Exposure and Dose Frequency or Duration Outcomes Reference Sprague Dawley, male In situ brain perfusion 2 minutes Significant amount of uranium found in Lemercier et brains of exposed rats al., 2003 5 Ã 10â8 to 5 Ã 10â5 M uranyl tricarbonate Sprague Dawley, male Intramuscular injection 1 injection per day for 7 Dose-related deficit in inclined-plane Abou-Donia days, followed by 30-day performance; reduced grip time, impaired et al., 2002 Uranyl acetate at 0.1, 1 mg/kg observation period beam-walk score and beam-walk time at both doses; significant increase in nitric oxide at 0.1 mg/kg in cortex, midbrain; significant increase at 1 mg/kg in AChE activity in the cortex; no change in ligand-binding densities for m2 muscarinic receptor Sprague Dawley, male Implanted in gastrocnemius muscle Animals euthanized 6, 12, 18 Exposure to DU led to neurophysiologic Pellmar et al., months after implantation changes in hippocampus (EPSP-spike 1999b DU pellets: 1 mm in diameter Ã 2 coupling); changes occurred at 6, 12 mm long, up to 10 pellets per thigh months, but not after 18 months Sprague Dawley, sex Implanted in gastrocnemius muscle Animals euthanized 1, 6, 12, Significant amounts of DU found in Pellmar et al., not specified 18 months after implantation motor cortex, frontal cortex, midbrain, 1999a DU pellets: 1 mm in diameter Ã 2 cerebellum, vermis compared with mm long, up to 10 pellets per thigh controls Sprague Dawley, male Ingestion (drinking water) Up to 91 days No signs of neurotoxicity related to Gilman et al., and female exposure 1998c Uranyl nitrate hexahydrate at up to 600 mg/L (37, 54 mg U/kg body weight per day for male, female rats, respectively)
Sprague Dawley, male Intragastric Single dose Acute cholinergic toxicity observed in Domingo et treated rats al., 1987 Uranyl acetate dehydrate at 11-717 mg U/kg body weight Mouse Phrenic nerve 0.2-0.8 mM uranyl nitrate More than 4-hour incubation Treatment facilitated release of Lin et al., diaphragm from acetylcholine from nerve terminals, 1988 ICR strain, male and potentiated muscle contraction female Other species Caenorhabditis 1, 10, 100 mM uranyl acetate 24-hour incubation period Exposure did not lead to significant Jiang et al., elegans (nematode) neurodegeneration 2007 Dog Inhalation 30 days Muscle weakness and instability of gait Dygert et al., beginning on day 13 at 18 mg U/m3 1949 Uranium hexafluoride gas at 0.5-18 mg U/m3 Dog Inhalation 30 days Exposure associated with anorexia Roberts, 1949 Uranyl nitrate hexahydrate at 9.5 mg U/m3 Cat Inhalation 30 days Muscle weakness and instability of gait Dygert et al., beginning on day 7 at 18 mg U/m3 1949 Uranium hexafluoride gas at 0.5-18 mg U/m3 NOTE: 5HIAA = 5-hydroxyindoleacetic acid, 5HTergic = serotoninergic, AChE = cholinergic acetylcholinesterase, DAergic = dopaminergic, DOPAC = 3,4 dihydroxyphenylacetic acid, DU = depleted uranium. 57
58 TABLE 3-7â Hepatic Effects Species Route of Exposure and Dose Frequency or Duration Outcomes Reference Rat Sprague Dawley rat, Subcutaneous administration Single administration Exposure led to changes in hepatic Guegeun et al., male metabolism of bile acids and 2006 Uranyl hexahydrate at 11.5 mg/kg Animals euthanized 1, 3 days xenobiotics; DU appeared to act after exposure through modulation of CYP enzymes Sprague Dawley rat, Ingestion (drinking water) 9 months, followed by Plasma acetaminophen concentrations Gueguen et al., male intraperitoneal injection of higher in DU group treated for 24 2007 Uranyl nitrate at 1 mg/day per acetaminophen at 400 mg/kg 2, hours compared with non-DU group; animal 24 hours before euthanasia DU-treated group had significantly increased ALT, AST after 2 hours of acetaminophen treatment and decreased CYP2A, 2B, 3A activity compared with controls Sprague Dawley rat, Ingestion (drinking water) 9 months mRNA concentrations of CYP3A1, Souidi et al., 2005 male CYP3A2, PXR significantly higher Uranyl nitrate: 1 mg/day per in the DU-exposed group compared animal with controls; mRNA concentrations of CYP1A1, CYP2B1, RXR, CAR unchanged; hepatic activity of CYP2A, CYP2B, CYP2C, CYP3A did not change significantly in treated vs control groups Other species Danio rerio DU: 1.5 Bq/L 3, 10, 20 days Decreased superoxide dismutase, Barillet et al., (zebrafish), male DU + 233U: 2376 Bq/L catalase activities, total glutathione 2007 adults content in liver extracts NOTE: ALT = alanine amino transferase, AST = aspartate amino transferase, CYP = cytochrome P450, DU = depleted uranium, i.p. = intraperitoneal, mRNA = messenger RNA.
TABLE 3-8â Reproductive and Developmental Effects Species Route of Exposure and Dose Frequency or Duration Outcomes Reference Rat Sprague Dawley, Ingestion in drinking water Exposure began 3 months before Exposure to uranium led to delayed Houpert et al., male and female mating and continued through hyperactivity, behavioral decrements in 2007b Enriched uranium nitrate at lactation performance (open-field, Y-maze, elevated 40 mg/L (about 1 mg/day per plus-maze tests) in 2-, 5-, 9-month-old rat) (P1 generation) offspring Sprague Dawley, Implanted in gastrocnemius P1 generation mated 30 days Exposure to DU did not lead to reproductive Arfsten et al., male and female muscle after implantation and monitored effects in P1 generation compared with 2005 during pregnancy; offspring (F1 controls; no developmental effects (birth DU pellets: 1 mm in diameter generation) assessed through weight, survival, litter size, gross physical Ã 2 mm long, up to six pellets adult stage abnormalities, neurodevelopmental effects, per calf (P1 generation) immune-function effects) in F1 generation Sprague Dawley, Implanted in gastrocnemius P1 generation mated 30, 120 days Exposure to DU did not adversely Arfsten et al., male and female muscle after implantation affect male reproductive success, sperm 2006 concentration, sperm velocity compared with DU pellets: 1 mm in diameter controls Ã 2 mm long, up to 10 pellets per calf (P1 generation) Sprague Dawley, Subcutaneous injection Administered on gestation days Exposure to DU led to maternal toxicity Albina et al., male and female 6-15 with or without restraint and embryotoxicity in high-dose group; 2003 Uranyl acetate dehydrate at stress for 2 hours/day; cesarean fetotoxicity (reduction in fetal body weight, 0.415, 0.830 mg/kg per day sections performed on gestation increase in total number of skeletally day 20 affected fetuses) observed in both groups; no teratogenic effects observed in either group; maternal restraint stress enhanced embryo, fetal toxicity in high-dose group Continued 59
60 TABLE 3-8â Continued Species Route of Exposure and Dose Frequency or Duration Outcomes Reference Mouse B6C3F1, male and Ingestion in drinking water Experiment 1: administered for Exposure to uranium hexahydrate led to Raymond-Whish female 30 days to immature 28-day-old estrogenic responses (selective reduction in et al., 2007 C57Bl/6, Uranium hexahydrate at 0.5 mice (B6C3F1) primary follicles, increase in uterine weight, ovariectomized Âµg/L (0.001 ÂµM) to 28 mg/L greater uterine luminal epithelial cell height, females (120 ÂµM) Experiment 2: administered to accelerated vaginal opening, persistent males and females for 30 days presence of cornified vaginal cells); pups before breeding, then to pregnant exposed in utero had significantly fewer females through gestation primordial follicles than unexposed (B6C3F1) Experiment 3: administered for 30 days starting 7 days after ovarectomy (C57Bl/6) Experiment 4: administered uranium or DES for 10 days beginning at age of 50 days (C57Bl/6), intraperitoneal injection of ICI 182,780 Swiss Webster, Ingestion in drinking water Exposed for 2 weeks, then mated; Exposure to uranium acetate did not lead to Briner and Byrd, female exposure of dams and offspring maternal toxicity; no gross malformations 2000 Uranium acetate at 19, 37, continued until sacrifice observed in pups 75 mg/L Exposed offspring developed more quickly than controls on behavior indexes (righting reflexes, forelimb placing and grasping, swimming development); hindlimb placing
was at first accelerated in exposed group, but timepoint was delayed later; on functional observation battery, exposed offspring had fewer spontaneous vocalizations and on touch-response test, more freezing and jerking behavior; exposed offspring gained weight more quickly than controls and at sacrifice had significantly higher body and brain weights, although brain as percentage of body weight was smaller in exposed groups (37 and 75 mg/L) Swiss, male Ingestion (drinking water) 64 days Testicular function and spermatogenesis Llobet et al., 1991 were not affected by exposure to uranium Uranyl acetate dihydrate: 0, 10, 20, 40, or 80 mg/kg/day Swiss, male and Intragastic administration Males: 60 days prior to mating Uranium exposure did not lead to adverse Paternain et al., female effects on fertility; increased embryolethality 1989 Uranyl acetate dihydrate: 0, 5, Females: 60 days prior to mating was observed in the highest dose group 10, 25 mg/kg/day and throughout mating, gestation, parturition, and nursing NOTE: DES = diethylstilbestrol, DU = depleted uranium, ICI 182,780 = a steroidal estrogen antagonist. 61
62 TABLE 3-9 Immunologic Effects Species or Cell Line Route of Exposure and Dose Frequency or Duration Outcomes Reference Animal studies Sprague Dawley, male Nose-only inhalation UO2: 30 minutes (190 mg/m3), Inflammatory cytokine expression Monleau et al., acute and repeated exposure; 2 increased 2006c UO2 at 190 Â± 41 mg/m3, 375 hours (375 mg/m3); or 3 hours Â± 70 mg/m3 (375 mg/m3) UO4 at 116 Â± 60 mg/m3 UO4: 30 minutes Sprague Dawley, male Ingestion in drinking water 3, 6, 9 months DU exposure led to decrease in intestinal Dublineau et al., mast cell number, increase in IL-1Î² and 2007 Uranyl nitrate at 40 mg/L IL-10 concentrations, decrease in mRNA (1 mg/day per animal) CCL-2 concentrations, decrease in intestinal macrophage density, increase in number of neutrophils Sprague Dawley, male Gavage Single administration DU appeared to modulate the expression Dublineau et al., and/or production of IFNÎ³, MCP-1 in 2006b Uranyl nitrate at 204 mg/kg Animals euthanized 1 or 3 days intestine after administration Sprague Dawley, male Ingestion in drinking water 3, 9 months DU preferentially accumulated in Peyerâs Dublineau et al., (harvested intestines) patches compared with epithelium; 2006a Uranyl nitrate at 1 mg/day per no induction of apoptosis pathway animal after chronic DU contamination in Peyerâs patches; no change in cytokine expression (Il-10, TGF-Î², IFN-Î³, TNF-Î±, MCP-1) in Peyerâs patches and mesenteric lymph nodes; no modification in uptake of yeast cells by Peyerâs patches
Sprague Dawley, male DU pellets: 1 Ã 2 mm P1 generation mated 30 days No significant difference among 8-week- Arfsten et al., and female after surgery; P1 females nursed old F1 treatment groups in mean thymus 2005 0, 4, 8, 12 implants/rat (P1 offspring until PND 20 and spleen mass, mean total number of generation) thymocytes per spleen Sprague Dawley, sex Implanted in gastrocnemius 1 day, 1, 6, 12, 18 months Significant concentrations of uranium Pellmar et al., not specified muscle found in spleen 1999a DU pellets: 1 mm in diameter Ã 2 mm long; up to 20 pellets per thigh In vitro studies Macrophages from Uranyl nitrate: 10-1,000 ÂµM Cells incubated for 2 hours Lymphoproliferation assay: uranyl Wan et al., 2006 BALB/c and DO11.10 nitrate exposure at 200 ÂµM for 2 hours T-cell receptor mice led to altered macrophage accessory-cell function Macrophage cell line, DU-uranyl chloride: 1, 10, Cells incubated for up to 24 hours Exposure at all concentrations led to Kalinich et al., J774 100 ÂµM decreased viability of cells; appeared to 2002 be apoptotic death NOTE: CCL = chemokine ligand 2, DU = depleted uranium, IFN = interferon, IL = interleukin, MCP = monocyte chemoattractant protein, PND = post natal day, TGF = transforming growth factor, TNF = tumor necrosis factor, UO 2 = uranium dioxide, UO4 = uranium peroxide. 63
64 TABLE 3-10 Musculoskeletal Effects Species Route of Exposure and Dose Frequency or Duration Outcomes Reference Rat Sprague Dawley, Ingestion in drinking water 9 months Decrease in vitamin D concentration Tissandie et al., male in plasma compared with controls; 2007 Uranyl nitrate at 1 mg/day per expression of CYP genes involved in animal vitamin D metabolism unaltered in liver of treated animals; significant decrease in cyp24a1 mRNA concentrations in kidneys of treated animals Sprague Dawley, Gavage Single dose Significant decreases in vitamin D Tissandie et al., male and PHT in plasma compared with 2006 Uranyl nitrate at 204 mg/kg of Animals were euthanized 1 or 3 controls; treatment modulated mRNA body weight (LD50 at 14 days) days after exposure concentrations and activity of CYP enzymes involved in vitamin D metabolism Wistar, sex not Oral Single dose No statistically significant difference Pujadas Bigi and specified in mandibular length observed in Ubios, 2007 Uranyl nitrate at 90 mg/kg of Animals were euthanized 7 or 27 treated vs control animals; mandibular body weight days after exposure area and height, tooth eruption, dental development decreased in treated animals at 7 days but similar to controls by 27 days Wistar, male Intramuscular injection Single dose Increases in concentrations of Fukuda et al., osteocalcin, tartrate-resistant acid 2006 DU nitrate at 0.2, 1.0, 2.0 mg/kg Animals euthanized 28 days after phosphatase, pyridinoline, parathyroid of body weight exposure hormone in all treated groups compared with controls
Sprague Dawley, Ingestion in drinking water 91 days No significant exposure-related effect on Gilman et al., male and female hematologic, biochemical endpoints 1998c Uranyl nitrate hexahydrate at 0.96, 4.8, 24, 120, 600 mg/L (0.09-53.56 mg/kg of body weight for females; 0.06-36.73 mg/kg of body weight for males) Wistar, male Intraperitoneal injection Single dose Alveolar bone volume (15 Ã 105 Âµm2 vs Guglielmotti et 34 Ã 105 Âµm2), total bone formation areas al., 1985 Uranyl nitrate at 2 mg/kg (4.85% vs 19.55%), volume density of bone in the alveolar apical third (0.26 vs 0.40) significantly lower in intoxicated animals compared with controls Rabbit New Zealand Uranyl nitrate hexahydrate at 91 days No significant exposure-related effect on Gilman et al., White, males and 0.96, 4.8, 24, 120, 600 mg/L hematologic, biochemical endpoints 1998b females (0.49-43.02 mg/kg of body weight for females; 0.05-28.7 mg/kg of body weight for males) NOTE: DU = depleted uranium, LD50 = dose required to kill half the test population, PHT = parathyroid hormone. 65
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