Transmission electron microscopic and X-ray absorption fine structure spectroscopic investigation of U repartition and speciation after accumulation in renal cells.

After environmental contamination, U accumulates in the kidneys and in bones, where it causes visible damage. Recent in vitro data prove that the occurrence of citrate increases U bioavailability without changing its speciation. Two hypotheses can explain the role of citrate: it either modifies the U intracellular metabolization pathway, or it acts on the transport of U through cell membrane. To understand which mechanisms lead to increased bioavailability, we studied the speciation of U after accumulation in NRK-52E kidney cells. U speciation was first identified in various exposure media, containing citrate or not, in which U was supplied as U carbonate. The influence of serum proteins was analyzed in order to detect the formation of macromolecular complexes of U. Transmission electron microscopy (TEM) was employed to follow the evolution of the U species distribution among precipitated and soluble forms. Finally, extended X-ray absorption fine structure spectroscopy (EXAFS) enabled the precipitates observed to be identified as U-phosphate. It also demonstrated that the intracellular soluble form of U is U carbonate. These results suggest that citrate does not change U metabolization but rather plays a role in the intracellular accumulation pathway. U speciation inside cells was directly and clearly identified for the first time. These results elucidate the role of U speciation in terms of its bioavailability and consequent health effects.

Uranium induces apoptosis and is genotoxic to normal rat kidney (NRK-52E) proximal cells.

Uranium (U) is a heavy metal used in the nuclear industry and for military applications. U compounds are toxic. Their toxicity is mediated either by their radioactivity or their chemical properties. Mammalian kidneys and bones are the main organs affected by U toxicity. Although the most characteristic response to U exposure is renal dysfunction, little information is available on the mechanisms of its toxicity at the molecular level. This report studied the genotoxicity of U. Apoptosis induction in normal rat kidney (NRK-52(E)) proximal cells was investigated as a function of exposure time or concentrations (0-800microM). In parallel, DNA damage was evaluated by several methods. In order to distinguish between the intrinsic and the extrinsic pathways of apoptosis, caspases-8, -9, -10 assays were conducted and the mitochondrial membrane potential was measured. Three methods were selected for their complementarities in the detection of genetic lesions. The comet assay was used for the detection of primary lesions of DNA. gamma-H2AX immunostaining was achieved to detect DNA double-strand breaks. The micronucleus assay was used to detect chromosomic breaks or losses. DNA damage and apoptosis were observed in a concentration-dependent manner. This study demonstrated that U is genotoxic from 300microM and induces caspase-dependent apoptosis cell death from 200microM mainly through the intrinsic pathway in NRK-52(E) cells. These results suggest that the DNA damage caused by U is reversible at low concentration (200-400microM) but becomes irreversible and leads to cell death for higher concentrations (500-800microM).

Effect of HMGCoA reductase inhibitors on cytochrome P450 expression in endothelial cell line.

Endothelial cells and smooth muscle cells are the major cells that constitute blood vessels, and endothelial cells line the lumen of blood vessels. These 2 types of cells also play an integral role in the regional specialization of vascular structure. On the basis of these observations, we designed our study to investigate the effect of various statins on CYP expression in endothelial cells. 3-hydroxymethyl coenzyme A reductase inhibitors play an important role in vascular function. The majority of the statins available on the market show extensive metabolism by cytochrome P450 (CYP) enzymes. Both cell types are involved in the bioconversion of arachidonic acid into vasoactive compounds. The aim of this study was to demonstrate the effect of statins on cytochrome P450 expression in endothelial cells. Our results show that endothelial cells expressed both CYPs involved in epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) production and the nuclear receptor implicated in cytochrome P450 regulation. Treatment of endothelial cells with lovastatin increased CYP2C9 expression. After 96 hours of treatment, fluvastatin and lovastatin clearly increased CYP2C9 protein level. CAR but not PXR was expressed in endothelial cells, indicating that the upregulating effect of statins on CYP2C9 in endothelial cells could be mediated through CAR only due to the lack of expression of PXR in these cells.

Citrate does not change uranium chemical speciation in cell culture medium but increases its toxicity and accumulation in NRK-52E cells.

Uranium (U), as a heavy metal, is a strong chemical toxicant, which induces the damage to proximal tubule kidney cells. In order to reproduce U toxicity in vitro and to avoid precipitation, it is necessary to complex it with a strong ligand such as bicarbonate before dilution with cell culture medium. It was recently shown, in vitro on the NRK-52E normal renal tubular epithelial cells, that citrate increased the toxicity of U(VI)-bicarbonate complexes. This property was attributed to a change in U speciation, characterized by the occurrence of U(VI)-citrate complexes, which were supposed to be more toxic than U(VI)-bicarbonate. Here, we present the results of extended X-ray absorption fine structure spectroscopy (EXAFS) analyses of the media that were used to expose cells in vitro. Resulting data show that even when citrate is added to the exposure medium, the predominant species is U(VI)-bicarbonate. Nonetheless, citrate increases U(VI) toxicity and accelerates its intracellular accumulation kinetics, without inducing precipitation. This study emphasizes another parameter that modulates U(VI) toxicity for renal tubule cells and further characterizes the mechanisms of U(VI) toxicity.

Cytochromes P450 are differently expressed in normal and varicose human saphenous veins: linkage with varicosis.

The expression of cytochrome P450 (CYP) enzymes and cyclo-oxygenases (COX) was investigated in human saphenous veins by reverse transcription-polymerase chain reaction analysis. Non-varicose veins were obtained from patients undergoing aortocoronary bypass grafting, whereas varicose veins were obtained from patients undergoing stripping removal of varicose saphenous veins. In non-varicose veins, CYP1B1, CYP2C, CYP2E1 and CYP4A11 were detected, whereas CYP2J2, CYP3A5, COX-1 and COX-2 were detected almost exclusively in varicose veins. CYP4F2 was not detectable. Except for CYP4A11, the levels of individual CYP mRNA were higher in varicose veins than in control veins. Smooth muscle cell volume, determined by a colour image-analysis system, was increased approximately 1.5-fold in varicose veins. Because CYPs and COXs produce various vasoactive compounds, increased expression of these enzymes could be involved in the impairment of vascular tone and may contribute to varicose pathology. Then, CYP or COX modulators may be potentially active in the treatment of chronic venous insufficiency.