Optimizing urothelial cell preparation for the human urinary micronucleus assay

Biological monitoring of early genotoxic effects in urothelial cells using the urinary micronucleus (MNu) assay is promising for early detection of cancer, such as bladder carcinoma. But many problems are encountered, the major being the poorly differential staining of cells, particularly in women having an important amount of squamous cells. We have optimized the protocol and obtained a differential staining of the cell types present in urine on 10 subjects. Following Carnoy I fixation and Papanicolaou staining, urothelial cells were blue while most squamous cells were pink. This differential staining allowed for optimization of the MNu assay on a single urine void, for both females and males. Even if our MNu means were comparable to the literature, the great variation in reported MNu results could reside in the ability of scorers to distinguish correctly between urothelial and squamous cells. When monitoring exposed populations, this erroneous distinction could largely influence the results, even more in women’s urine samples. Given a situation where exposure would not increase micronuclei frequency in vaginal squamous cells, their erroneous analysis in the MNu assay could mask an early genotoxic effect. Therefore, as transitional cell carcinoma of the bladder originates from transformed urothelial cells, restricting micronuclei analysis to urothelial cells could yield a more precise estimate of cancer risk in exposed populations. Moreover, it is hoped that the improvements proposed in this paper will allow for an easier implementation of the MNu assay in various set-ups and enhance its specificity, since MNu are considered a suitable biomarker

Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters

Reabsorption of filtered solutes from the glomerular filtrate and excretion of waste products and xenobiotics are the main functions of the renal proximal tubular (PT) epithelium. A human PT cell line expressing a range of functional transporters would help to augment current knowledge in renal physiology and pharmacology. We have established and characterized a conditionally immortalized PT epithelial cell line (ciPTEC) obtained by transfecting and subcloning cells exfoliated in the urine of a healthy volunteer. The PT origin of this line has been confirmed morphologically and by the expression of aminopeptidase N, zona occludens 1, aquaporin 1, dipeptidyl peptidase IV and multidrug resistance protein 4 together with alkaline phosphatase activity. ciPTEC assembles in a tight monolayer with limited diffusion of inulin-fluorescein-isothiocyanate. Concentration and time-dependent reabsorption of albumin via endocytosis has been demonstrated, together with sodium-dependent phosphate uptake. The expression and activity of apical efflux transporter p-glycoprotein and of baso-lateral influx transporter organic cation transporter 2 have been shown in ciPTEC. This established human ciPTEC expressing multiple endogenous organic ion transporters mimicking renal reabsorption and excretion represents a powerful tool for future in vitro transport studies in pharmacology and physiology

Staying focused: A functional account of perceptual suppression during binocular rivalry

Presenting different images to either eye can induce perceptual switching, with alternating disappearances of each image—a phenomenon called binocular rivalry. We believe that disappearances during binocular rivalry can be driven by a process that facilitates visibility near the point of fixation. As the point of fixation is tied neither to a particular stimulus nor to a specific eye, indifference to both would be an essential characteristic for the process we envisage. Many factors that influence disappearances during binocular rivalry scale with distance in depth from fixation. Of these, here we use blur. We break the links between this cue and both eye of origin and stimulus type. We find that perceptual dominance can track a better focused image as it is swapped between the eyes and that perceptual switches can be driven by alternating the focus of images fixed in each eye. This implies that, as a determinant of suppression selectivity, blur is functionally independent from both eye of origin and stimulus type. Our data and theoretical account suggest that binocular rivalry is not an irrelevant laboratory curiosity but, rather, that it is a product of a functional adaptation that promotes visibility in cluttered environments

Binocular switch suppression: A new method for persistently rendering the visible 'invisible'

Rendering the usually visible ‘invisible’ has long been a popular experimental manipulation. With one notable exception, ‘continuous flash suppression’ [Tsuchiya, N., & Koch, C. (2005). Continuous flash suppression reduces negative afterimages. Nature Neuroscience, 8, 1096–1101], existing methods of achieving this goal suffer from being either unable to suppress stimuli from awareness for prolonged periods, from being unable to reliably suppress stimuli at specific epochs, or from a combination of both of these limitations. Here we report a new method, binocular switch suppression (BSS), which overcomes these restrictions. We establish that BSS is novel as it taps a different causal mechanism to the only similar pre-existing method. We also establish that BSS is superior to pre-existing methods both in terms of the depth and duration of perceptual suppression achieved. BSS should therefore prove to be a useful tool for the large number of researchers interested in exploring the neural correlates and functional consequences of conscious visual awareness

Ochratoxin A and T-2 Toxin Induce Clonogenicity and Cell Migration in Human Colon Carcinoma and Fetal Lung Fibroblast Cell Lines.

IF 3.02International audienceT-2 toxin and Ochratoxin A (OTA) are toxic secondary metabolites produced by various fungi, and together they contaminate feedstuffs worldwide. T-2 toxin and OTA may exert carcinogenic action in rodent. Despite the various in vivo experiments, carcinogenicity of these two mycotoxins has not yet been proven for human. In this current study, we proposed to investigate, in Human colon carcinoma cells and fetal lung fibroblast-like cells transfected with MYC, the effect of T-2 toxin and OTA on cell clonogenicity and cell migration. Results of the present investigation showed that T2-toxin as well as OTA has an important clonogenic effect in all cell lines, suggesting that these mycotoxins could promote the transcription of c-myc gene. Furthermore, T-2 toxin and OTA enhanced the migration effect of HCT116 cells at very low concentrations, proposing that these mycotoxins may exhibit carcinogenesis-like properties in the studied cells