Research ArticleThorium decorporation efficacy of rationally-selected biocompatible compounds with relevance to human application
Graphical abstract
Introduction
Despite good safety standards in nuclear industries, a risk of internalization of actinides in humans exists during handling of radioactive materials or accidents at nuclear facilities like Chernobyl and Fukushima Dai-ichi reactors [[1], [2], [3]]. In addition, nuclear war or terrorism also present serious concerns of internal contamination of actinides to general public. The radioactive actinides enter the body through inhalation, absorption through skin breaks/open wounds or by ingestion of contaminated water/food [4]. Studies on French CEA-AREWA centres (Alternative Energies and Atomic Energy Commission) between 1970–2003 revealed that both inhalation and wound routes are responsible for ∼81% of total internal contamination in humans [4]. Within few hours (1–3 h) of internalization, depending on the size (1–20 μm) and solubility/chemical form (nitrate, chloride, dioxide), actinides are distributed by the blood and deposited in the liver, bone and lungs (in case of inhalation), where they are retained by the tissues for several years (biological half-life ∼5 to 20 years) [5]. They cause both chemical as well as radiological toxicity due to their complexation with biomolecules and emission of high energy alpha-particles (4–5 Mev) [6]. Therefore, there is a need to develop efficient countermeasures for medical management of actinide contamination.
Among the actinides, uranium (U) has been extensively used for nuclear energy generation either due to the natural occurrence of its fissile form (U-235) or its potential to be converted into fissile fuel (U-238 to Pu-239) [3]. Internal contamination with U and plutonium (Pu) in humans have been reported under occupational, accidental or as a result of nuclear weapons test fallout [2,5,7]. In addition, the threat of nuclear terrorism involving Pu poses a significant risk of contamination to the general public [8]. In view of depletion of natural U-235, several countries including India have planned to develop thorium (Th)-based nuclear reactors due to several potential advantages such as i) large natural Th-232 reserves, ii) resistance for weapons use, and iii) significantly less generation of fission products and long-lived alpha-emitters in the nuclear waste [9,10]. The large-scale handling of Th-containing materials such as monazite [ThO2 ∼2.5–10%] may increase the risks of occupational and accidental exposures of Th to nuclear workers and general public in future [[11], [12], [13], [14], [15]]. Notably, the long-term inhalation of Th-containing dust was found to cause pneumoconiosis and lung carcinogenesis in Chinese miners (average Th in lung, 0.4 mg) as compared to unexposed workers [15].
Th-232, an alpha particle emitter (radiological half-life 14 × 109 years) may internalize in the form of nitrate or oxide during various steps of fuel cycle (mining, milling, extraction, fabrication and disposal) [5,16]. Under physiological conditions, Th is stable in + IV oxidation state, which forms strong complexes with biological ligands in blood/tissues [16]. Due to the closeness of charge-to-ionic-radii-ratio of Th (0.042) with iron (0.046), Th binds iron(Fe)-binding sites and mimics the mechanism of Fe-transport and Fe-storage/utilization in liver and bone [17]. Our recent study showed that the charge-to-ionic-radii-ratio of actinides plays an important role in determining their interactions with Fe-binding sites in hemoglobin (Hb) [18]. Our previous animal studies and other’s report on human data showed that following injection, Th mainly accumulates in liver, spleen and bone [19]. Studies on patients, receiving Thorotrast (a contrast agent used in 1930–1950) injections were found to have liver and bone cancers after ∼20 years [20]. Our animal studies showed that Th induces oxidative-stress-mediated toxicity in liver [19]. Together, these results suggested a clinical need for efficient decorporation of Th to significantly reduce chemical and radiation toxicities. For internal contamination, the FDA-approved diethylenetriaminepentaacetic acid (DTPA), which forms stable complex with Pu4+, americium (Am3+) and curium (Cm3+), is recommended to enhance the excretion of actinides [3,4]. However, it has several limitations: i) short retention time in blood plasma (T1/2 ∼1.5 h), ii) inability to reach actinide-depository organs and iii) needs intravenous administration. Also, its long-term use leads to calcium and magnesium deficiency, which causes neurotoxicity, nephrotoxicity, hematopoiesis suppression, etc [5]. Although, some national [21] and international guidelines [22] still recommend DTPA to treat contamination with Th, DTPA is reportedly ineffective for Th decorporation [23]. Therefore, there is a need to develop efficient, and less toxic Th-chelators, which can be used alone or in combination with DTPA.
In recent past, attempts have been made to improve decorporation efficacy of DTPA by either chemical or pharmacological modifications [4,24,25]. Alternatively, a new class of chelating agent i.e. siderophore analogue has also been synthesized and tested for actinide (mainly Pu) decorporation [26]. However, no efforts have been made to explore the Th-decorporation potential in rationally-selected natural or synthetic compounds, which could be efficiently evaluated for clinical application. In the present study, in order to explore more efficient and biocompatible (less toxic) agent(s) than DTPA, ten natural/synthetic compounds/formulation (Scheme 1) were selected on the basis of the following rationales: i) compounds with known Fe-binding or other metal-binding property viz. tiron, desferioxamine, d-penicillamine [27], as Th behaves similar to Fe(III) in vivo; or ii) phosphate-containing biomolecule, phytic acid (PA) as Th forms strong complexes with phosphate ligands [28]; or iii) compounds/formulations with known hepatoprotective action for human viz. Liv.52® (L52), silibin (SLB) [29,30] as Th preferentially accumulates in liver; or iv) compounds structurally similar to SLB viz. rutin, quercetin, myricetin and kaempferol [31]. Since, Th induces lysis of erythrocytes (hemolysis) [32], this biological end point was used to screen the relative efficacies of the selected compounds. The effective compounds were further assessed for their ability to decorporate Th from human erythrocytes, blood and liver cells, which were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS) and transmission electron microscopy (TEM). Furthermore, density functional theory (DFT)-based calculations and extended X-ray absorption fine structure (EXAFS) spectroscopy were performed to investigate the complexation of Th with the most effective agents.
Section snippets
Materials
232Th-nitrate was obtained from the Radiochemistry Division of our Centre. DTPA (Cat. No. 6518), desferioxamine (D9533), d-penicillamine (P4875), tiron (172553), silibin (S0417), rutin (R5143), kaempferol (60010), quercetin (Q0125), myricetin (M6760), phytic acid (68388) were purchased from Sigma, MO, USA. Polyherbal hepatoprotective formulation, Liv.52® syrup (L52) was purchased from The Himalaya Drug Company, India.
Preparation of solutions
232Th-nitrate stock solutions (10–100 mM), prepared in 0.01 N HNO3 were
Screening of agents for Th decorporation using hemolysis assay
The IC50 values (i.e. concentration of test agent required to prevent hemolysis by 50%) of the anti-hemolysis activities of the test agents, under both pre- and post-treatment conditions were determined from the graphs shown in Fig. 1 and listed in Table 1. The results revealed that among the selected agents, tiron, SLB, L52 and PA, but not others (based on their IC50 values) were more effective than the FDA-approved actinide decorporation agent, DTPA, under the pre-treatment conditions (Table 1
Discussion
In the present study, screening of ten rationally-selected compounds/formulation using hemolysis assay has identified tiron, SLB, PA and L52 as potential physiological Th decorporating agents. Our previous work suggested that Th induces hemolysis by acting on surface sialic acid of human erythrocytes [32]. Therefore, this assay was applied to evaluate the relative Th-complexing abilities of test agents vis-à-vis that of DTPA. In the pre-treatment experiments (Fig. 1), the effectiveness of test
Conclusions
The present study screened the ten rationally-selected compounds/formulation, which has identified tiron, PA, SLB and L52, as potential biocompatible agents for Th decorporation. The Th-decorporation ability of these agents was validated using relevant human cell models and blood as compared to DTPA, a FDA-approved actinide decorporation drug. TEM studies provided the evidence about their Th decorporation ability in liver cells. Furthermore, EXAFS and DFT studies confirmed their Th complexation
Conflicts of interest
There are no conflict of interest.
Acknowledgements
BARC, Department of Atomic Energy, Government of India supported the research work. Authors thank Dr. Vinay Kumar, Head, RB&HSD, BARC and Dr. S. Chattopadhyay, Ex-Director, Biosciences Group, BARC for their intellectual support and guidance during the work. We acknowledge Dr. S. N. Jha, AMPD, BARC for his support in EXAFS at RRCAT, Indore and Smt. Sharda Sawant, ACTREC, Navi Mumbai for TEM. BS thanks Dr. R. K. Gopalakrishnan and Dr. K. S. Pradeepkumar, HSEG, BARC for their support.
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