Renal adaptive response to exposure to low doses of uranyl nitrate and sodium fluoride in mice.

BACKGROUND: Despite their differences in physicochemical properties, both uranium (U) and fluoride (F) are nephrotoxicants at high doses but their adverse effects at low doses are still the subject of debate. METHODS: This study aims to improve the knowledge of the biological mechanisms involved through an adaptive response model of C57BL/6 J mice chronically exposed to low priming doses of U (0, 10, 20 and 40 mg/L) or F (0, 15, 30 and 50 mg/L) and then challenged with acute exposure of 5 mg/kg U or 7.5 mg/kg NaF. RESULTS: We showed that an adaptive response occurred with priming exposures to 20 mg/L U and 50 mg/L F, with decreased levels of the biomarkers KIM-1 and CLU compared to those in animals that received the challenge dose only (positive control). The adaptive mechanisms involved a decrease in caspase 3/7 activities in animals exposed to 20 mg/L U and a decrease in in situ VCAM expression in mice exposed to 50 mg/L F. However, autophagy and the UPR were induced independently of priming exposure to U or F and could not be identified as adaptive mechanisms to U or F. CONCLUSION: Taken together, these results allow us to identify renal adaptive responses to U and F at doses of 20 and 50 mg/L, probably through decrease apoptosis and inflammatory cell recruitment.

[Study of the mechanisms of cytotoxic effect of uranyl nitrate].

The mechanisms of cytotoxic effect of uranyl nitrate were studied. It was shown that uranyl nitrate induced HEp-2 cell death, mainly by necrotic way. In the experiments in vitro, uranyl nitrate caused an appearance of 8-oxoguanine in DNA, indicating the induction of oxidative stress. The experiments with isolated rat liver mitochondria revealed that 1 mM uranyl nitrate decreased the respiration rates of mitochondria in state 3 and DNP-induced respiration. At the same time, uranyl nitrate had no influence on the opening of the mitochondrial permeability transition pore and decreased the rate of formation of H2O2 by mitochondria. Possible molecular mechanisms of uranyl-induced necrosis are discussed.

Ex vivo decrease in uranium diffusion through intact and excoriated pig ear skin by a calixarene nanoemulsion.

Cutaneous contamination by radionuclides is a major concern in the nuclear industry. In case of skin exposure to uranium, no efficient emergency treatment is available to remove the actinide from the skin. For this purpose, we developed a nanoemulsion containing calixarene molecules displaying good chelating properties towards uranium. In this paper, we describe the ability of this formulation to trap uranium and limit its transfer from the cutaneous contaminated site into the blood. Uranium percutaneous diffusion kinetics was assessed with Franz cells over 24 h through intact and excoriated pig ear skin biopsies, after or without application of the nanoemulsion. Uranium distribution in the skin layers was analysed by SIMS microscopy. The results showed that prompt application of the calixarene nanoemulsion allows a 94% and 98% reduction of the amount of uranium diffused respectively through intact and excoriated skin. The formulation is still efficient in case of delayed application up to 30 minutes since the 24 h-uranium transfer through excoriated skin is reduced by 71%. Besides, no accumulation of uranium or uranium-calixarene chelate was observed in the different skin layers. In conclusion, this study demonstrated the efficiency of the calixarene nanoemulsion, which can be regarded as a promising treatment for uranium cutaneous contamination.

Uranyl nitrate inhibits lactate gluconeogenesis in isolated human and mouse renal proximal tubules: a 13C-NMR study.

As part of a study on uranium nephrotoxicity, we investigated the effect of uranyl nitrate in isolated human and mouse kidney cortex tubules metabolizing the physiological substrate lactate. In the millimolar range, uranyl nitrate reduced lactate removal and gluconeogenesis and the cellular ATP level in a dose-dependent fashion. After incubation in phosphate-free Krebs-Henseleit medium with 5 mM L-[1-13C]-, or L-[2-13C]-, or L-[3-13C]lactate, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods. In the presence of 3 mM uranyl nitrate, glucose production and the intracellular ATP content were significantly reduced in both human and mouse tubules. Combination of enzymatic and NMR measurements with a mathematical model of lactate metabolism revealed an inhibition of fluxes through lactate dehydrogenase and the gluconeogenic enzymes in the presence of 3 mM uranyl nitrate; in human and mouse tubules, fluxes were lowered by 20% and 14% (lactate dehydrogenase), 27% and 32% (pyruvate carboxylase), 35% and 36% (phosphoenolpyruvate carboxykinase), and 39% and 45% (glucose-6-phosphatase), respectively. These results indicate that natural uranium is an inhibitor of renal lactate gluconeogenesis in both humans and mice.

Bisphosphonate-containing supramolecular hydrogels for topical decorporation of uranium-contaminated wounds in mice.

PURPOSE: Personnel experiencing accidents that involve radionuclides or victims of potential malicious radioactive attacks may suffer injuries with wounds contaminated by radionuclides. The current treatment for contamination from uranium in external injuries is the use of saline solution to wash the wounds, which has the drawback of further spreading of the contaminants due to the flow of water. To minimize the cost of storage of contaminated liquids and to improve the efficiency of treatment, we propose the use of hydrogels as a form of decorporation agent. METHODS: Mice with uranium-contaminated wounds on their backs were treated with bisphosphonate or diethylene triamine pentaacetic acid (DTPA) containing supramolecular hydrogels. Survival rates of the treated mice and changes in the body weight of the mice were observed and compared to those of mice without hydrogel treatment. Distribution of uranium in the mice was also explored as proof of the effectiveness of the hydrogel treatment. RESULTS: The survival rate of the hydrogel-treated mice was significantly higher than that of the mice without treatment. The body weights of the hydrogel-treated mice showed significant recovery after 10 days while the body weight of mice without hydrogel treatment continuously decreased. The amount of uranyl ions in the organs (mainly concentrated in the kidney) of the hydrogel-treated mice was much smaller than that of the mice without hydrogel treatment. CONCLUSIONS: By incorporating uranium chelating agents, we developed new supramolecular hydrogels that could effectively and conveniently decorporate uranium ions from the contaminated wound sites of mice, with the highest efficacy achieved by our pamidronate-based molecular hydrogel.

Effect of uranyl nitrate on enzymes of carbohydrate metabolism and brush border membrane in different kidney tissues.

Uranium, the heaviest of the naturally occurring elements is widely present as environmental contaminant from natural deposits, industrial emissions and most importantly from modern weapons. Histopathological examinations revealed that uranyl nitrate (UN) exposure caused severe damage to pars recta of renal proximal tubule. However, biochemical events involved in cellular response to renal injury are not completely elucidated. We hypothesized that UN exposure would severely damage kidney tissues and alter their metabolic functions. Rats were treated with a single nephrotoxic dose of UN (0.5mg/kg body weight) i.p. After 5d, effect of UN was studied on the activities of various enzymes of carbohydrate metabolism, brush border membrane (BBM) and oxidative stress in different kidney tissues. Activity of lactate dehydrogenase increased whereas activities of isocitrate, succinate and malate dehydrogenases, glucose-6-phosphatase and fructose-1,6-bisphosphatase significantly decreased by UN exposure. Activity of glucose-6-phosphate dehydrogenase decreased whereas that of NADP-malic enzyme increased. The activities of BBM enzymes were significantly lowered and after dissociation from BBM excreted in urine. Lipid peroxidation and the activities of superoxide dismutase and glutathione peroxidase increased whereas catalase activity decreased by UN. UN treatment caused specific alterations in the activities of metabolic and membrane enzymes and perturbed antioxidant defenses.

Ultrastructural and metabolic changes in osteoblasts exposed to uranyl nitrate.

Exposure to uranium is an occupational hazard to workers who continually handle uranium and an environmental risk to the population at large. Since the cellular and molecular pathways of uranium toxicity in osteoblast cells are still unknown, the aim of the present work was to evaluate the adverse effects of uranyl nitrate (UN) on osteoblasts both in vivo and in vitro. Herein we studied the osteoblastic ultrastructural changes induced by UN in vivo and analyzed cell proliferation, generation of reactive oxygen species (ROS), apoptosis, and alkaline phosphatase (APh) activity in osteoblasts exposed to various UN concentrations (0.1, 1, 10, and 100 microM) in vitro. Cell proliferation was quantified by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, ROS was determined using the nitro blue tetrazolium test, apoptosis was morphologically determined using Hoechst 3332 and APh activity was assayed spectrophotometrically. Electron microscopy revealed that the ultrastructure of active and inactive osteoblasts exposed to uranium presented cytoplasmic and nuclear alterations. In vitro, 1-100 microM UN failed to modify cell proliferation ratio and to induce apoptosis. ROS generation increased in a dose-dependent manner in all tested doses. APh activity was found to decrease in 1-100 microM UN-treated cells vs. controls. Our results show that UN modifies osteoblast cell metabolism by increasing ROS generation and reducing APh activity, suggesting that ROS may play a more complex role in cell physiology than simply causing oxidative damage.

Chronic ingestion of uranyl nitrate perturbs acetylcholinesterase activity and monoamine metabolism in male rat brain.

Recent animal studies have shown that uranium can reach the brain after chronic exposure. However, little information is available on the neurological effects of chronic long-term exposure to uranium. In the present study, the effects during 1.5, 6 and 9-month periods of chronic ingestion of uranyl nitrate (UN) in drinking water (40 mg of uranium per litre) on cholinergic acetylcholinesterase (AChE) activity and on dopaminergic and serotoninergic metabolisms were investigated in several areas of male Srague Dawley rat brains. Uranium brain accumulation and distribution was also investigated after 1.5 and 9 months. Both after 1.5, 6 and 9 months of exposure, AChE activity was unaffected in the striatum, hippocampus and frontal cortex. Nevertheless, AChE activity was transitionally perturbed in the cerebellum after 6 months of exposure. After 1.5 months of exposure, DA level increased in hypothalamus. After 6 months of exposure, a tiny but significant modification of the DAergic turnover ratio was detected in the frontal cortex. And after 9 months, UN produced a significant decrease in the 5HIAA level and the 5HTergic turn-over ratio in the frontal cortex and also a decrease in the DOPAC level and DAergic turn-over ratio in the striatum. Uranium brain accumulation was statistically significant in striatum after 1.5 months and in striatum, hippocampus and frontal cortex after 9 months of exposure. Although neurochemical changes did not always correlated with increased accumulation of uranium in specific areas, these results suggest that chronic ingestion of UN can cause chronic and progressive perturbations of physiological level of neurotransmitter systems. Considering previous reports on behavioural uranium-induced effects and the involvement of neurotransmitters in various behavioural processes, it would be crucial to determine whether these neurochemical disorders were accompanied by neurobehavioral deficits even at 40 mg of uranium per litre exposure.

Alterations in water and electrolyte absorption in the rat colon following neutron irradiation: influence of neutron component and irradiation dose.

PURPOSE: To study the absorptive function of rat colon following whole-body exposure to neutron irradiation, either to the same total dose with varying proportion of neutrons or to the same neutron proportion with an increasing irradiation dose. MATERIALS AND METHODS: Different proportions of neutron irradiation were produced from the reactor SILENE using a fissile solution of uranium nitrate (8, 47 and 87% neutron). Water and electrolyte fluxes were measured in the rat in vivo under anaesthesia by insertion into the descending colon of an agarose gel cylinder simulating the faeces. Functional studies were completed by histological analyses. In the first set of experiments, rats received 3.8 Gy with various neutron percentages and were studied from 1 to 14 days after exposure. In the second set of experiments, rats were exposed to increasing doses of irradiation (1-4Gy) with a high neutron percentage (87%n) and were studied at 4 days after exposure. RESULTS AND CONCLUSIONS: The absorptive capacity of rat colon was diminished by irradiation at 3-5 days, with a nadir at 4 days. The results demonstrate that an increase in the neutron proportion is associated with an amplification of the effects. Furthermore, a delay in the re-establishment of normal absorption was observed with the high neutron proportion (87%n). A dose-dependent reduction of water absorption by rat colon was also observed following neutron irradiation (87%n), with a 50% reduction at 3 Gy. Comparison of this dose-effect curve with the curve obtained following gamma (60)Co-irradiation indicates an RBE of 2.2 for absorptive colonic function in rat calculated at 4 days after exposure.

Uranyl nitrate-induced proximal tubule alterations in rabbits: a quantitative analysis.

Naturally occurring uranium in drinking water is a significant health concern in several areas of North America. Because the kidney is a known target organ to examine the effects of uranium or its compounds, the objective of this study was to determine whether kidney repair occurs after exposure to, and withdrawal of, uranyl nitrate (UN). This work, part of a larger study to establish safe levels of uranium in drinking water supplies, examined the ultrastructural changes in proximal tubule cells of New Zealand white rabbits following subchronic exposure to UN in water and for 91 days after exposure ended. The rabbit was chosen as the experimental animal because of its high sensitivity to uranium. Animals were exposed to 24 or 600 mg UN per liter (UN/L) in drinking water for 91 days, with no recovery or recovery periods of 45 or 91 days. Ultrastructural changes, quantified by a stereological image analysis system based on point counting, were observed in renal proximal tubules (PTs). Each electron micrograph was statistically considered an experimental unit. The severity of lesions was directly proportional to the dose. Animals exposed to 600 mg UN/L had the most severe lesions; nevertheless, alterations were remarkable in animals exposed to the low dose. At both recovery periods, the lesions were significantly more severe than those in animals of the no-recovery group, which may result from the kidney's ability to store uranium. The PT cells had increased lysosomal and vacuolar mass as well as variations in mitochondrial mass. In addition, there was epithelial cell degeneration with a focal loss of brush borders, thickening and splitting of tubular basement membrane, and occasionally cell necrosis. Interstitial fibrosis of the renal cortex persisted as the recovery period increased in the animals of UN-dosed groups. Alterations may be due to disturbed fluid transport across the PT and other cells and decreased cell respiration resulting from damaged cell constituents. Cell damage caused by UN in drinking water persisted throughout the 91-day recovery period. By eventually determining the no observable effect level for the kidney by UN, this study may assist in devising a model to ascertain the safe levels of uranium in water.

The effects of uranyl ions on neuromuscular transmission in the urinary bladder of the normal and streptozotocin-diabetic mouse.

The depressant effects of uranyl nitrate on the nerve-evoked muscle contraction of urinary bladder isolated from normal and streptozotocin-diabetic mice were compared. The non-cholinergic component of the evoked bladder contraction (in the presence of atropine) was specifically sensitive to the suppressive effect of uranyl nitrate. In contrast, the cholinergic component remaining after treatment with alpha, beta-methylene ATP was rather insensitive to uranyl nitrate. The contractile responses induced by KCl, acetylcholine and ATP were also not affected by uranyl nitrate, indicating a presynaptic site of action. High Ca2+ and calmodulin inhibitors (trifluoperazine, diltiazem and W7) antagonized the suppressive effects of uranyl ions. These results suggest that the depressant effects of uranyl nitrate is mediated by a reduction of prejunctional non-cholinergic transmitter release through the calcium-calmodulin pathway. In contrast to the normal bladder, the urinary bladder of streptozotocin-diabetic mice revealed not only weaker neurogenic contractile responses to electrical field stimulation, but also a profound reduction in the depressant effect of uranyl nitrate. These findings suggest that the Ca2+ regulation of non-cholinergic neurotransmission in mouse urinary bladder may be impaired in the diabetic state.

Enhanced uranyl photocleavage across the minor groove of all (A/T)4 sequences indicates a similar narrow minor groove conformation.

The uranyl(VI)-mediated photocleavage of a Drew-Dickerson sequence oligonucleotide (5'-dGATCACGC-GAATTCGCGT) either as the (self-complementary) duplex or cloned into the BamH1 site of pUC19 has been studied. At pH 6.5 in acetate buffer relatively enhanced photocleavage is observed at the 3'-end of the AATT sequence corresponding to maximum cleavage across the minor groove in the A/T tract. Thus maximum cleavage correlates with minimum minor groove width in the crystal structure and also with the largest electronegative potential according to computations. Using plasmid constructs with cloned inserts of the type [CGCG(A/T4)]n, we also analysed all possible sequence combinations of the (A/T)4 tract and in all cases we observed maximum uranyl-mediated photocleavage across the minor groove in the (A/T)4 tract without any significant differences between the various sequences. From these results we infer that DNA double helices of all (A/T)4 sequences share the same narrow minor groove helix conformation.

Enhancement of a slow potassium current component by uranyl nitrate and its relation to the antagonism on beta-bungarotoxin in the mouse motor nerve terminal.

Uranyl nitrate (UO2(NO3)2) has been shown to be capable of increasing transmitter release from the motor nerve accompanied by the potentiation of nerve evoked muscle contraction. In this paper, we have demonstrated that UO2(2+) induced an initial twitch depression followed by a later twitch potentiation in low (0.35 mM) Ca2+ medium. Although UO2(2+) has been identified as a K(+)-channel blocker, we have found it only partially blocked the fast K(+)-current (IK(f) as recorded in the perineurial sheath of the mouse triangularis sterni preparation. Increasing the concentration of UO2(2+) to a high concentration of 0.4 mM did not further inhibit IK(f) but markedly prolonged the duration of the outward current of the nerve terminals. From the time course of its appearance together with the specific inhibition by 4-aminopyridine, dendrotoxin and beta-bungarotoxin, which has been shown to be capable of blocking the K(+)-current of the motor nerve terminal, it was proposed that UO2(2+) prolonged the duration of the nerve terminal spikes by an enhancement of an IK(s)-like current, which was further characterized by its susceptibility to be enhanced by low K+, low Ca2+ and Cd2+ but attenuated by high K+ and high Ca2+. These cation effects not only supported UO2(2+)-induced IK(s) current but also excluded the possibility of an enhancement of Ca(2+)-activated K(+)-current induced by UO2(2+) plus TEA. The significance of this enhancement of IK(s) induced by UO2(2+) has been elucidated by the finding that dendrotoxin inhibited but tetraethylammonium potentiated not only UO2(2+)-induced IK(s) but also UO2(2+)-induced twitch depression.(ABSTRACT TRUNCATED AT 250 WORDS)

Uranyl photoprobing of a four-way DNA junction: evidence for specific metal ion binding.

Metal ions are very important in mediating the folding of nucleic acids, as exemplified by the folding of the four-way DNA junction into the stacked X-conformation. Uranyl ion-mediated photocleavage provides a method for the localization of high-affinity ion binding sites in nucleic acids, and we have applied this to the four-way DNA junction. We have made the following observations. (i) Uranyl ions (UO2(2+)) suppressed the reactivity of junction thymine bases against attack by osmium tetroxide, indicating that the uranyl ion induces folding of the junction into a stacked conformation. (ii) DNA located immediately at the point of strand exchange on the two exchanging strands was hypersensitive to uranyl photocleavage. The relative hypersensitivity was considerably accentuated when the photocleavage was carried out in the presence of citrate ions. This suggests the presence of a tight binding site for the uranyl ion in the junction. (iii) The same positions were significantly protected from uranyl cleavage by the presence of hexamminecobalt (III) or spermidine. These ions are known to induce the folded conformation of the four-way junction with high efficiency, suggesting a direct competition between the ions. By contrast, magnesium ions failed to generate a similar protection against photocleavage. These results suggest that the uranyl, hexamminecobalt (III) and spermidine ions compete for the same high affinity binding site on the junction. This site is located at the centre of the junction, at the point where the exchanging strands pass between the stacked helices. We believe that we have observed the first known example of a metal ion 'footprint' on a folded nucleic acid structure.

Renal handling of alendronate in rats. An uncharacterized renal transport system.

Alendronate (4-amino-1-hydroxybutylidene-1,1-bisphosphonate), an antiosteolytic agent, is currently under investigation in the treatment of a variety of bone diseases. Earlier studies from this laboratory have demonstrated that systemically administered alendronate is rapidly either taken up by bone tissues or excreted by the kidney, and that renal excretion is the only route of elimination. The purpose of this study is to characterize the renal handling of alendronate in rats by standard clearance procedures with inulin as a marker of glomerular filtration rate. Alendronate is highly bound to rat serum protein. The excretion of alendronate by the kidney is concentration-and dose-dependent, and saturable, indicating that it is secreted by an active transport mechanism. The secretory mechanism exhibits limitation of transport, with an apparent Tm of approximately 25 micrograms/min/kg. However, high doses of cimetidine, quinine, probenecid, and p-aminohippuric acid had no effect on the renal excretion of alendronate, suggesting that alendronate is not secreted by anionic or cationic transport systems. In contrast, alendronate clearance is inhibited by etidronate, another bisphosphonate, in a dose-dependent manner, implying that these two bisphosphonates compete for an as yet uncharacterized renal transport system. As expected, the renal excretion of alendronate is drastically reduced in rats with acute renal failure. As a consequence of renal impairment, alendronate accumulates in plasma, and the concentration of the drug in bone tissues increases significantly.

The effects of the platinum anti-tumour agents on renal cell kinetics and the response to a second cytotoxic agent.

Following 15 mg kg-1 cisplatin to mice, the labelling index (LI) in the kidney decreased from 0.4% to less than 0.01% at 1-3 d, increased to 1.9% at day 16 and returned to control levels by day 30. Cytotoxicity was assessed by counts of viable tubule cross-sections and recovery was incomplete up to 14 months after treatment. Cisplatin treatment impaired the regeneration response to a dose of 16 mg kg-1 uranyl nitrate (UN) given 14 d after cisplatin when assessed by an increase in LI and sub-capsular tubule count. However, there was recovery with greater time intervals. At nine months after cisplatin there was no difference in response to UN of controls and mice previously treated by cisplatin. Prior treatment with paraplatin or iproplatin at LD50 doses produced not only no histopathological changes but also no impairment of the subsequent responses to UN.