Self-assembled monolayer on mesoporous supports (SAMMS™) are hybrid materials created from attachment of organic moieties onto very high surface area mesoporous silica. SAMMS with surface chemistries including three isomers of hydroxypyridinone, diphosphonic acid, acetamide phosphonic acid, glycinyl urea, and diethylenetriamine pentaacetate (DTPA) analog were evaluated for chelation of actinides (239Pu, 241Am, uranium, thorium) from blood. Direct blood decorporation using sorbents does not have toxicity or renal challenges associated with traditional chelation therapy and may have potential applications for critical exposure cases, reduction of nonspecific dose during actinide radiotherapy, and for sorbent hemoperfusion in renal insufficient patients, whose kidney clear radionuclides at very slow rate. Sorption affinity (Kd), sorption rate, selectivity, and stability of SAMMS were measured in batch contact experiments. An isomer of hydroxypyridinone (3,4-HOPO) on SAMMS demonstrated the highest affinity for all four actinides from blood and plasma and greatly outperformed the DTPA analog on SAMMS and commercial resins. In batch contact, a fifty percent reduction of actinides in blood was achieved within minutes, and there was no evidence of protein fouling or material leaching in blood after 24 hr. The engineered form of SAMMS (bead format) was further evaluated in a 100-fold scaled-down hemoperfusion device and showed no blood clotting after 2 hr. A 0.2 g quantity of SAMMS could reduce 50 wt.% of 100 ppb uranium in 50 mL of plasma in 18 min and that of 500 dpm mL−1 in 24 min. 3,4-HOPO-SAMMS has a long shelf-life in air and at room temperature for at least 8 years, indicating its feasibility for stockpiling in preparedness for an emergency. The excellent efficacy and stability of SAMMS materials in complex biological matrices suggest that SAMMS can also be used as orally administered drugs and for wound decontamination. By changing the organic groups of SAMMS, they can be used not only for actinides but also for other radionuclides. By using the mixture of these SAMMS materials, broad spectrum decorporation of radionuclides is very feasible.
actinide; plutonium; americium; uranium; thorium; chelation therapy; SAMMS
Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small angle X-ray scattering, receptor binding assays, and synchrotron X-ray fluorescence microscopy we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway, receptor-mediated endocytosis of the iron transport protein serum transferrin; however only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small angle scattering demonstrate that only transferrin with plutonium bound in the protein’s C-terminal lobe and iron bound in the N-lobe (PuCFeNTf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin’s two lobes act to restrict, but not eliminate, cellular Pu uptake.
To study the cellular responses to molecular and polymeric forms of plutonium using PC12 cells derived from rat adrenal glands.
Materials and methods
Serum starved PC12 cells were exposed to polymeric and molecular forms of plutonium for three hours. Cells were washed with 10 mM EGTA, 100 mM NaCl at pH 7.4 to remove surface sorbed plutonium. Localization of plutonium in individual cell was quantitatively analyzed by synchrotron X-ray fluorescence (XRF) microscopy.
Molecular plutonium complexes introduced to cell growth media in the form of NTA, citrate, or transferrin complexes were taken up by PC12 cells, and mostly co-localized with iron within the cells. Polymeric plutonium prepared separately was not internalized by PC12 cells but it was always found on the cell surface as big agglomerates; however polymeric plutonium formed in situ was mostly found within the cells as agglomerates.
PC12 cells can differentiate molecular and polymeric forms of plutonium. Molecular plutonium is taken up by PC12 cells and mostly co-localized with iron but aged polymeric plutonium is not internalized by the cells.
PC12 Cells; plutonium polymer; molecular plutonium; X-ray fluorescence microscopy
We examined the ability of the metal-reducing bacteria Geobacter metallireducens GS-15 and Shewanella oneidensis MR-1 to reduce Pu(VI) and Pu(V). Cell suspensions of both bacteria reduced oxidized Pu [a mixture of Pu(VI) and Pu(V)] to Pu(IV). The rate of plutonium reduction was similar to the rate of U(VI) reduction obtained under similar conditions for each bacteria. The rates of Pu(VI) and U(VI) reduction by cell suspensions of S. oneidensis were slightly higher than the rates observed with G. metallireducens. The reduced form of Pu was characterized as aggregates of nanoparticulates of Pu(IV). Transmission electron microscopy images of the solids obtained from the cultures after the reduction of Pu(VI) and Pu(V) by S. oneidensis show that the Pu precipitates have a crystalline structure. The nanoparticulates of Pu(IV) were precipitated on the surface of or within the cell walls of the bacteria. The production of Pu(III) was not observed, which indicates that Pu(IV) was the stable form of reduced Pu under these experimental conditions. Experiments examining the ability of these bacteria to use Pu(VI) as a terminal electron acceptor for growth were inconclusive. A slight increase in cell density was observed for both G. metallireducens and S. oneidensis when Pu(VI) was provided as the sole electron acceptor; however, Pu(VI) concentrations decreased similarly in both the experimental and control cultures.
Unfertilized eggs and fertilized embryos from Drosophila mothers mutant for the plutonium (plu) gene contain giant polyploid nuclei resulting from unregulated S-phase. The PLU protein, a 19-kDa ankyrin repeat protein, is present in oocytes and early embryos but is not detectable after the completion of the initial rapid S-M cycles of the embryo. The persistence of the protein during the early embryonic divisions is consistent with a direct role in linking S-phase and M-phase. When ectopically expressed in the eye disc, PLU did not perturb the cell cycle, suggesting that PLU regulates S-phase only in early embryonic development. The pan gu (png) and giant nuclei (gnu) genes also affect the S-phase in the unfertilized egg and early embryo. We show that functional png is needed for the presence of PLU protein. By analyzing png mutations of differing severity, we find that the extent of the png mutant phenotype inversely reflects the level of PLU protein. Our data suggest that PLU protein is required at the time of egg activation and the completion of meiosis.
The purpose was to obtain quantitative data on plutonium microdistribution in different structural elements of human bone tissue for local dose assessment and dosimetric models validation. A sample of the thoracic vertebra was obtained from a former Mayak worker with a rather high plutonium burden. Additional information was obtained on occupational and exposure history, medical history, and measured plutonium content in organs. Plutonium was detected in bone sections from its fission tracks in polycarbonate film using neutron-induced autoradiography. Quantitative analysis of randomly selected microscopic fields on one of the autoradiographs was performed. Data included fission fragment tracks in different bone tissue and surface areas. Quantitative information on plutonium microdistribution in human bone tissue was obtained for the first time. From these data, quantitative relationship of plutonium decays in bone volume to decays on bone surface in cortical and trabecular fractions were defined as 2.0 and 0.4, correspondingly. The measured quantitative relationship of decays in bone volume to decays on bone surface does not coincide with recommended models for the cortical bone fraction by the International Commission on Radiological Protection. Biokinetic model parameters of extrapulmonary compartments might need to be adjusted after expansion of the data set on quantitative plutonium microdistribution in other bone types in human as well as other cases with different exposure patterns and types of plutonium.
plutonium; skeleton; neutron-induced autoradiography
Mice were exposed to plutonium dioxide (PuO2) aerosols 2 weeks before or after urethan injection. Both exposures reduced the number and size of adenomas. The incidence of arrested metaphases showed no consistently significant differences between plutonium-exposed and mock-exposed animals. The results are discussed in relation to recent electron microscopic evidence of degenerative changes in the type II epithelial cells of the mouse lung following PuO2 inhalation. It is concluded that damage at the cellular level may account for the observed reduction in growth of pulmonary adenomas in mice whose lungs contained plutonium particles.
We have investigated the possibility that transgenerational effects from preconceptional paternal irradiation (PPI) may render offspring more vulnerable to secondary exposure to an unrelated carcinogen. 239Pu (0, 128 or 256 Bq g(-1)) was administered by intravenous injection to male mice, 12 weeks before mating with normal females. Two strains of mouse were used -- CBA/H and BDF1. Haemopoietic spleen colony-forming units (CFU-S) and fibroblastoid colony-forming units (CFU-F), a component of their regulatory microenvironment, were assayed independently in individual offspring at 6, 12 and 19 weeks of age. Bone marrow and spleen from each of these mice were grown in suspension culture for 2 or 7 days for assessment of chromosomal aberrations. Female BDF1 were injected with methyl-nitroso-urea (MNU) as a secondary carcinogen at 10 weeks of age and monitored for onset of leukaemia/lymphoma. Mean values of CFU-S and CFU-F were unaffected by preconceptional paternal plutonium-239 (PP-239Pu), although for CFU-F in particular there was an apparent increase in variation between individual animals. There was significant evidence of an increase in chromosomal aberrations with dose in bone marrow but not in spleen. By 250 days, 68% of MNU-treated control animals (no PPI) had developed thymic lymphoma (62%) or leukaemia (38%). The first case arose 89 days after MNU administration. In the groups with PPI, leukaemia/lymphoma developed from 28 days earlier, rising to 90% by 250 days. Leukaemia (65%) now predominated over lymphoma (35%). This second generation excess of leukaemia appears to be the result of PPI and may be related to inherited changes that affect the development of haemopoietic stem cells.
This study describes the effect of intratracheal instillations (2 X 5 mg) of benzo(a)pyrene (B(a)P) on lung carcinogenesis in rats which had previously inhaled different levels of 239 plutonium oxide (220, 630, 6300 Bq, initial lung burden). Survival decreased with increasing PuO2 exposure and additional B(a)P exposure. The incidence of malignant lung tumours, adjusted for differences in survival, increased in a dose-related fashion with PuO2 dose and was elevated in the presence of additional B(a)P exposure. A multiplicative relative risk model was found to describe reasonably well the observed joint effect. The practical implications of these findings are discussed.
The Fukushima Daiichi nuclear power plant (DNPP) accident caused massive releases of radioactivity into the environment. The released highly volatile fission products, such as 129mTe, 131I, 134Cs, 136Cs and 137Cs were found to be widely distributed in Fukushima and its adjacent prefectures in eastern Japan. However, the release of non-volatile actinides, in particular, Pu isotopes remains uncertain almost one year after the accident. Here we report the isotopic evidence for the release of Pu into the atmosphere and deposition on the ground in northwest and south of the Fukushima DNPP in the 20–30 km zones. The high activity ratio of 241Pu/239+240Pu (> 100) from the Fukushima DNPP accident highlights the need for long-term 241Pu dose assessment, and the ingrowth of 241Am. The results are important for the estimation of reactor damage and have significant implication in the strategy of decontamination.
VERA, the Vienna Environmental Research Accelerator, is especially equipped for the measurement of actinides, and performs a growing number of measurements on environmental samples. While AMS is not the optimum method for each particular plutonium isotope, the possibility to measure 239Pu, 240Pu, 241Pu, 242Pu and 244Pu on the same AMS sputter target is a great simplification. We have obtained a first result on the global fallout value of 244Pu/239Pu = (5.7 ± 1.0) × 10−5 based on soil samples from Salzburg prefecture, Austria. Furthermore, we suggest using the 242Pu/240Pu ratio as an estimate of the initial 241Pu/239Pu ratio, which allows dating of the time of irradiation based solely on Pu isotopes. We have checked the validity of this estimate using literature data, simulations, and environmental samples from soil from the Salzburg prefecture (Austria), from the shut down Garigliano Nuclear Power Plant (Sessa Aurunca, Italy) and from the Irish Sea near the Sellafield nuclear facility. The maximum deviation of the estimated dates from the expected ages is 6 years, while relative dating of material from the same source seems to be possible with a precision of less than 2 years. Additional information carried by the minor plutonium isotopes may allow further improvements of the precision of the method.
Minor plutonium isotopes; Plutonium dating; Environmental plutonium; AMS
Microprobe X-ray absorption near edge structure (μ-XANES) measurements were used to determine directly, for the first time, the oxidation state of intracellular plutonium in individual 0.1 μm2 areas within single rat pheochromocytoma cells (PC12). The living cells were incubated in vitro for 3 hours in the presence of Pu added to the media in different oxidation states (Pu(III), Pu(IV), and Pu(VI)) and in different chemical forms. Regardless of the initial oxidation state or chemical form of Pu presented to the cells, the XANES spectra of the intracellular Pu deposits was always consistent with tetravalent Pu even though the intracellular milieu is generally reducing.
Plutonium can enter the body through different routes and remains there for decades; however its specific biochemical interactions are poorly defined. We, for the first time, have studied plutonium-binding proteins using a metalloproteomic approach with rat PC12 cells. A combination of immobilized metal ion chromatography, 2D gel electrophoresis, and mass spectrometry were employed to analyze potential plutonium-binding proteins. Our results show that several proteins from PC12 cells show affinity towards Pu4+-NTA (plutonium bound to nitrilotriacetic acid). Proteins from seven different spots in the 2D gel were identified. In contrast to the previously known plutonium-binding proteins transferrin and ferritin, which bind ferric ions, most identified proteins in our experiment are known to bind calcium, magnesium, or divalent transition metal ions. The identified plutonium interacting proteins also have functional roles in downregulation of apoptosis and other pro-proliferative processes. MetaCore analysis based on this group of proteins produced a pathway with a statistically significant association with development of neoplastic diseases.
PC12 cells; 2-D gel electrophoresis; proteomics; plutonium-binding proteins; IMAC; anti-apoptotic; GO process
The contamination of Japan after the Fukushima accident has been investigated mainly for volatile fission products, but only sparsely for actinides such as plutonium. Only small releases of actinides were estimated in Fukushima. Plutonium is still omnipresent in the environment from previous atmospheric nuclear weapons tests. We investigated soil and plants sampled at different hot spots in Japan, searching for reactor-borne plutonium using its isotopic ratio 240Pu/239Pu. By using accelerator mass spectrometry, we clearly demonstrated the release of Pu from the Fukushima Daiichi power plant: While most samples contained only the radionuclide signature of fallout plutonium, there is at least one vegetation sample whose isotope ratio (0.381 ± 0.046) evidences that the Pu originates from a nuclear reactor (239+240Pu activity concentration 0.49 Bq/kg). Plutonium content and isotope ratios differ considerably even for very close sampling locations, e.g. the soil and the plants growing on it. This strong localization indicates a particulate Pu release, which is of high radiological risk if incorporated.
Occupational risks, the present nuclear threat, and the potential danger associated with nuclear power have raised concerns regarding the metabolism of plutonium in pregnant women.
We measured plutonium levels in the milk teeth of children born between 1951 and 1995 to assess the potential risk that plutonium incorporated by pregnant women might pose to the radiosensitive tissues of the fetus through placenta transfer.
We used milk teeth, whose enamel is formed during pregnancy, to investigate the transfer of plutonium from the mother’s blood plasma to the fetus. We measured plutonium using sensitive sector field inductively coupled plasma mass spectrometry techniques. We compared our results with those of a previous study on strontium-90 (90Sr) released into the atmosphere after nuclear bomb tests.
Results show that plutonium activity peaks in the milk teeth of children born about 10 years before the highest recorded levels of plutonium fallout. By contrast, 90Sr, which is known to cross the placenta barrier, manifests differently in milk teeth, in accordance with 90Sr fallout deposition as a function of time.
These findings demonstrate that plutonium found in milk teeth is caused by fallout that was inhaled around the time the milk teeth were shed and not from any accumulation during pregnancy through placenta transfer. Thus, plutonium may not represent a radiologic risk for the radiosensitive tissues of the fetus.
fetus exposure; milk teeth; nuclear bomb test fallout; placenta transfer; plutonium; plutonium metabolism
The bacterial reduction of actinides has been suggested as a possible remedial strategy for actinide-contaminated environments, and the bacterial reduction of Pu(VI/V) has the potential to produce highly insoluble Pu(IV) solid phases. However, the behavior of plutonium with regard to bacterial reduction is more complex than for other actinides because it is possible for Pu(IV) to be further reduced to Pu(III), which is relatively more soluble than Pu(IV). This work investigates the ability of the metal-reducing bacteria Geobacter metallireducens GS15 and Shewanella oneidensis MR1 to enzymatically reduce freshly precipitated amorphous Pu(IV) (OH)4 [Pu(IV)(OH)4(am)] and soluble Pu(IV)(EDTA). In cell suspensions without added complexing ligands, minor Pu(III) production was observed in cultures containing S. oneidensis, but little or no Pu(III) production was observed in cultures containing G. metallireducens. In the presence of EDTA, most of the Pu(IV)(OH)4(am) present was reduced to Pu(III) and remained soluble in cell suspensions of both S. oneidensis and G. metallireducens. When soluble Pu(IV)(EDTA) was provided as the terminal electron acceptor, cell suspensions of both S. oneidensis and G. metallireducens rapidly reduced Pu(IV)(EDTA) to Pu(III)(EDTA) with nearly complete reduction within 20 to 40 min, depending on the initial concentration. Neither bacterium was able to use Pu(IV) (in any of the forms used) as a terminal electron acceptor to support growth. These results have significant implications for the potential remediation of plutonium and suggest that strongly reducing environments where complexing ligands are present may produce soluble forms of reduced Pu species.
As a first phase in an investigation of the role of the soil microflora in Pu complex formation and solubilization in soil, the effects of Pu concentration, form, and specific activity on microbial types, colony-forming units, and CO2 evolution rate were determined in soils amended with C and N sources to optimize microbial activity. The effects of Pu differed with organism type and incubation time. After 30 days of incubation, aerobic sporeforming and anaerobic bacteria were significantly affected by soil Pu levels as low as 1 μg/g when Pu was added as the hydrolyzable 239Pu(NO3)4 (solubility, <0.1% in soil). Other classes of organisms, except the fungi, were significantly affected at soil Pu levels of 10 μg/g. Fungi were affected only at soil Pu levels of 180 μg/g. Soil CO2 evolution rate and total accumulated CO2 were affected by Pu only at the 180 μg/g level. Because of the possible role of resistant organisms in complex formation, the mechanisms of effects of Pu on the soil fungi were further evaluated. The effect of Pu on soil fungal colony-forming units was a function of Pu solubility in soil and Pu specific activity. When Pu was added in a soluble, complexed form [238Pu2(diethylenetriaminepentaacetate)3], effects occurred at Pu levels of 1 μg/g and persisted for at least 95 days. Toxicity was due primarily to radiation effects rather than to chemical effects, suggesting that, at least in the case of the fungi, formation of Pu complexes would result primarily from ligands associated with normal (in contrast to chemically-induced) biochemical pathways.