Uranium Toxicity and Chelation Therapy

Home , Chelation therapy, Uranyl acetate

Pure Appl. Chem. 2014; 86(7): 1105–1110

Conference paper

Glen D. Lawrence*, Kamalkumar S. Patel and Aviva Nusbaum Uranium toxicity and chelation therapy

Abstract: Uranium toxicity has been a concern for more than 100 years. The toxicology of many forms of uranium, ranging from dust of several oxides to soluble uranyl ion, was thoroughly studied during the Man- hattan Project in the United States in the 1940s. The development of depleted uranium kinetic penetrators as armor-piercing incendiary weaponry produced a novel form of uranium environmental contamination, which led to greater susceptibility to the adverse health effects of the toxic heavy metal after its use in various military conflicts. The aerosol from burning uranium penetrator fragments is rapidly dissolved in biological fluids and readily absorbed from the lungs, leading to a wide range of toxic effects. We have studied some chelating agents for uranyl ion, including citrate ion and desferal (desferrioxamine B), which may be effective for minimizing the toxic effects of this insidious heavy metal. Some characteristics of the desferrioxamine complex are presented, along with information about the use of citrate as an effective chelating agent for therapy of uranium toxicity.

Keywords: bioactivity; environmental chemistry; IUPAC Congress-44; metal complexes; solubility; toxicology; uranium.

DOI 10.1515/pac-2014-0109

Introduction

Uranium is the heaviest naturally occurring element in the earth’s crust; it is more abundant than gold or silver. Although discovered in 1789, it found few commercial uses for a century and a half beyond its use as a coloring agent for pottery and glass. Radioactivity was first discovered in uranium in 1896, soon after the discovery of X-rays. In the late 1930s it was found that bombarding uranium with neutrons resulted in lighter elements being formed, i.e., nuclear fission. 235U, the fissionable isotope, is only 0.71 % natural abundance, whereas 238U is 99.28 % of natural uranium. 238U undergoes spontaneous fission but cannot sustain a chain reaction and is transmuted into heavier elements in a reactor. It is necessary to enrich uranium in the 235U isotope for a sustained nuclear reaction. This produces large quantities of uranium that are depleted in fissionable 235U but have a higher percentage of 238U. About eight tons of natural uranium are needed to produce one ton of enriched uranium for reactor fuel, leaving seven tons or more of depleted uranium as waste. One of the most spectacular developments in ballistics has been the DU (depleted uranium) kinetic pene trator. It is able to pierce armor and as it slices through hard materials it heats up, fragments and ignites. Burning at high temperatures, it completely destroys its target, whether an armored tank, a concrete bunker or conventional vehicle. As the nearly pure uranium metal in the penetrator burns, it sends up smoke

Article note: A collection of invited papers based on presentations on the Environmental Chemistry theme at the 44th IUPAC Congress, Istanbul, Turkey, 11–16 August 2013.

*Corresponding author: Glen D. Lawrence, Department of Chemistry and Biochemistry, Long Island University, Brooklyn, NY 11201, USA, e-mail: [email protected] Kamalkumar S. Patel and Aviva Nusbaum: Department of Chemistry and Biochemistry, Long Island University, Brooklyn, NY 11201, USA

© 2014 IUPAC & De Gruyter 1106 G. D. Lawrence et al.: Uranium toxicity and chelation therapy containing fine particles of uranium oxide aerosol. Chazel et al. [1] showed that 22 % of the aerosol activity was from uranium oxide particles with less than 0.4 μm diameter, which penetrate deep into lung alveoli and dissolve rapidly in aqueous media. The effect that DU shells have on their targets lures the curious to see what amazing destruction it can do. Just walking or rummaging around a DU penetrator destroyed vehicle can resuspend the fine particles of uranium oxide dust, making it available for breathing it in and having it cling to skin and clothing. Uranium is not absorbed well from the digestive tract; less than 2 % of uranium oxides of > 1.3 μm dia meter taken in by mouth get absorbed and enter the blood, with the bulk of it passing through the GI tract in feces [2]. A lethal dose of uranium does not kill immediately like cyanide or strychnine, but instead can take several days and effects of a sublethal dose may not be noticed until days after poisoning has occurred. Kidney failure is generally the ultimate cause of death from a lethal dose. The acute toxic effects of a non- lethal dose of uranium that causes kidney damage within the first 2 weeks are somewhat reversible, with restoration of most kidney function after several months [3]. However, a substantial amount of uranium that enters the body gets distributed to several different organs where it can have long term effects. Craft et al. [4] reviewed the chemistry and toxicological effects of uranium. They included some background on uranium chemistry, especially regarding uranium oxides, but did not mention the greater solubility and toxicity of submicron particle sizes.

Uranium aerosol bioavailability

The most likely route for uranium absorption into the systemic circulation as a result of environmental contamination from DU weapons would be inhalation and particle size will strongly influence the relative toxicity. Early studies on UO2 toxicity following inhalation by rats showed little change in toxic response for particle sizes in the range of 10–3 μm. However, when 1 μm particle sizes were used, signs of kidney damage began to appear, and when 0.6 and 0.2 μm particle diameters were used, there were more pronounced signs of toxicity, including weight loss, large increases in blood nonprotein nitrogen and much greater retention (less excretion) of phenolsulfonphthalein, indicative of kidney failure [5]. Studies by Wilson et al. showed that 0.5 μm mass median diameter UO2 particles suspended in water or aqueous buffers are rapidly oxidized in air to UO3 and U3O8, which dissolve readily to produce uranyl ion in solution [6]. Dissolution of uranium oxides is catalyzed by acids. UO2 [7] or U3O8 [8] particles with 0.5 μm mass median diameter were absorbed as rapidly as soluble uranyl aerosols and produced the same toxic effects as soluble uranyl nitrate aerosols. All subsequent studies in the United States on inhaled uranium oxides worked with particles greater than 1.3 μm diameter and the studies by Wilson et al. were rarely cited in the literature. One study in Great Britain, published in 1982, reported that uranium oxide particles with diameter less than 0.025 μm dissolve rapidly in body fluids and are rapidly absorbed from the lungs of rats [9]. A major contention regarding toxicity of uranium oxides in the environment following conflicts where DU munitions have been used is not whether uranium is toxic, but whether those exposed would receive sufficient amounts to produce a toxic effect. Numerous animal studies indicate about a 10-fold range for species susceptibility to lethal uranium poisoning. An injected dose of about 0.08 mg per kg body weight in rabbits is sufficient to produce observable toxic effects, whereas 0.4 mg/kg or more is needed to produce similar toxic effects in mice [10]. The LD50 for intravenous injection of uranyl nitrate to several different species was: rabbits, 0.1 mg U/kg; guinea pigs, 0.3 mg U/kg; rats, 1.0 mg U/kg; and mice, 10–20 mg U/kg [10, 11]. Other routes of administration of the same dose would be less lethal. One study in humans showed that injection of greater than 0.1 mg/kg body weight resulted in increased urinary excretion of catalase and albumin, casts in the urine and elevated nonprotein nitrogen in blood [12]. Clearly, an absorbed dose of less than 1 mg/kg in humans will produce toxic effects, meaning 50–100 mg of submicron uranium oxide particles inhaled can result in toxic symptoms. It has been widely assumed that the dominant oxides in uranium oxide dust are relatively insoluble in water and body fluids. Biokinetic models generally assume biological half-times on the order of a year for G. D. Lawrence et al.: Uranium toxicity and chelation therapy 1107

UO2 particles based on particle sizes greater than 1 μm diameter [13]. These assumptions are not valid for DU residues in conflict areas in view of an abundance of uranium oxide particles with less than 0.4 μm diameter and evidence that uranium in these smaller diameter particles are rapidly absorbed and far more toxic than particles with diameters greater than 1 μm.

Biochemical and physiological studies of uranium toxicity

Once uranium has been absorbed into the bloodstream, it will be predominantly in the form of uranyl ion, which is firmly associated with the albumin fraction of serum [14]. About 50 % of the uranyl ion in blood of rats was bound to transferrin, the iron transport protein found in the albumin fraction of serum. The other half was bound to small molecules, including citrate and bicarbonate [9]. There was a similar distribution in the blood of dogs injected with uranyl ion, with about 40 percent as the transferrin complex in this species and about 60 percent as the bicarbonate complex [15]. The uranyl ion forms a 2:1 U:transferrin complex with a binding constant of about 1016 at pH 7.4, compared with the iron(III) ion binding constant of about 1020 under similar conditions [16]. Uranyl ion speciation in simulated biological fluids shows complex interactions with small molecules such as bicarbonate and citrate [17]. In the 1960s uranyl ion was used to highlight DNA in electron microscopy but other heavy metals replaced uranyl even though they were more expensive. It became apparent that uranyl ion causes DNA strand break- age that can lead to chromosomal instability [18]. Uranium was also shown to induce free radical muta- tions in DNA bases forming thymine glycol and 8-deoxyguanosine in hydroxyl radical mediated processes [19]. The chemical generation of hydroxyl radical was 106 times greater than radiolytic generation by the DU source of uranyl nitrate in that study. The same lab found that uranyl nitrate induced genomic instability in human osteoblast cell cultures causing delayed micronuclei formation and increased lethality [20]. Exposure to uranium also causes cultured human cells to be transformed into cancerous cells [21]. When DU metal fragments were implanted in rats they developed soft tissue sarcomas [22]. Uranyl nitrate decreased viability of Chinese hamster ovary cells in culture with an IC50 of 0.049 mM [23]. At 0.01–0.3 mM uranyl nitrate decreased cell cycle kinetics, increased frequency of micronuclei and sister chromatid exchange and augmented chromosomal aberrations were observed. Treatment of mouse J774 mac- rophage cells with 1, 10, or 100 μM uranyl chloride decreased viability of cells within 24 h and changes in membrane structure were indicative of apoptosis; changes were evident in 2 h with 100 μM concentration [24]. It is clear that uranium causes genotoxicity and cytotoxicity. Uranium inhibits bone formation [25] as well as bone modeling and remodeling [26]. Treatment of preg- nant mice with uranyl acetate (5 mg/kg) resulted in decreased maternal weight gain and food consumption and increased liver weight. There was no change in the number of fetuses, fetal resorptions or dead fetuses, but there were dose related fetal effects, including reduced body weight and body length, and increased skeletal malformations, such as cleft palate, bipartitie sternebrae, reduced ossification and ossified skeletal variations [27]. A U.S. soldier who was found to have been exposed to DU in Iraq after the 2003 invasion later fathered a child with bone deformities. There was an increase in birth defects, childhood leukemia and other cancers in southern Iraq after the 1991 Operation Desert Storm, in which hundreds of tons of DU weapons were used [28]. There were also marked increases in birth defects, childhood leukemia, and other cancers in Fallujah after the 2004 assaults by U.S. forces on that city [29]. Daily injections for 7 days of 0.10 and 1.0 mg/kg uranyl acetate into rats caused neurological deficits with respect to inclined plane performance, grip time, beam walk score and beam walk time. There were some spe- cific changes in nitric oxide in cortex and midbrain of the lowest dose group and increased acetylcholinest- erase activity in cortex of the 1.0 mg/kg dose group [30]. Rats imbedded with DU metal fragments developed soft tissue sarcomas at the implantation sites and showed changes in electrophysiological measurements of hippocampal slices at 6 months after implantation [31]. ATPases are important in maintaining ion gradients in all cells and electrolyte balance in the kidney. Uranyl ion inhibits the Na/K ATPase (sodium-potassium pump) by about 50 % at 20 μM, but there is nearly 20 % inhibition at 2 μM [32]. 1108 G. D. Lawrence et al.: Uranium toxicity and chelation therapy

Chelation therapy

In the 1940s a study showed that sodium citrate administered either intravenously (230 mg/kg) or by oral gastric intubation (1.15 g/kg) gave dramatic protection from uranium poisoning. All 7 dogs given sodium citrate orally and all 3 given sodium citrate intravenously survived a lethal dose of uranyl nitrate (i.v.), whereas 5 of the 10 dogs not receiving sodium citrate died within 10 days from uremia and the other 5 were put to death at various times between 2.5 and 7 days for kidney histology. Renal lesions in citrate treated animals were much less severe and recovery was rapid compared to animals receiving uranyl nitrate only [33]. In another study of that era, dogs given 5 mg/kg uranyl nitrate alone showed acute signs of toxicity and 12 of 13 dogs died between 9 and 13 days after uranium treatment. When 230 mg sodium citrate was given (i.v.) each day for 5 days prior and 5 days after uranium treatment, only 1 dog died and survivors showed little, if any signs of intoxication from the same dose of uranium. There was also significant tubular regeneration in the citrate treated animals [34]. It appears that sodium citrate was as effective when administered orally as when administered intravenously. It is interesting to note that uranium poisoned rats show increased excretion of citrate in urine without exogenous citrate administration [35]. Ethylenediaminetetraacetic acid (EDTA) was found to increase urinary excretion of uranyl ion, but was not effective at mobilizing uranium bound to bone [36]. Sixteen chelating agents were tested for their efficacy as antidotes for acute uranium poisoning in mice, with eight producing significantly increased survival rate: Tiron, gallic acid, diethylenetriaminepentaacetic acid (DTPA), p-aminosalicylic acid, sodium citrate, EDTA, 5-aminosalicylic acid and ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA). Mice received the chelating agents at one-fourth their LD50 (i.p. injection) 10 min after subcutaneous administration of various doses of uranyl acetate [37]. It is important to note that d-fructose, one of the tested chelating agents, dramatically increased the lethality of uranium. Both Tiron and gallic acid, given up to 1 h after uranyl acetate (s.c.) injection, were effective at increasing uranium excretion and significantly reduced tissue uranium concentrations in bone and kidney 4 days after exposure [38]. DTPA was less effective for increasing uranium excretion. Although Tiron and gallic acid have low toxicity, their chemical nature makes it necessary to inject them for effective chelation therapy rather than oral administration. Desferrioxamine B (DFO) is a trihydroxamic acid siderophore used in the treatment of acute iron poisoning and chronic iron or aluminum overload [39]. DFO is available for clinical use as the methanesulfonate salt, known as desferal mesylate. Other transition metals bind to DFO with substantially weaker binding constants than iron(III) [40]. Uranyl ion has also been shown to bind to DFO with a somewhat weaker binding constant than iron(III) [41], but its efficacy in removing uranyl ion from animals or humans has not been reported. We found that uranyl ion forms a strong complex with DFO at pH 7.5 in HEPES buffer. The complex has maximum absorbance at about 360 nm between pH 7 and 9, with molar absorptivity around 1400 cm–1 M–1. Fluoride, citrate, and bicarbonate at 0.10 M were found to interfere with DFO complex formation, whereas bromide, chloride, nitrate, and sulfate were without any significant effect at 0.10 M concentration. In view of the information on chelation therapy for uranium poisoning, it would seem that citrate salts or citric acid would be the therapy of choice in conflict areas or areas where sophisticated medical technologies and chelating agents are not available. Since citric acid and citrate salts appear to be effective when taken orally, the aim for efficacious prophylactic or post-exposure treatment for uranium poisoning would be to consume foods and beverages that contain ample citric acid and citrate salts. Citric acid and citrate salts are relatively nontoxic and are found in many fruits and vegetables, as well as in medical dosage form such as potassium citrate prescribed for kidney stones. When workers or others are in uranium contaminated areas it would be wise for them to consume citrates prior to exposure, if possible, and continue consuming high levels of citrates for days after exposure to minimize toxicity. We have measured citric acid/citrate in several fruits and beverages (Table 1). It is important to warn that d-fructose can exacerbate uranium toxicity [37] and should be avoided. Consequently, some products that have significant amounts of citric acid, such as juice cocktails, iced tea and lemonade, should be checked to make sure they do not have added sucrose or fructose. Other sugars were not tested for their potential effects on uranium toxicity and probably all sugars should be avoided until studies are performed. G. D. Lawrence et al.: Uranium toxicity and chelation therapy 1109

Table 1 Citric acid/citrate content of fruits and drinks.

Serving size Fruit or drink Citric acid/citrate (g/serving)

1 Lemon 3.0 1 Lime 1.8 1/2 Grapefruit 2.7 1 Orange 1.5 236 mL Orange juice, fresh 4.0 236 mL Orange juice, from concentrate 3.2 236 mL Grapefruit juice, from concentrate 5.5 236 mL Lemonade 3.0 236 mL Cranberry juice cocktail 2.5 236 mL Iced tea (commercial brands with lemon) 1.5 236 mL Powerade (various flavors) 1.2 236 mL Gatorade (orange) 2.0 236 mL Minute Maid cranberry/apple/raspberry drink 2.3 236 mL Nantucket Nectars (various flavors, average) 1.8 236 mL Tropicana Twister passionfruit 2.0 236 mL Snapple raspberry 1.4 236 mL Vitamin Water, power C 0.5 236 mL Sprite soda 0.8 236 mL Mountain Dew 0.8 236 mL Seagram’s ginger ale 0.9 236 mL America’s Choice lemon-lime soda 0.9 236 mL Diet Coke 0.07 236 mL Coke/Pepsi, regular < 0.03

Conclusions

The U.S. military has developed DU weapons that have been used in conflicts, particularly in Iraq. Studies have shown that significant amounts ( > 20 % of activity) of uranium oxide aerosols from DU penetrators strik- ing tanks have particle diameters less than 0.4 μm, which are easily inhaled, readily dissolve in biological fluids and are rapidly absorbed from the lung. Once absorbed as uranyl ion it can damage kidney tubules, bind to bone, enter liver and inhibit transport proteins and enzymes. Increases in birth defects and several cancers in Southern Iraq following the 1991–92 use of DU weapons on a large scale in that area and following the U.S. assaults on Fallujah in 2005 might be due to exposure to uranium oxide aerosols produced in those military actions. Of the chelation therapies that have been studied in animals, it appears that citric acid and citrate salts might be the most practical to employ in conflict areas where DU weapons have been used. Citric acid/citrate consumption should be recommended to anyone in areas where uranium aerosols might be found. It is important to avoid fructose and/or sucrose that may be in some beverages that contain high levels of citrates.

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