Chelation therapy with 3,4,3-Li(1,2-HOPO) after pulmonary exposure to plutonium in rats.

Internal exposure to plutonium can occur through inhalation for the nuclear worker, but also for the public if the radionuclide was released into the atmosphere in the context of a nuclear accident or terrorist attack. DieThylenetriaminePentaAcetic acid (DTPA) is currently still the only authorized chelator that can be used to decorporate internalized plutonium. The Linear HydrOxyPyridinOne-based ligand named 3,4,3-Li(1,2-HOPO) remains the most promising drug candidate to replace it in the hopes of improving chelating treatment. This study aimed to assess the efficacy of 3,4,3-Li(1,2-HOPO) in removing plutonium from rats exposed to the lungs, depending on the timing and route of treatment, and almost always compared to DTPA at a ten-fold higher dose used as a reference chelator. First, early intravenous injection or inhalation of 3,4,3-Li(1,2-HOPO) demonstrated superior efficacy over DTPA in preventing plutonium accumulation in liver and bone in rats exposed by injection or lung intubation. However, this superiority of 3,4,3-Li(1,2-HOPO) was much less pronounced with delayed treatment. In rats given plutonium in the lungs, the experiments also showed that 3,4,3-Li-HOPO reduced pulmonary retention of plutonium more effectively than DTPA only when the chelators were injected early but not at delayed times, while it was always the better of the two chelators when they were inhaled. Under our experimental conditions, the rapid oral administration of 3,4,3-Li(1,2-HOPO) was successful in preventing systemic accumulation of plutonium, but not in decreasing lung retention. Thus, after exposure to plutonium by inhalation, the best emergency treatment would be the rapid inhalation of a 3,4,3-Li(1,2-HOPO) aerosol to limit pulmonary retention of plutonium and prevent extrapulmonary deposition of plutonium in target systemic tissues.

Lauriston S. Taylor Lecture: the quest for therapeutic actinide chelators.

All of the actinides are radioactive. Taken into the body, they damage and induce cancer in bone and liver, and in the lungs if inhaled, and U(VI) is a chemical kidney poison. Containment of radionuclides is fundamental to radiation protection, but if it is breached accidentally or deliberately, decontamination of exposed persons is needed to reduce the consequences of radionuclide intake. The only known way to reduce the health risks of internally deposited actinides is to accelerate their excretion with chelating agents. Ethylendiaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) were introduced in the 1950's. DTPA is now clinically accepted, but its oral activity is low, it must be injected as a Ca(II) or Zn(II) chelate to avoid toxicity, and it is structurally unsuitable for chelating U(VI) or Np(V). Actinide penetration into the mammalian iron transport and storage systems suggested that actinide ions would form stable complexes with the Fe(III)-binding units found in potent selective natural iron chelators (siderophores). Testing of that biomimetic approach began in the late 1970's with the design, production, and assessment for in vivo Pu(IV) chelation of synthetic multidentate ligands based on the backbone structures and Fe(III)-binding groups of siderophores. New efficacious actinide chelators have emerged from that program, in particular, octadentate 3,4,3-LI(1,2-HOPO) and tetradentate 5-LIO(Me-3,2-HOPO) have potential for clinical acceptance. Both are much more effective than CaNa3-DTPA for decorporation of Pu(IV), Am(III), U(VI), and Np(IV,V), they are orally active, and toxicity is acceptably low at effective dosage.

Actinide and metal toxicity to prospective bioremediation bacteria.

Bacteria may be beneficial for alleviating actinide contaminant migration through processes such as bioaccumulation or metal reduction. However, sites with radioactive contamination often contain multiple additional contaminants, including metals and organic chelators. Bacteria-based bioremediation requires that the microorganism functions in the presence of the target contaminant, as well as other contaminants. Here, we evaluate the toxicity of actinides, metals and chelators to two different bacteria proposed for use in radionuclide bioremediation, Deinococcus radiodurans and Pseudomonas putida, and the toxicity of Pu(VI) to Shewanella putrefaciens. Growth of D. radiodurans was inhibited at metal concentrations ranging from 1.8 microM Cd(II) to 32 mM Fe(III). Growth of P. putida was inhibited at metal concentrations ranging from 50 microM Ni(II) to 240 mM Fe(III). Actinides inhibited growth at mM concentrations: chelated Pu(IV), U(VI) and Np(V) inhibit D. radiodurans growth at 5.2, 2.5 and 2.1 mM respectively. Chelated U(VI) inhibits P. putida growth at 1.7 mM, while 3.6 mM chelated Pu(IV) inhibits growth only slightly. Pu(VI) inhibits S. putrefaciens growth at 6 mM. These results indicate that actinide toxicity is primarily chemical (not radiological), and that radiation resistance does not ensure radionuclide tolerance. This study also shows that Pu is less toxic than U and that actinides are less toxic than other types of metals, which suggests that actinide toxicity will not impede bioremediation using naturally occurring bacteria.

Plutonium targets the p16 gene for inactivation by promoter hypermethylation in human lung adenocarcinoma.

Lung cancer from radon or (239)plutonium exposure has been linked to alpha-particles that damage DNA through large deletions and point mutations. We investigated the involvement of an epigenetic mechanism, gene inactivation by promoter hypermethylation in adenocarcinomas from plutonium-exposed workers at MAYAK, the first Russian nuclear enterprise established to manufacture weapons plutonium. Adenocarcinomas were collected retrospectively from 71 workers and 69 non-worker controls. Lung adenocarcinomas were examined from workers and non-worker controls for methylation of the CDKN2A (p16), O(6)-methylguanine-DNA methyltransferase (MGMT), death associated protein kinase (DAP-K), and Ras effector homolog 1 genes (RASSF1A). The prevalence for methylation of the MGMT or DAP-K genes did not differ between workers and controls, while a higher prevalence for methylation of the RASSF1A gene was seen in tumors from controls. In marked contrast, the prevalence for methylation of p16, a key regulator of the cell cycle, was increased significantly (P = 0.03) in tumors from workers compared with non-worker controls. Stratification of plutonium exposure into tertiles also revealed a striking dose response for methylation of the p16 gene (P = 0.008). Workers in the plutonium plant where exposure to internal radiation was highest had a 3.5 times (C.I. 1.5, 8.5; P = 0.001) greater risk for p16 methylation in their tumors than controls. This increased probability for methylation approximated the 4-fold increase in relative risk for adenocarcinoma in this group of workers exposed to plutonium. In addition, a trend (P = 0.08) was seen for an increase in the number of genes methylated (> or =2 genes) with plutonium dose. Here we demonstrate that exposure to plutonium may elevate the risk for adenocarcinoma through specifically targeting the p16 gene for inactivation by promoter methylation.

Review of 239Pu and 226Ra effects in beagles.

A long term biological study has been completed that was designed to assess the predicted effects in humans of internally deposited 239Pu by comparison with 226Ra in beagles. Herein we summarize for the first time results of several previous reports about the effects of these two radionuclides in our beagles in an attempt to elucidate what has been learned since the beginning of the study in the early 1950's. Perhaps the most important finding was that bone surface-seeking plutonium is more toxic at equal mean skeletal radiation doses (<3 Gy for 239Pu, <20 Gy for 226Ra) than bone volume-seeking radium for the induction of skeletal malignancy by about a factor of 16 for a single intravenous injection of monomeric 239Pu. In addition, ancillary studies have shown that when plutonium transfers continuously onto bone surfaces from a depot of particulate 239Pu in phagocytic cells, its relative toxicity per Gy average skeletal dose is enhanced by about a factor of 2. Juvenile animals or dogs injected as mature adults were only about half as sensitive for equal mean skeletal doses as dogs injected as young adults. Male and female dogs were about equally sensitive to radiation of the skeleton by either radionuclide. Findings about radiation-induced fractures are summarized as well as data on the induction of soft-tissue malignancies by 239Pu or 226Ra. Natural survival was not affected at the lower dosage levels of either 226Ra or 239Pu as compared with control dogs given no radioactivity, but the survival of animals at higher levels was reduced. No additional life-shortening effects beyond those attributable to occurrence of radiation-induced malignancies or other radiation-induced effects were suggested by analysis of data for low dosage levels.

[Early ultrastructural lesions of kidney cells after intravenous administration of 239 plutonium citrate in rats]. / Lésions ultrastructurales précoces des cellules rénales après administration intraveineuse de citrate de plutonium 239 chez le rat.

After intravenous administration of (5 x 2.3 kBq and 5 x 9.25 kBq) plutonium citrate in adult male Sprague Dawley rats, their kidneys are withdrawn and prepared for observation under a transmission electron microscope. Seven days after the first injections, deep cellular alterations are observed in the proximal convoluted tubules. These alterations are mainly mitochondrial. The affected mitochondria are of swollen aspect and have their cristae partially or completely destroyed. Nevertheless within the same tubule we observe non altered cells directly in contact with deeply altered cells. In all the cases the lysosomes of the altered cells appear to be perfectly normal. The cell nuclei are mostly unaltered but a few cases of nuclear fragmentation exist. We also notice some architectural modifications in the brush border and in the betacytomembranes of the proximal convoluted tubule. Equally important mitochondrial alterations are also noticed in the different varieties of glomerular cells. We observe no other glomerular alterations. The major subcellular alterations in the proximal convoluted tubules and in the different varieties of glomerular cells deeply contrast with the distal convoluted tubules which are found to be totally unaltered. These mitochondrial alterations may be due to the alpha particle disintegration of plutonium which may either directly react with the mitochondria or, through the products of radiolysis of water react with the mitochondria respiration process. However the direct chemotoxicity of plutonium cannot be neglected.

Frequencies of complex chromosome exchange aberrations induced by 238Pu alpha-particles and detected by fluorescence in situ hybridization using single chromosome-specific probes.

We undertook an analysis of chromosome-type exchange aberrations induced by alpha-particles using fluorescence in situ hybridization (FISH) with whole chromosome-specific probes for human chromosomes 1 or 4, together with a pan-centromeric probe. Contact-inhibited primary human fibroblasts (in G1) were irradiated with 0.41-1.00 Gy 238Pu alpha-particles and aberrations were analysed at the next mitosis following a single chromosome paint. Exchange and aberration painting patterns were classified according to Savage and Simpson (1994a). Of exchange aberrations, 38-47% were found to be complex derived, i.e. resulting from three or more breaks in two or more chromosomes, and the variation with dose was minimal. The class of complex aberrations most frequently observed were insertions, derived from a minimum of three breaks in two chromosomes. There was also an elevated frequency of rings. The high level of complex aberrations observed after alpha-particle irradiation indicates that, when chromosome domains are traversed by high linear energy transfer alpha-particle tracks, there is an enhanced probability of production of multiple localized double-strand breaks leading to more complicated interactions.

Chromatid damage induced by 238Pu alpha-particles in G2 and S phase Chinese hamster V79 cells.

The comparative induction of chromatid aberrations by 238Pu alpha-particles, or by 250 kVp X-rays was investigated in V79 Chinese hamster cells. Metaphases were sampled at hourly intervals postirradiation up to 8 h and BrdU/FPG staining methods were used to distinguish G2, S and G1 phase cells. Two experiments were performed. In the first, an alpha-particle dose of 0.41 Gy was compared with an X-ray dose of 1.5 Gy used in a previously published study. In the second, an X-ray dose of 1.2 Gy was used in parallel with 0.41 Gy of alpha-particles to produce a similar overall frequency of interchanges, and allow comparative ratios to be derived for other aberration types. At these isoexchange doses, alpha-particles produce relatively less gaps and breaks, particularly in late G2, and significantly more isochromatid deletions. A very high proportion of the isochromatid deletions were incomplete after alpha-particles compared with X-rays, but no difference in incompleteness was found for interchanges. With X-rays, about 6% of interchanges are complex intra-interchange forms. At similar exchange frequencies this increases to 26.7% for alpha-irradiation, suggesting increased multiple lesion interaction. Differences in dose distribution between alpha-particles and X-rays are discussed and mitotic delay is examined after separation of the analysed cells into damaged and undamaged classes.

A relationship between residual stromal damage in hematopoietic tissue and the functional activity of granulocytes.

This paper analyzes the function of mouse granulocytes in the long term, after external irradiation with x- and gamma-rays and 239Pu contamination at different gestational ages and in a variety of culture conditions. These treatments can produce persistent defects in the stroma, which regulates hematopoiesis. Superoxide-anion production has been measured in granulocytes from peripheral blood and from long-term bone marrow cultures (LTBMC). A significant enhancement of O2- is produced using single or fractionated doses of x-rays; however, little or no increase is observed with gamma-rays. With 239Pu, enhancement of O2- depends on gestational age at contamination. The absence of hydrocortisone (HC) in LTBMC and the irradiation of the adherent layer with 15 Gy stimulate O2- production. The increased production of O2- appears to be correlated with an excess of colony-stimulating factors (CSFs) released to the supernatant by stromal cells. Neutralization with anti-granulocyte-macrophage CSF (anti-GM-CSF) monoclonal antibody shows that GM-CSF is the main factor produced. In summary, conditions that lead to residual stromal damage also result in the generation of granulocytes that are functionally primed for excess superoxide-anion production.

Effects of inhaled alpha-emitting actinides on mouse alveolar macrophages.

The effects of inhaled alpha-emitting actinides on the alveolar macrophage (AM) population of the rodent lung are reviewed and, in particular, of the effects of 239PuO2 on murine AM. The effects discussed include changes the AM pool size, macrophage diameter, mobility, phagocytic competence, and enzyme content. Finally, similarities in the dose-response relationships for the induction of nuclear aberrations by alpha emitters and in the induction of lung tumors by the same materials are noted.

[The significance of modifying factors in evaluating the effect of radionuclides]. / Znachenie modifitsiruiushchikh faktorov pri otsenke vozdeistviia radionuklidov.

The authors provide the data obtained in experimental animals, pertaining to the influence of certain modifying factors on metabolism and biological effects induced by radioactive isotopes of iodine, by transuranium elements and beryllium. Out of all the modifying factors investigated, the form of the introduced compound and age turned out the most significant ones.

[The effective and ineffective levels of the joint action of 239Pu and gamma radiation according to the change in the ECG indices of dogs]. / Effektivnye i neéffektivnye urovni sovmestnogo vozdeistviia 239Pu i gamma-izlucheniia po izmeneniiu nekotorykh pokazatelei EKG u sobak.

Effective and ineffective levels of the isolated and combined action of inhaled 239Pu and external gamma radiation on dogs were estimated by some changes in the ECG. Specific share of each factor in the combined effect and the quality coefficient of 239Pu were estimated by two criteria of changes in the ECG. The data were extrapolated to man by the most reliable criterion, that is, the decrease in the ECG voltage indicated by the sum of R I, II, III waves.

[The antibody-producing cell count of rats exposed to polymeric 239Pu]. / Soderzhanie antiteloobrazuiushchikh kletok u krys pri deistvii polimernogo 239Pu.

In experiments with Wistar rats a study was made of the content of antibody-forming cells (AFC) in the spleen at remote times (3 to 12 months) after intravenous injection of 239Pu(IV) in doses of 166, 55, and 18 kBq/kg body mass. The doses absorbed in the central and peripheral immunity organs were defined. Pronounced spleen hypoplasia and profound inhibition of humoral immunity were displayed 1 year after the injection of a small amount of the radionuclide. AFC deficiency in animals was amounted to 11-32 per cent throughout the entire period of observation.

Primary liver tumors in beagle dogs exposed by inhalation to aerosols of plutonium-238 dioxide.

Primary liver tumors developed in Beagle dogs exposed by inhalation to aerosols of 238PuO2. Initial deposition of 238PuO2 in the respiratory tract was followed by translocation of a portion of the 238Pu to the liver and skeleton, which resulted in a large dose commitment and tumor risk to all three tissues. In a population of 144 dogs exposed to 238PuO2, 112 dogs died or were killed 4000 days after 238Pu exposure, 100 dogs had osteosarcoma, and 28 dogs had lung cancers. At increasing times after exposure, however, liver lesions have become more pronounced. Ten primary liver tumors in nine animals were diagnosed in the dogs dying before 4000 days after exposure. An additional five primary liver tumors in three dogs occurred in 9 animals killed after 4000 days after exposure. The majority of these tumors have been fibrosarcomas. The liver tumors were usually not the cause of death, and rarely metastasized. The occurrence of liver tumors in this study indicates that 238Pu is an effective hepatic carcinogen. Liver carcinogenesis is assuming an increasing importance in this study at late times after inhalation exposure. These results suggest that the liver may be an important organ at risk for the development of neoplasia in humans at time periods long after inhalation of 238Pu.

[Alkaline phosphatase activity of the blood serum in dogs exposed jointly to external gamma irradiation and alpha radiation]. / Aktivnost' shchelochnoi fosfatazy v syvorotke krovi sobak pri sovmestnom vozdeistvii vneshnego gamma-oblucheniia i al'fa-izlucheniia.

Alkaline phosphatase activity in dog blood serum was studied for two years following separate and combined exposure to gamma radiation (6.45 to 51.6 mC/kg) and inhaled submicron 239Pu oxide, containing 25 per cent of 241Am, in chronically effective amounts (approximately 7-10 kBq/kg). Alkaline phosphatase activity was of an undulatory character, and the significance of changes depended upon the kind and the level of radiation as well as the time lapsed from the start of the exposure. With the combined exposure to gamma and alpha radiation in the doses used, no enhancement of the effect was noted as compared with the action of each factor applied separately.

[Cytogenetic effect of 238Pu alpha radiation on the germ cells of male mice]. / Tsitogeneticheskii éffekt al'fa-izlucheniia 238Pu v polovykh kletkakh samtsov myshi.

A study was made of the yield of reciprocal translocations in stem spermatogonia of mice exposed to alpha-radiation (238Pu) and whole-body acute and chronic gamma-radiation within a wide range of doses and dose-rates. The frequency of reciprocal translocations induced by a single intraperitoneal administration of plutonium nitrate was relatively low and independent of the dose 1.5-18 months after the effect. The yield of the reciprocal translocations induced by chronic effect of gamma-radiation was appreciably lower than that observed after acute irradiation with the same dose and grew linearly with dose. The RBE of alpha-radiation was 10-20 with respect to chronic effect of gamma-radiation.

[Absorbed doses and hematological shifts in dogs inhaling submicron 239Pu dioxide]. / Pogloshchennye dozy i gematologicheskie sdvigi u sobak pri ingaliatsii submikronnoi dvuokisi 239Pu.

In dogs breathing submicron 239Pu dioxide, the absorbed doses were determined in 12 organs and tissues where the radionuclide was deposited; the integral doses to a whole body were also determined by the sum of the exposed organs. The relationship of the hematologic changes not only with the doses for "critical" tissues but also with the integral dose was studied.

[Alkaline phosphatase and transaminase activity in the liver and blood serum of rats in the late periods following combined exposure to external gamma radiation and alpha radiation from plutonium-239]. / Aktivnost' shchelochnoi fosfatazy i transaminaz v pecheni i syvorotke krovi krys v otdalennye sroki posle sochetannogo vozdeistviia vneshnego gamma-izlucheniia i al'fa-izlucheniia plutoniia-239.

A study was made of activity of alkaline phosphatase and alanine- and aspartate aminotransferase in rat liver and blood serum at remote times after external gamma-irradiation combined with internal exposure to 239Pu nitrate delivered in two chronically effective doses. The radionuclide was shown to be mainly responsible for the changes observed in activity of the enzymes under study. The degree to which the changes were manifest depended upon dose of plutonium administered.

Population and ultrastructural changes in murine alveolar cells following 239PuO2 inhalation.

Early changes affecting the principal cellular components of pulmonary alveoli after inhaling 239plutonium dioxide were followed by quantitative and qualitative electron microscopy in mice and rats. Different accumulated doses of a irradiation were achieved. The numbers of alveolar macrophages and interstitial mononuclear cells rose in mice especially after a higher dose of radiation, whilst in rats interstitial fibroblasts were increased. The evidence from mice suggested pronounced secretory activity of type II epithelial cells with subsequent uptake of phospholipid by alveolar macrophages, which developed large cytoplasmic inclusions, but rats were much less severely affected. Pneumonitis was not a feature and with the dosage of radiation employed endothelium escaped structural damage. Sensitivity between species differed, both according to cell type and to intensity of exposure, so demanding caution in the application of experimental findings to man.