carbocyanines and diacetylfluorescein

To assess the potential effects of environmental pollutants belonging to the musk fragrances group in the physiology of aquatic animal species, in this work we treated rainbow trout RTG-2 cells with the polycyclic ketone tonalide (AHTN) at dilutions ranging from 3.5 to 500 ng/ml. The following parameters were monitored: intracellular ATP concentration (energy production), mitochondrial membrane potential (early apoptosis marker), cell viability (vital staining with DFP), quantitative expression of genes coding for the cytochrome P450 detoxifying enzymes CYP1A1 and CYP3A27, and of genes coding for the immunoregulatory peptides IL-1β, IL-8, TNFα, Cox-2 and TGF-β. Obtained results showed that incubation with tonalide induced in RTG-2 cells no effects on cell viability, a slight increase of mitochondrial membrane potential activity, and a significant increase in intracellular ATP concentration. However, dramatic effects were observed in transcription levels of some tested genes, with upregulation levels of 300 and 600 times measured for TGF-β and TNFα, respectively and of 150 times for the CYP3A27 gene. Our results show for the first time the potent effects exerted by tonalide on immunoregulatory genes of RTG-2 cells and also indicate that the measured sensitivity of RTG-2 towards tonalide was in the same range of that currently available using chemical methods. A possible use of the panel of genes we employed as a tool for the monitoring of musk fragrances in biological samples is discussed.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Aryl Hydrocarbon Hydroxylases; Carbocyanines; Cell Line; Cell Survival; Cyclooxygenase 2; Cytotoxins; Fluoresceins; Fluorescent Dyes; Interleukin-1beta; Interleukin-8; Oncorhynchus mykiss; Perfume; RNA; Tetrahydronaphthalenes; Transcription, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Water Pollutants, Chemical

Perfluorinated (aliphatic) acids (PFAs) and congeners have many applications in various industrial fields and household for decades. Years later they have been detected in wildlife and this has spurred interest in environmental occurrence as well as influencing living organisms. PFAs were established as peroxisome proliferators and hepatocarcinogens. Amphipatic structure suggests that they may alter cell membrane potential (mbDeltaPsi) and/or induce changes in cytosolic pH (pHi). The aim of this study was to examine the correlation between changes of above parameters and PFAs structure (CF(6)-CF(12)) in human colon carcinoma HCT116 cells. mbDeltaPsi and pHi were measured by flow cytometry using fluorescence polarization of the plasma membrane probe 3,3'-dipentyloxacarbocyanine (DiOC(5)(3)) and fluorescein diacetate (FDA), respectively. Dose- and time-dependent manner analysis revealed relatively fast depolarization of plasma membrane and acidification of cytosol both positively correlated with fluorocarbon chain length. mbDeltaPsi depletion after 4 h of incubation reached 8.01% and 30.08% for 50 muM PFOA and 50 muM PFDoDA, respectively. Prolonged treatment (72 h) led to dramatic dissipation of membrane potential up to 21.65% and 51.29% and strong acidification to pHi level at 6.92 and 6.03 at the presence of above compounds, respectively. The data demonstrate that PFAs can alter plasma membrane protonotrophy with the mode dependent on the compound hydrophobicity.

Topics: Carbocyanines; Carcinogens, Environmental; Cell Membrane; Cytosol; Dose-Response Relationship, Drug; Fatty Acids; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Fluorocarbons; HCT116 Cells; Humans; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Membrane Potentials; Molecular Structure; Structure-Activity Relationship; Time Factors

The ability of a flow cytometer to rapidly assess microbial viability was investigated using three vital stains: rhodamine 123 (Rh123); 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] and fluorescein diacetate (FDA). Rh123 was found to clearly differentiate viable from non-viable bacteria. The methodology for staining bacteria with this dye was optimised. Rh123 was shown to stain and discriminate several different species of viable bacteria although this was not universal. Viable cells of Bacillus subtilis were found to stain better with FDA than with Rh123. The results demonstrate the ability of flow cytometry to rapidly detect and estimate the viability of bacterial populations.

Topics: Bacterial Physiological Phenomena; Carbocyanines; Evaluation Studies as Topic; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Rhodamine 123; Rhodamines; Staining and Labeling