Role of the sodium-dependent phosphate co-transporters and of the phosphate complexes of uranyl in the cytotoxicity of uranium in LLC-PK1 cells.

Although uranium is a well-characterized nephrotoxic agent, very little is known at the cellular and molecular level about the mechanisms underlying the uptake and toxicity of this element in proximal tubule cells. The aim of this study was thus to characterize the species of uranium that are responsible for its cytotoxicity and define the mechanism which is involved in the uptake of the cytotoxic fraction of uranium using two cell lines derived from kidney proximal (LLC-PK(1)) and distal (MDCK) tubule as in vitro models. Treatment of LLC-PK(1) cells with colchicine, cytochalasin D, concanavalin A and PMA increased the sodium-dependent phosphate co-transport and the cytotoxicity of uranium. On the contrary, replacement of the extra-cellular sodium with N-methyl-D-glucamine highly reduced the transport of phosphate and the cytotoxic effect of uranium. Uranium cytotoxicity was also dependent upon the extra-cellular concentration of phosphate and decreased in a concentration-dependent manner by 0.1-10 mM phosphonoformic acid, a competitive inhibitor of phosphate uptake. Consistent with these observations, over-expression of the rat proximal tubule sodium-dependent phosphate co-transporter NaPi-IIa in stably transfected MDCK cells significantly increased the cytotoxicity of uranium, and computer modeling of uranium speciation showed that uranium cytotoxicity was directly dependent on the presence of the phosphate complexes of uranyl UO(2)(PO(4))(-) and UO(2)(HPO(4))(aq). Taken together, these data suggest that the cytotoxic fraction of uranium is a phosphate complex of uranyl whose uptake is mediated by a sodium-dependent phosphate co-transporter system.

Octadentate hydroxypyridinonate (HOPO) ligands for plutonium (i.v.): pharmacokinetics and oral efficacy.

Linear octadentate spermine based 3,4,3-LI(1,2-HOPO) and the mixed ligand, 3,4,3-LI(1,2-Me-3,2-HOPO), are the most effective agents for decorporation of Pu prepared so far; they are effective at low dosage, orally active, and of low toxicity at effective injected dosage. Their pharmacological properties are favourable for in vivo Pu chelation--penetration of extracellular water, useful residence in the circulation, substantial hepato-biliary excretion, low but useful GI absorption, and transitory residence in the kidneys. Reductions of body Pu were significant, compared with controls, when oral administration to normally fed mice (30 or 100 micromol kg(-1)) was delayed as long as 24 h after i.v. Pu injection. The HOPO ligands (10-100 micromol kg(-1)) or CaNa3-DTPA (100 or 300 micromol kg(-1)) were given orally to normally fed mice starting at 4 h after an i.v. Pu injection and continued 5 d per week for 3 weeks. 3,4,3-LI(1,2-HOPO) (100 micromol kg(-1)) reduced Pu in skeleton, liver, and body, to 44 +/- 9, 18 +/- 8, and 38 +/- 7% of controls, respectively, reductions significantly greater than with the mixed HOPO ligand or with three times more CaNa3-DTPA.

Effects of cadmium and uranium on some in vitro renal targets.

Metals are major pollutants not only in occupational settings but also in the general environment. Chronic exposure of workers has been related to severe damage, especially at the renal level. While toxic compounds such as metals are well known to severely impair tubular functions, it is clear that nephrotoxicants can act on various other renal targets, i.e., vascular and glomerular ones. In vitro models are available to assess these toxicities and can be used to better understand the different cell targets. This paper summarizes data obtained in our laboratory after exposure of isolated renal structures such as glomeruli, and cell cultures such as glomerular mesangial and tubular epithelial cells, to cadmium and uranium. Morphometric studies by image analysis of isolated glomeruli and mesangial cultured cells showed that cadmium and uranium induced a dose- and time-dependent glomerular contraction accompanied by disorganization of the cytoskeleton. Classical viability tests demonstrated various factors influencing the metal toxicity. The important roles of pH, extracellular protein concentrations and the nature of the anion accompanying the metal were demonstrated. These data obtained in in vitro models provide better understanding of the cytotoxicity after metal uptake and accumulation in glomerular and tubular cells. Moreover, the glomerular and tubular cytotoxicity they induce may be correlated with severe renal hemodynamic changes in vivo. Finally, we briefly present eventual improvements for in vitro renal models by the use of new cell models such as immortalized human cell lines or by the introduction of porous supports and perifusion devices.

Determination of the physical and chemical properties, biokinetics, and dose coefficients of uranium compounds handled during nuclear fuel fabrication in France.

The introduction of new ICRP recommendations, especially the new Human Respiratory Tract Model (HRTM) in ICRP Publication 66 led us to focus on some specific parameters related to industrial uranium aerosols collected between 1990 and 1999 at French nuclear fuel fabrication facilities operated by COGEMA, FBFC, and the CEA. Among these parameters, the activity median aerodynamic diameter (AMAD), specific surface area (SSA), and parameters describing absorption to blood f(r), s(r) and s(s) defined in ICRP Publication 66 were identified as the most relevant influencing dose assessment. This study reviewed the data for 25 pure and impure uranium compounds. The average value of AMAD obtained was 5.7 microm (range 1.1-8.5 microm), which strongly supports the choice of 5 microm as the default value of AMAD for occupational exposures. The SSA varied between 0.4 and 18.3 m2 g(-1). For most materials, values of the absorption parameters f(r), s(r), and s(s) derived from the in vitro experiments were generally consistent with those derived from the in vivo experiments. Using average values for each pure compound allowed us to classify UO2 and U3O8 as Type S, mixed oxides, UF4, UO3 and ADU as Type M, and UO4 as Type F based on the ICRP Publication 71 criteria. Dose coefficients were also calculated for each pure compound, and average values for each type of pure compound were compared with those derived using default values. Finally, the lung retention kinetics and urinary excretion rates for inhaled U03 were compared using material-specific and default absorption parameters, in order to give a practical example of the application of this study.

Reduction of uranium transfer by local chelation in simulated wounds in rats.

The aim of the paper is to develop a new approach to treat uranium-contaminated wounds. The efficacy of a local uranium chelator, carballylic amido bis phosphonic acid (CAPBP) was assessed using two different uranium compounds. Rats were contaminated by intramuscular injections of uranyl nitrate or an industrial U04 compound to simulate wound contamination. CAPBP was injected intramuscularly (i.m.) or intraperitoneally (i.p.) at a dosage of 30 micromol kg(-1). In one experiment, the local administration of CAPBP was combined with a systemic administration of ethane-1-hydroxy-1,1-biphosphonate (EHBP). The local CAPBP treatment resulted in increased retention of uranium at the wound site: about 30% for uranyl nitrate or U04 after the first day and about 15% of UO4 after the third day. Consequently, it reduced uranium translocation into the blood and deposition in the kidneys and bone. The combined treatment reduced the uranium deposits in the kidneys, bone and carcass to about one-half of those observed in controls 3 days after U04 contamination. The local CAPBP treatment increased the interval of time between contamination and uranium deposit in the target organs. Thus, it can increase the efficacy of nonspecific local treatments or specific systemic treatments. It could be given rapidly through spray or gel after an accident.

Efficacy of ethane-1-hydroxy-1,1-bisphosphonate (EHBP) for the decorporation of uranium after intramuscular contamination in rats.

PURPOSE: To obtain compounds that will effectively reduce the fixation of uranium in its main target organs: bone and kidney. There is an urgent need for a chelating agent that is suitable and available for human use. MATERIALS AND METHODS: The efficacy of ethane-1-hydroxy-1,1-bisphosphonate (EHBP), already in use as a therapeutic agent, was investigated in animal experiments. The effect of different treatment regimens was investigated on rats (EHBP: 50-100 micromol kg(-1); ligand/uranium ratio 2500 to 5000). RESULTS: The present study shows that one prompt injection of EHBP reduced uranium deposition in kidneys by a factor of five after acute intramuscular contamination in rats. At the same time, the total body uranium in the treated animals was 70% of controls. When the treatment was delayed 30 min after contamination, the kidney content was still reduced by a factor of two. CONCLUSIONS: EHBP has the advantage of clinical acceptance as a therapeutic agent for other purposes and its toxicity has been well studied. It therefore has a role in the treatment of human contamination with uranium.

Review and critical analysis of available in vitro dissolution tests.

The procedures recommended in Publications 30 and 66 by ICRP for calculating radiation doses from inhaled or ingested radionuclides include classification of material on the basis of different parameters, among which transportability plays a major role, The allocation of transportable Classes or absorption Types should, whenever possible, be based on animal or human data. However, when such in vivo data are unavailable, it becomes appropriate to consider the use of other approaches, among which in vitro dissolution techniques are reasonable alternatives. This paper reviews and critically analyzes in vitro dissolution techniques that have been described historically and recommends methods shown to be useful in estimating the in vivo solubility of radioactive particles.

The effects of the initial lung deposit on uranium biokinetics after administration as UF4 and UO4.

This study was designed to assess the effect of the initial lung deposit (ILD) on uranium biokinetics in rats after intracheal instillation of biologically soluble uranium compounds. Rats received various doses of either UO4 or UF4 dust. The uranium content was determined in the kidneys, lungs, remaining carcass, urine and faeces at intervals of up to 30 days. The percentages of uranium absorbed into blood, transferred to tissues, and excreted in urine were independent of the uranium lung deposit for the two compounds. The K/K + U ratio 24 h after installation (K is the per cent of uranium retained in the kidneys and U the per cent excreted in urine) which can be used to evaluate kidney function, was essentially constant in the range from 0.02 to 12.5 microg U g(-1) kidneys.

Intracellular behaviour of uranium(VI) on renal epithelial cell in culture (LLC-PK1): influence of uranium speciation.

The main objective of this work was to assess the potentiality of in vitro models to study and understand the uranium-induced cytotoxicity on renal cells. Cytotoxicity and morphological studies were performed in a tubular proximal original established cell line (LLC-PK1 cell line). Dose-dependent cytotoxicity response was obtained with the uranium bicarbonate complex. In vitro experiments revealed a toxicity of uranium-bicarbonate complexes after a 24-h exposition and for concentrations ranging from 7 x 10(-4) M to 10(-3) M. In contrast, a lack of cytotoxicity of uranium(VI) citrate complexes studied using the same experimental conditions was noticed. Furthermore, electron transmission microscopy and X-ray microanalysis studies, after exposition of LLC-PK1 cells to the uranium-bicarbonate system ([U] = 8 x 10(-4) M) revealed that uranium entered into the cells and it was precipitated within the cytoplasmic compartment as uranyl phosphate needles. Similar morphological studies conducted with citrate complexes did not show any intake of uranium by LLC-PK1 cells. Experiments conducted in phosphate free culture medium showed that uranium was incorporated as a soluble material and that the association of the metal with phosphate ions occurred in the cytoplasmic compartment of LLC-PK1 cells.

Efficacy of 3,4,3-LIHOPO for reducing the retention of uranium in rat after acute administration.

Decorporation therapy is the only known effective method of reducing the radiation dose to persons following accidental internal contamination with transportable radionuclides. Deposits of actinides in bone should be minimized because development of osteosarcoma appears to be related to internal exposure. In contrast with other actinides, such as plutonium or americium where chelating agent treatment is efficient, the therapeuric approaches used for cases of uranium contamination are widely ineffective. This is the first report on in vivo efficacy of a chelating agent, a siderophore analogue code named 3,4,3-LIHOPO, after systematic exposure to natural uranium in the rat. Using the classical antidotal therapy (sodium bicarbonate) for comparison, this ligand has been investigated for its ability to remove uranium from rats after intravenous or intramuscular injection as nitrate. Following an immediate single intramuscular or intravenous injection of 3,4,3-LIHOPO (30 mumol.kg-1) urinary excretion of uranium was greatly enhanced with a corresponding reduction 24 h later in kidney and bone uranium content (to about 20 and 50% of the control rat respectively). Under identical experimental conditions, sodium bicarbonate (640 mumol.kg-1) reduced the uranium content in kidney in kidney and bone only to about 90 and 70% of controls respectively, and there was less enhancement of uranium excretion. However, when treatment was delayed by 30 min and administered intraperitoneally, there was no marked difference in retention and excretion of uranium between the two compounds. As this ligand showed no apparent irreversible toxicity at effective dosages, it is concluded that the administration of the 3,4,3-LIHOPO chelating agent represents potentially a most significant advance for prompt treatment of uranium contamination, while a more detailed investigation is necessary on the possible advantage when treatment delayed.

In vitro chemical and cellular tests applied to uranium trioxide with different hydration states.

A simple and rapid in vitro chemical solubility test applicable to industrial uranium trioxide (UO3) was developed together with two in vitro cellular tests using rat alveolar macrophages maintained either in gas phase or in alginate beads at 37 degrees C. Industrial UO3 was characterized by particle size, X-ray, and IR spectra, and chemical transformation (e.g., aging and hydration of the dust) was also studied. Solvents used for the in vitro chemical solubility study included carbonates, citrates, phosphates, water, Eagle's basal medium, and Gamble's solution (simulated lung fluid), alone, with oxygen, or with superoxide ions. Results, expressed in terms of the half-time of dissolution, according to International Commission on Radiological Protection (ICRP) classification (D,W,Y), varied for different hydration states of UO3, showing a lower solubility of hydrated UO3 in solvents compared to basic UO3 or UO3 heated at 450 degrees C. Two in vitro cellular tests on cultured rat alveolar macrophages (cells maintained in gas phase and cells immobilized in alginate beads) were used on the same UO3 samples and generally showed a lower solution transfer rate in the presence of macrophages than in the culture medium alone. The results of in vitro chemical and cellular tests were compared, with four main conclusions: a good reproducibility of the three tests in Eagle's basal medium the effect of hydration state on solubility, the classification of UO3 in terms of ICRP solubility criteria, and the ability of macrophages to decrease uranium solubility in medium.

In vitro solubility of uranium tetrafluoride with oxidizing medium compared with in vivo solubility in rats.

A simple in vitro solubility test for uranium tetrafluoride (UF4) was developed to investigate the effects of addition of enzymes, proteins or gases such as O2 to synthetic biological fluid or Gamble solvent. The tests were conducted concomitantly with an in vivo inhalation experiment using male rats. In the presence of Gamble solvent alone, UF4 exhibited class Y behaviour with a dissolution half-time of 300-500 days. However, when oxygen or carbonates were added to the Gamble solvent, UF4 displayed class W behaviour, with a half-time of 25-50 days. Lastly, in the presence of oxygen and pyrogallol, which simulates the action of the enzyme NADPH, the superoxide ion (O2-) was formed, which appears to have a dominant role in the oxidation of U4+ to U6+. Under these conditions UF4 behaved like a class D compound, and had a dissolution half-time of only 2-3 days. These latter results correlated with those of the inhalation experiment in which the dissolution half-time was calculated to be between 2.5 and 5.2 days. The data are also in agreement with urine monitoring data obtained for workers exposed to UF4 over a 20-year period.