Unusual Versatility of the Filamentous, Diazotrophic Cyanobacterium Anabaena torulosa Revealed for Its Survival during Prolonged Uranium Exposure.

Reports on interactions between cyanobacteria and uranyl carbonate are rare. Here, we present an interesting succession of the metabolic responses employed by a marine, filamentous, diazotrophic cyanobacterium, Anabaena torulosa for its survival following prolonged exposure to uranyl carbonate extending up to 384 h at pH 7.8 under phosphate-limited conditions. The cells sequestered uranium (U) within polyphosphates on initial exposure to 100 µM uranyl carbonate for 24 to 28 h. Further incubation until 120 h resulted in (i) significant degradation of cellular polyphosphates causing extensive chlorosis and cell lysis, (ii) akinete differentiation followed by (iii) extracellular uranyl precipitation. X-ray diffraction (XRD) analysis, fluorescence spectroscopy, X-ray absorption near edge structure (XANES), and extended X-ray absorption fine structure (EXAFS) spectroscopy established the identity of the bioprecipitated uranium as a U(VI) autunite-type mineral, which settled at the bottom of the vessel. Surprisingly, A. torulosa cells resurfaced as small green flakes typical of actively growing colonies on top of the test solutions within 192 to 240 h of U exposure. A consolidated investigation using kinetics, microscopy, and physiological and biochemical analyses suggested a role of inducible alkaline phosphatase activity of cell aggregates/akinetes in facilitating the germination of akinetes leading to substantial regeneration of A. torulosa by 384 h of uranyl incubation. The biomineralized uranium appeared to be stable following cell regeneration. Altogether, our results reveal novel insights into the survival mechanism adopted by A. torulosa to resist sustained uranium toxicity under phosphate-limited oxic conditions.IMPORTANCE Long-term effects of uranyl exposure in cyanobacteria under oxic phosphate-limited conditions have been inadequately explored. We conducted a comprehensive examination of the metabolic responses displayed by a marine cyanobacterium, Anabaena torulosa, to cope with prolonged exposure to uranyl carbonate at pH 7.8 under phosphate limitation. Our results highlight distinct adaptive mechanisms harbored by this cyanobacterium that enabled its natural regeneration following extensive cell lysis and uranium biomineralization under sustained uranium exposure. Such complex interactions between environmental microbes such as Anabaena torulosa and uranium over a broader time range advance our understanding on the impact of microbial processes on uranium biogeochemistry.

Mechanism of Attenuation of Uranyl Toxicity by Glutathione in Lactococcus lactis.

UNLABELLED: Both prokaryotic and eukaryotic organisms possess mechanisms for the detoxification of heavy metals, and these mechanisms are found among distantly related species. We investigated the role of intracellular glutathione (GSH), which, in a large number of taxa, plays a role in protection against the toxicity of common heavy metals. Anaerobically grown Lactococcus lactis containing an inducible GSH synthesis pathway was used as a model organism. Its physiological condition allowed study of putative GSH-dependent uranyl detoxification mechanisms without interference from additional reactive oxygen species. By microcalorimetric measurements of metabolic heat during cultivation, it was shown that intracellular GSH attenuates the toxicity of uranium at a concentration in the range of 10 to 150 µM. In this concentration range, no effect was observed with copper, which was used as a reference for redox metal toxicity. At higher copper concentrations, GSH aggravated metal toxicity. Isothermal titration calorimetry revealed the endothermic binding of U(VI) to the carboxyl group(s) of GSH rather than to the reducing thiol group involved in copper interactions. The data indicate that the primary detoxifying mechanism is the intracellular sequestration of carboxyl-coordinated U(VI) into an insoluble complex with GSH. The opposite effects on uranyl and on copper toxicity can be related to the difference in coordination chemistry of the respective metal-GSH complexes, which cause distinct growth phase-specific effects on enzyme-metal interactions. IMPORTANCE: Understanding microbial metal resistance is of particular importance for bioremediation, where microorganisms are employed for the removal of heavy metals from the environment. This strategy is increasingly being considered for uranium. However, little is known about the molecular mechanisms of uranyl detoxification. Existing studies of different taxa show little systematics but hint at a role of glutathione (GSH). Previous work could not unequivocally demonstrate a GSH function in decreasing the presumed uranyl-induced oxidative stress, nor could a redox-independent detoxifying action of GSH be identified. Combining metabolic calorimetry with cell number-based assays and genetics analysis enables a novel and general approach to quantify toxicity and relate it to molecular mechanisms. The results show that GSH-expressing microorganisms appear advantageous for uranyl bioremediation.

Toxic influence of silver and uranium salts on activated sludge of wastewater treatment plants and synthetic activated sludge associates modeled on its pure cultures.

Toxic impact of silver and uranium salts on activated sludge of wastewater treatment facilities has been studied. Some dominating cultures (an active nitrogen fixer Agrobacterium tumifaciens (A.t) and micromyces such as Fusarium nivale, Fusarium oxysporum, and Penicillium glabrum) have been isolated and identified as a result of selection of the activated sludge microorganisms being steadiest under stressful conditions. For these cultures, the lethal doses of silver amounted 1, 600, 50, and 300 µg/l and the lethal doses of uranium were 120, 1,500, 1,000, and 1,000 mg/l, respectively. A.tumifaciens is shown to be more sensitive to heavy metals than micromyces. Synthetic granular activated sludge was formed on the basis of three cultures of the isolated micromyces steadiest against stress. Its granules were much more resistant to silver than the whole native activated sludge was. The concentration of silver causing 50 % inhibition of synthetic granular activated sludge growth reached 160-170 µg/l as far as for the native activated sludge it came only to 100-110 µg/l.

Rapid phenotypic changes in Caenorhabditis elegans under uranium exposure.

Pollutants can induce selection pressures on populations, and the effects may be concentration-dependant. The main ways to respond to the stress are acclimation (i.e. plastic changes) and adaptation (i.e. genetic changes). Acclimation provides a short-term response to environmental changes and adaptation can have longer-term implications on the future of the population. One way of studying these responses is to conduct studies on the phenotypic changes occurring across generations in populations experimentally subjected to a selective factor (i.e. multigenerational test). To our knowledge, such studies have not been performed with uranium (U). Here, the phenotypic changes were explored across three generations in experimental Caenorhabditis elegans populations exposed to different U-concentrations. Significant negative effects of U were detected on survival, generation time, brood size, body length and body bend. At lower U-concentrations, the negative effects were reduced in the second or the third generation, indicating an improvement by acclimation. In contrast, at higher U-concentrations, the negative effects on brood size were amplified across generations. Consequently, under high U-concentrations acclimation may not be sufficient, and adaptation of individuals would be required, to permit the population to avoid extinction. The results highlight the need to consider changes across generations to enhance environmental risk assessment related to U pollution.

Consequences of a multi-generation exposure to uranium on Caenorhabditis elegans life parameters and sensitivity.

The assessment of toxic effects at biologically and ecologically relevant scales is an important challenge in ecosystem protection. Indeed, stressors may impact populations at much longer term than the usual timescale of toxicity tests. It is therefore important to study the evolutionary response of a population under chronic stress. We performed a 16-generation study to assess the evolution of two populations of the ubiquitous nematode Caenorhabditis elegans in control conditions or exposed to 1.1 mM of uranium. Several generations were selected to assess growth, reproduction, survival, and dose-responses relationships, through exposure to a range of concentrations (from 0 to 1.2 mM U) with all endpoints measured daily. Our experiment showed an adaptation of individuals to experimental conditions (increase of maximal length and decrease of fecundity) for both populations. We also observed an increase of adverse effects (reduction of growth and fertility) as a function of uranium concentration. We pointed out the emergence of population differentiation for reproduction traits. In contrast, no differentiation was observed on growth traits. Our results confirm the importance of assessing environmental risk related to pollutant through multi-generational studies.

Homeostatic regulation of elemental stoichiometry by Lemna gibba L. G3 when nutrient interact with toxic metals.

We investigated responses of Lemna gibba L. to exposure to UO(2)(2+) and AsO(4)(3-) under variable PO(4)(3-) concentration. Total plant phosphorus (P(tot)) in L. gibba and accumulation of dissolved organic carbon (DOC) in the media were quantified and tested for correlation with plant yield and initial concentrations of PO(4)(3-), UO(2)(2+) and AsO(4)(3-). The accumulation of DOC in medium was high under low PO(4)(3-) supply and increased loading of either UO(2)(2+) or AsO(4)(3-). The P(tot) was low in high initial concentration of UO(2)(2+) and AsO(4)(3-) as well under acute low PO(4)(3-) supply. The DOC accumulation correlated negatively to the P(tot). This reveals interaction between PO(4)(3-) and UO(2)(2+) or AsO(4)(3-) in the medium interferes with the uptake process of PO(4) (3-). Hence, the DOC accumulation is exudation of low molecular weight organic substance by L. gibba in response to the reduced P(tot): biomass ratio (carbon in the yield) due to delimited acquisition of phosphorus from the medium. It is a homeostatic regulation of the stoichiometry, which is disturbed during the interaction between PO(4)(3-) and UO(2)(2+) or AsO(4)(3-). Further investigations are necessary to relate these interactions to traditional resource stoichiometry elements of C, N, and P.

Particulate depleted uranium is cytotoxic and clastogenic to human lung epithelial cells.

Depleted uranium (DU) is commonly used in military applications and consequently exposure to soldiers and non-combatants is potentially frequent and widespread. DU is suspected to be a carcinogen, potentially affecting the bronchial cells of the lung. Few studies have considered DU in human bronchial cells. Accordingly, we determined the cytotoxicity and clastogenicity of particulate DU in human bronchial epithelial cells (BEP2D cells). DU-induced concentration-dependent cytotoxicity in human bronchial epithelial cells, and was not clastogenic after 24h but induced chromosomal aberrations after 48h. These data indicate that if DU is a human bronchial carcinogen, it is likely acting through a mechanism that involves DNA breaks after longer exposures.

UO(2) 2+ speciation determines uranium toxicity and bioaccumulation in an environmental Pseudomonas sp. isolate.

In the present study, experiments were performed to investigate how representative cellulosic breakdown products, when serving as growth substrates under aerobic conditions, affect hexavalent uranyl cation (UO(2) (2+)) toxicity and bioaccumulation within a Pseudomonas sp. isolate (designated isolate A). Isolate A taken from the Cold Test Pit South (CTPS) region of the Idaho National Laboratory (INL), Idaho Falls, ID, USA. The INL houses low-level uranium-contaminated cellulosic material and understanding how this material, and specifically its breakdown products, affect U-bacterial interactions is important for understanding UO(2) (2+) fate and mobility. Toxicity was modeled using a generalized Monod expression. Butyrate, dextrose, ethanol, and lactate served as growth substrates. The potential contribution of bicarbonate species present in high concentrations was also investigated and compared with toxicity and bioaccumulation patterns seen in low-bicarbonate conditions. Isolate A was significantly more sensitive to UO(2) (2+) and accumulated significantly more UO(2) (2+) in low-bicarbonate concentrations. In addition, UO(2) (2+) growth inhibition and bioaccumulation varied depending on the growth substrate. In the presence of high bicarbonate concentrations, sensitivity to UO(2) (2+) inhibition was greatly mitigated, and did not vary between the four substrates tested. The extent of UO(2) (2+) accumulation was also diminished. The observed patterns were related to UO(2) (2+) aqueous complexation, as predicted by MINTEQ (ver. 2.52) (Easton, PA, USA). In the low- bicarbonate medium, the presence of positively charged and unstable UO(2) (2+)-hydroxide complexes explained both the greater sensitivity of isolate A to UO(2) (2+), and the ability of isolate A to accumulate significant amounts of UO(2) (2+). The exclusive presence of negatively charged and stable UO(2) (2+)-carbonate complexes in the high bi-carbonate medium explained the diminished sensitivity of isolate A to UO(2) (2+) toxicity, and limited ability of isolate A to accumulate UO(2) (2+).

A nested case-control study of multiple myeloma risk and uranium exposure among workers at the Oak Ridge Gaseous Diffusion Plant.

The primary risk factors of multiple myeloma are age, race and sex, but several studies have found an association between radiological hazards and multiple myeloma. The purpose of this nested case-control study was to investigate whether workers with chronic low-level exposure to internally deposited uranium at the Oak Ridge Gaseous Diffusion Plant in eastern Tennessee were at higher risk of dying of multiple myeloma than those without occupational exposure to uranium, with the consideration of potential confounders of external ionizing radiation and occupational chemical hazards such as mercury, nickel and trichloroethylene. The main analyses were carried out using conditional logistic regression on 98 cases and 490 controls (five controls matched to each case on gender, race and age at risk). Our study showed a weak association between internal uranium dose estimated from urinalysis results and multiple myeloma risk: OR = 1.04 (95% CI 1.00-1.09) at 10 microGy with the inclusion of other risk factors. The parameter estimates and the corresponding odds ratios were very similar when internal doses were imputed for subjects without urine samples. Further studies that include updating this cohort and combining with workers from other gaseous diffusion plants are needed to investigate the relationship between multiple myeloma risk and radiation or other chemical exposures.

Cytotoxic and phenotypic effects of uranium and lead on osteoblastic cells are highly dependent on metal speciation.

Bone is one of the main retention organs for uranium (U) and lead (Pb). The clinical effects of U or Pb poisoning are well known: acute and chronic intoxications impair bone formation. However, only few studies dealt with the cellular and molecular mechanisms of their toxicity. The purpose of this study was to investigate acute cytotoxicity of U and Pb and their phenotypic effects on rat and human osteoblasts, the cells responsible for bone formation. The most likely species of the toxicants in contact with cells after blood contamination were selected for cell exposure. Results showed that the cytotoxic effect of U and Pb is highly dependent on their speciation. Thus, Pb was cytotoxic when left free in the exposure medium or when complexed with carbonate, cysteine or citrate, but not when complexed with albumin or phosphate, under an insoluble form. U was cytotoxic whatever its speciation, but differences in sensitivity were observed as a function of speciation. Population growth recovery could be obtained after exposure to low doses of U or Pb, except for some U-carbonate complexes which had irreversible effects whatever the dose. The activation of two markers of bone formation and mineralization, osteocalcin and bone sialoprotein (BSP), was observed after exposure to non-toxic doses or non-toxic species of U or Pb while their inhibition was observed after toxic exposure to both metals. This work provides new elements to better understand the complex mechanisms of U and Pb toxicity to osteoblasts. Our results also illustrate the importance of a strictly controlled speciation of the metals in toxicological studies.

Acute chemical toxicity of uranium.

Although human experience with uranium spans more than 200 years, the LD50 for acute intake in humans has not been well established. Large acute doses of uranium can produce death from chemical toxicity in rats, guinea pigs, and other small experimental animals, with variation in sensitivity among species. However, there has never been a death attributable to uranium poisoning in humans, and humans seem to be less sensitive to both acute and chronic toxic effects of uranium than other mammalian species studied. Highly relevant data on uranium toxicity in humans are available from the experience of persons administered large doses of uranium for therapy of diabetes and from acute accidental inhalation intakes. Although the data on which to establish oral and inhalation acute LD50 for uranium in humans are sparse, they are adequate to conclude that the LD50 for oral intake of soluble uranium compounds exceeds several grams of uranium and is at least 1.0 g for inhalation intakes. For intakes of uranium compounds of lesser solubility, acute LD50 values are likely to be significantly greater. It is suggested that 5 g be provisionally considered the acute oral LD50 for uranium in humans. For inhalation intakes of soluble compounds of uranium, 1.0 g of uranium is proposed as the provisional acute inhalation LD50.

Hot dog? Toxicological concerns and The Hound of the Baskervilles.

The terrifying dog in the Hound of the Baskervilles is described as having 'blazing eyes' and a 'luminous muzzle', appearances attributed by Watson and Holmes to the application of phosphorus. Review of the toxicity and flammability of white phosphorus make this improbable. It is suggested that Conan Doyle's description was probably influenced by knowledge of the recent and much publicized discovery of luminescence due to the radioactivity of uranium salts.

Distribution and genotoxic effects after successive exposure to different uranium oxide particles inhaled by rats.

In nuclear fuel cycle facilities, workers may inhale airborne uranium compounds that lead to internal contamination, with various exposure scenarios depending on the workplace. These exposures can be chronic, repeated, or acute, and can involve many different compounds. The effect of uranium after multiple scenarios of exposure is unknown. The aim of this study, therefore, was to investigate the genotoxic and biokinetics consequences of exposure to depleted insoluble uranium dioxide (UO2) by repeated or acute inhalation on subsequent acute inhalation of moderately soluble uranium peroxide (UO4) in rats. The results show that UO2 repeated preexposure by inhalation increases the genotoxic effects of UO4 inhalation, assessed by comet assay, in different cell types, when UO4 exposure alone has no effect. At the same time, the study of UO4 bioaccumulation showed that the UO4 biokinetics in the kidneys, gastrointestinal tract, and excreta, but not in the lungs, were slightly modified by previous UO2 exposures. All these results show that both genotoxic and biokinetics effects of uranium may depend on preexposure and that repeated exposure induces a potentiation effect compared with acute exposure.

Magnetic beads-based DNAzyme recognition and AuNPs-based enzymatic catalysis amplification for visual detection of trace uranyl ion in aqueous environment.

We herein developed a novel biosensor for the visual detection of trace uranyl ion (UO2(2+)) in aqueous environment with high sensitivity and specificity by using DNAzyme-functionalized magnetic beads (MBs) for UO2(2+) recognition and gold nano-particles (AuNPs)-based enzymatic catalysis oxidation of TMB (3,3',5,5'-tetramethylbenzidine sulfate) for signal generation. The utilization of MBs facilitates the magnetic separation and collection of sensing system from complex sample solution, which leads to more convenient experimental operation and more strong resistibility of the biosensor to the matrix of sample, and the utilization of AuNPs-based enzymatic catalysis amplification greatly improved the sensitivity of the biosensor. Compared with the previous DNAzyme-based UO2(2+) sensors, the proposed biosensor has outstanding advantages such as relative high sensitivity and specificity, operation convenience, low cost and more strong resistibility to the matrix of sample. It can be used to detect as low as 0.02 ppb (74 pM) of UO2(2+) in aqueous environment by only naked-eye observation and 1.89 ppt (7.0 pM) of UO2(2+) by UV-visible spectrophotometer with a recovery of 93-99% and a RSD ≤ 5.0% (n=6) within 3h. Especially, the visual detection limit of 0.02 ppb (74 pM) is much lower than the maximum allowable level of UO2(2+) (130 nM) in the drinking water defined by the U.S. Environmental Protection Agency (EPA), indicating that our method meets the requirement of rapid and on-site detection of UO2(2+) in the aqueous environment by only naked-eye observation.

Self-assembly of small molecules affords multifunctional supramolecular hydrogels for topically treating simulated uranium wounds.

Two types of therapeutic agents, which have discrete yet complementary functions, self-assemble into nanofibers in water to formulate a new supramolecular hydrogel as a self-delivery biomaterial to reduce the toxicity of uranyl oxide at the wound sites.

In vivo effects of chronic contamination with depleted uranium on CYP3A and associated nuclear receptors PXR and CAR in the rat.

In addition to its natural presence at high concentrations in some areas, uranium has several civilian and military applications that could cause contamination of human populations, mainly through chronic ingestion. Reports describe the accumulation of this radionuclide in some organs (including the bone, kidney, and liver) after acute or chronic contamination and show that it produces chemical or radiological toxicity or both. The literature is essentially devoid of information about uranium-related cellular and molecular effects on metabolic functions such as xenobiotic detoxification. The present study thus evaluated rats chronically exposed to depleted uranium in their drinking water (1mg/(ratday)) for 9 months. Our specific aim was to evaluate the hepatic and extrahepatic mRNA expression of CYP3A1/A2, CYP2B1, and CYP1A1 as well as of the nuclear receptors PXR, CAR, and RXR in these rats. CYP3A1 mRNA expression was significantly higher in the brain (200%), liver (300%), and kidneys (900%) of exposed rats compared with control rats, while CYP3A2 mRNA levels were higher in the lungs (300%) and liver (200%), and CYP2B1 mRNA expression in the kidneys (300%). Expression of CYP1A1 mRNA did not change significantly during this study. PXR mRNA levels increased in the brain (200%), liver (150%), and kidneys (200%). Uranium caused CAR mRNA expression in the lungs to double. Expression of RXR mRNA did not change significantly in the course of this study, nor did the hepatic activity of CYP2C, CYP3A, CYP2A, or CYP2B. Uranium probably affects the expression of drug-metabolizing CYP enzymes through the PXR and CAR nuclear receptors. These results suggest that the stimulating effect of uranium on these enzymes might lead to hepatic or extrahepatic toxicity (or both) during drug treatment and then affect the entire organism.

The neurotoxicology of uranium.

The brain is a target of environmental toxic pollutants that impair cerebral functions. Uranium is present in the environment as a result of natural deposits and release by human applications. The first part of this review describes the passage of uranium into the brain, and its effects on neurological functions and cognitive abilities. Very few human studies have looked at its cognitive effects. Experimental studies show that after exposure, uranium can reach the brain and lead to neurobehavioral impairments, including increased locomotor activity, perturbation of the sleep-wake cycle, decreased memory, and increased anxiety. The mechanisms underlying these neurobehavioral disturbances are not clearly understood. It is evident that there must be more than one toxic mechanism and that it might include different targets in the brain. In the second part, we therefore review the principal mechanisms that have been investigated in experimental models: imbalance of the anti/pro-oxidant system and neurochemical and neurophysiological pathways. Uranium effects are clearly specific according to brain area, dose, and time. Nonetheless, this review demonstrates the paucity of data about its effects on developmental processes and the need for more attention to the consequences of exposure during development.

Transformation of human osteoblast cells to the tumorigenic phenotype by depleted uranium-uranyl chloride.

Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Although the health effects of occupational uranium exposure are well known, limited data exist regarding the long-term health effects of internalized DU in humans. We established an in vitro cellular model to study DU exposure. Microdosimetric assessment, determined using a Monte Carlo computer simulation based on measured intracellular and extracellular uranium levels, showed that few (0.0014%) cell nuclei were hit by alpha particles. We report the ability of DU-uranyl chloride to transform immortalized human osteoblastic cells (HOS) to the tumorigenic phenotype. DU-uranyl chloride-transformants are characterized by anchorage-independent growth, tumor formation in nude mice, expression of high levels of the k-ras oncogene, reduced production of the Rb tumor-suppressor protein, and elevated levels of sister chromatid exchanges per cell. DU-uranyl chloride treatment resulted in a 9.6 (+/- 2.8)-fold increase in transformation frequency compared to untreated cells. In comparison, nickel sulfate resulted in a 7.1 (+/- 2.1)-fold increase in transformation frequency. This is the first report showing that a DU compound caused human cell transformation to the neoplastic phenotype. Although additional studies are needed to determine if protracted DU exposure produces tumors in vivo, the implication from these in vitro results is that the risk of cancer induction from internalized DU exposure may be comparable to other biologically reactive and carcinogenic heavy-metal compounds (e.g., nickel).

Suppression of depleted uranium-induced neoplastic transformation of human cells by the phenyl fatty acid, phenyl acetate: chemoprevention by targeting the p21RAS protein pathway.

Depleted uranium is a dense heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that exposure to depleted uranium in vitro can transform immortalized human osteoblast (HOS) cells to the tumorigenic phenotype (associated with aberrant RAS oncogene expression and tumor suppressor protein production). Since depleted uranium is used in military applications, it would therefore be beneficial to identify and test potential antitumor-promoting agents. Chemopreventive interventions that target deregulated signal transduction pathways may be effective strategies to prevent carcinogenesis. Since the RAS protein plays a key role in signal transduction, disruption of its signaling pathway may be particularly effective. The phenyl fatty acid, phenyl acetate, a differentiation inducer that affects post-translational processing of RAS, was tested for its ability to prevent depleted uranium-induced neoplastic transformation using HOS cells. After a 24-h exposure to insoluble depleted uranium-uranium dioxide (1 mg/ml), cells were incubated for 1 day to 6 weeks with 2.5 mM phenyl acetate. Treatment with depleted uranium resulted in transformation to the tumorigenic phenotype. In contrast, HOS cells exposed to depleted uranium and then treated with phenyl acetate did not exhibit transformation to the tumorigenic phenotype. These data suggest that depleted uranium-induced neoplastic transformation in vitro can be prevented by targeting the RAS protein.

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.