Fluorescence labeling of mitochondria in living cells by the cationic photosensitizer ZnTM2,3PyPz, and the possible roles of redox processes and pseudobase formation in facilitating dye uptake.

The study of labeling selectivity and mechanisms of fluorescent organelle probes in living cells is of continuing interest in biomedical sciences. The tetracationic phthalocyanine-like ZnTM2,3PyPz photosensitizing dye induces a selective violet fluorescence in mitochondria of living HeLa cells under UV excitation that is due to co-localization of the red signal of the dye with NAD(P)H blue autofluorescence. Both red and blue signals co-localize with the green emission of the mitochondria probe, rhodamine 123. Microscopic observation of mitochondria was improved using image processing and analysis methods. High dye concentration and prolonged incubation time were required to achieve optimal mitochondrial labeling. ZnTM2,3PyPz is a highly cationic, hydrophilic dye, which makes ready entry into living cells unlikely. Redox color changes in solutions of the dye indicate that colorless products are formed by reduction. Spectroscopic studies of dye solutions showed that cycles of alkaline titration from pH 7 to 8.5 followed by acidification to pH 7 first lower, then restore the 640 nm absorption peak by approximately 90%, which can be explained by formation of pseudobases. Both reduction and pseudobase formation result in formation of less highly charged and more lipophilic (cell permeant) derivatives in equilibrium with the parent dye. Some of these are predicted to be lipophilic and therefore membrane-permeant; consequently, low concentrations of such species could be responsible for slow uptake and accumulation in mitochondria of living cells. We discuss the wider implications of such phenomena for uptake of hydrophilic fluorescent probes into living cells.

Porphycenes: facts and prospects in photodynamic therapy of cancer.

The photodynamic process induces cell damage and death by the combined effect of a photosensitizer (PS), visible light, and molecular oxygen, which generate singlet oxygen ((1)O(2)) and other reactive oxygen species that are responsible for cytotoxicity. The most important application of this process with increasing biomedical interest is the photodynamic therapy (PDT) of cancer. In addition to hematoporphyrin-based drugs, 2nd generation PSs with better photochemical properties are now studied using cell cultures, experimental tumors and clinical trials. Porphycene is a structural isomer of porphyrin and constitutes an interesting new class of PS. Porphycene derivatives show higher absorption than porphyrins in the red spectral region (lambda > 600 nm, epsilon > 50000 M-(1)cm(-1)) owing to the lower molecular symmetry. Photophysical and photobiological properties of porphycenes make them excellent candidates as PSs, showing fast uptake and diverse subcellular localizations (mainly membranous organelles). Several tetraalkylporphycenes and the tetraphenyl derivative (TPPo) induce photodamage and cell death in vitro. Photodynamic treatments of cultured tumor cells with TPPo and its palladium(II) complex induce cytoskeletal changes, mitotic blockage, and dose-dependent apoptotic or necrotic cell death. Some pharmacokinetic and phototherapeutic studies on experimental tumors after intravenous or topical application of lipophilic alkyl-substituted porphycene derivatives are known. Taking into account all these features, porphycene PSs should be very useful for PDT of cancer and other biomedical applications.

Uptake and localization mechanisms of fluorescent and colored lipid probes. Part 3. Protocols for predicting intracellular localization of lipid probes using QSAR models.

We provide detailed protocols for applying the QSAR decision-rule models described in Part 2 of this paper. These procedures permit prediction of the intracellular localization of fluorescent probes or of any small molecular xenobiotic whether fluorescent or not. Also included is a set of notes that give practical advice on various possible problems and limitations of the methods, together with a flow chart that provides a graphical algorithmic summary of the QSAR models.

Uptake and localization mechanisms of fluorescent and colored lipid probes. Part 2. QSAR models that predict localization of fluorescent probes used to identify ("specifically stain") various biomembranes and membranous organelles.

We discuss a variety of biological targets including generic biomembranes and the membranes of the endoplasmic reticulum, endosomes/lysosomes, Golgi body, mitochondria (outer and inner membranes) and the plasma membrane of usual fluidity. For each target, we discuss the access of probes to the target membrane, probe uptake into the membrane and the mechanism of selectivity of the probe uptake. A statement of the QSAR decision rule that describes the required physicochemical features of probes that enable selective staining also is provided, followed by comments on exceptions and limits. Examples of probes typically used to demonstrate each target structure are noted and decision rule tabulations are provided for probes that localize in particular targets; these tabulations show distribution of probes in the conceptual space defined by the relevant structure parameters ("parameter space"). Some general implications and limitations of the QSAR models for probe targeting are discussed including the roles of certain cell and protocol factors that play significant roles in lipid staining. A case example illustrates the predictive ability of QSAR models. Key limiting values of the head group hydrophilicity parameter associated with membrane-probe interactions are discussed in an appendix.

A specific fluorogenic reaction for tryptophan residues using isatin in organic solvents.

The development of new fluorogenic reactions for specific chemical groups is of increasing interest in cytochemistry. We describe the application of the nonfluorescent compound isatin on methanol-fixed blood smears. When treated with 0.1% isatin in absolute ethanol or acetone for 30 min and observed under violet-blue exciting light, eosinophil leucocyte granules show a bright green fluorescence. This fluorogenic reaction is abolished after blocking of tryptophan residues by performic acid oxidation. Spectrofluorometric studies with amino acids in vitro reveal that isatin forms a unique and specific fluorescent product with tryptophan.

Cytochemical application of tris (2,2'-bipyridine) ruthenium (II): fluorescence reaction with sulfated polyanions of mast cell granules.

We describe the use of tris (2,2'-bipyridine) ruthenium (II) (Rubipy) as a cationic fluorochrome for cytochemical and histochemical studies. After staining with Rubipy, mast cell granules (MCGs) and lymphocyte nuclei (LN) from mouse peritoneal cavity and human breast carcinoma showed intense orange fluorescence and no fading under blue or blue-violet exciting light. Staining at low pH (< 2) or pre-treatment with Al3+ ions strongly diminished the fluorescence of LN, whereas that of MCG was less affected. Ca2+ and Ba2+ ions only diminished MCG fluorescence. Blots of DNA, pectic acid, heparin, and other sulfated polysaccharides stained with Rubipy showed high emission, which was reduced in DNA and pectic acid staining at low pH. Studies with chemically modified heparins suggested that O-sulfates were more important than N-sulfates in Rubipy-heparin interactions. These results are in agreement with an ionic binding mode between Rubipy and heparin. A very suitable method for mast cell detection was found with Mayer's hematoxylin before Rubipy staining, which could be of great value for histopathological studies. This procedure allowed visualization of the mast cells by fluorescence microscopy, and nuclei and tissue morphology were easily visualized under brightfield illumination.

A new fluorescence reaction in DNA cytochemistry: microscopic and spectroscopic studies on the aromatic diamidino compound M&B 938.

We describe the fluorescence properties and cytochemical applications of the aromatic diamidine M&B 938. Treatment of cell smears (chicken blood, Ehrlich ascites tumor, rat bone marrow, mouse mast cells, and Trypanosoma cruzi epimastigotes) with aqueous solutions of M&B 938 (0.5-1 microgram/ml at pH 6-7; UV excitation) induced bright bluish-white fluorescence in DNA-containing structures (interphase and mitotic chromatin, AT-rich kinetoplast DNA of T. cruzi), which was abolished by previous DNA extraction. DNA was the unique fluorescent polyanion after staining with M&B 938 at neutral or alkaline pH, other polyanions such as RNA and heparin showing no emission. M&B 938-stained mouse metaphase chromosomes revealed high fluorescence of the AT-rich centromeric heterochromatin, and strong emission of heterochromatin in human chromosomes 1, 9, 15, 16, and Y was found after distamycin A counterstaining. On agarose gel electrophoresis, M&B 938-stained DNA markers appeared as fluorescent bands. The 1.635-KBP fragment from DNA ladder revealed a higher emission value than that expected from linear regression analysis. Spectroscopic studies showed bathochromic and hyperchromic shifts in the absorption spectrum of M&B 938 complexed with DNA, as well as strong enhancement of fluorescence at 420 nm. In the presence of poly(dA)-poly(dT), the emission of M&B 938 was 4.25-fold higher than with DNA; no fluorescence was observed with poly(dG)-poly(dC). Experimental results and considerations of the chemical structure suggest that the minor groove of AT regions of DNA could be the specific binding site for M&B 938, which shows interesting properties and useful applications as a new DNA fluorochrome.

The osmium tetroxide-p-phenylenediamine procedure reveals the chromatid cores and kinetochores of meiotic chromosomes by light and electron microscopy.

We analyzed first-metaphase meiotic chromosomes of the grasshopper Chorthippus jucundus by two different methods, i.e., a silver impregnation technique and the osmium tetroxide-p-phenylenediamine (Os-PPD) procedure. The former was applied on squashed testes previously fixed in ethanol-acetic acid, whereas for Os-PPD the material was not subjected to any previous extraction treatment but was fixed in OsO4, treated with PPD, and embedded in Epon 812. Both techniques revealed chromatid cores and kinetochores regardless of the processing of the material (squashed or sectioned). Unstained Os-PPD sections were analyzed by light microscopy and transmission electron microscopy (TEM). The Os-PPD technique provided a high contrast of chromatid cores and kinetochores in relation to the chromatin, which revealed a low electron density. To determine the Os-PPD reaction mechanism, the PAS procedure, as well as scanning electron microscopy (SEM) backscattering and SEM X-ray microanalysis, was performed on sections. By use of the Os-PPD-PAS procedure, glycol groups formed by oxidation of osmium bound to aromatic substrates were detected in chromatid cores and kinetochores by brightfield and fluorescence microscopy. A high Z contrast was detected in these structures by backscattered electron imaging. SEM X-ray microanalysis showed osmium and phosphorus to be the main elements present on the chromatid cores. Taking into account the known reactivity of OsO4 and the present results, the possible participation of nucleic acids as well as proteins in the Os-PPD reaction mechanism and in the composition of chromatid cores and kinetochores is discussed.

Meso-tetraphenylporphyrin: photosensitizing properties and cytotoxic effects on cultured tumor cells.

In this study we analyzed photosensitizing and photodamaging properties of the hydrophobic meso-tetraphenylporphyrin (TPP, incorporated into liposomes) on HeLa cells. Under the fluorescence microscope, red fluorescence by TPP was detected on the cell surface. TPP followed by violet-blue or red irradiation led to cell death, blebs and plasma membrane deformations appearing immediately after photodynamic treatment. Production of singlet oxygen by TPP was studied by analyzing tryptophan photodegradation, which increased in the presence of D2O and was abolished by NaN3. Present results suggest that the plasma membrane is the main cellular target for TPP, which could be a valuable photosensitizing drug in studies on photodynamic therapy of cancer.

The pesticide malathion induces alterations in actin cytoskeleton and in cell adhesion of cultured breast carcinoma cells.

We have studied the effects of the organophosphorous pesticide malathion on cell viability, actin cytoskeleton, cell adhesion complex E-cadherin/beta-catenin, and Rho and Rac1 GTPases from the human mammary carcinoma cell line MCF-7. Malathion induced cell lethality, determined by the MTT assay, depending on the treatment conditions. Cells incubated with low concentrations of malathion, 16-32 microg/ml, showed high survival rates (>95%) at any evaluated time (1-5 days), whereas complete cell lethality was found using 512 microg/ml and 5 days of treatment. Deep morphological changes were induced with high doses of 64 and 128 microg/ml, and long incubation time (5 days); cells showed perinuclear vacuoles, rounding, shrinkage, and a gradual loss of adhesion. These changes were related to a decrease in the expression of the adhesion molecules, E-cadherin and beta-catenin, and to the distribution and reactivity of actin microfilaments to TRITC-phalloidin. Disruption of microfilaments, accompanied by the collapse of actin to perinuclear region, were characteristic of cells with loss of adhesion. At lower concentrations, some cells presented deformations on the plasma membrane as lamellipodia-like structures, which were particularly evident from 32 to 128 microg/ml. Conversely, we observed an increase in the expression of Rho and Rac1 GTPases, modulators of actin cytoskeleton and cell adhesion.

Necrotic cell death induced by photodynamic treatment of human lung adenocarcinoma A-549 cells with palladium(II)-tetraphenylporphycene.

In this study we describe photodamaging and photokilling effects of palladium(II)-tetraphenylporphycene (PdTPPo) (previously incorporated into dipalmitoylphosphatidylcholine liposomes) on the human lung adenocarcinoma A-549 cell line. No dark cytotoxicity was found when the drug was applied at 10(-6) M or 5 x 10(-7) M for 1 or 18 h, respectively. After 1-h treatment with 10(-7) M or 5 x 10(-7) M PdTPPo followed by red light irradiation for variable times, dose-dependent lethal effects were observed in A-549 cells. Apoptosis was not found after the above photodynamic treatments or under even milder sublethal conditions. In contrast to HeLa cells subjected to PdTPPo photosensitization where either apoptosis or necrosis were induced, morphological analysis and electrophoretical DNA pattern of A-549 cells always revealed a clearly necrotic death mechanism. However, A-549 cells died by apoptosis after serum and L-glutamine deprivation, indicating that only the photodynamically induced apoptosis was inhibited. Immunofluorescent labeling revealed that microtubules and actin microfilaments were immediately and strongly damaged by photodynamic treatments with PdTPPo. No metaphase arrest and/or mitotic alterations were observed after phototreatments. Present results show that the cell type plays a fundamental role in relation to the apoptotic or necrotic response to photosensitization, and that cytoskeletal components are important targets implicated in cell death processes.

Photodynamic damage to HeLa cell microtubules induced by thiazine dyes.

The aim of this study was to analyze possible alterations of the microtubule cytoskeleton of cultured cells subjected to photodynamic treatments with the thiazine dyes methylene blue or toluidine blue. Indirect immunofluorescence labeling of alpha-tubulin was performed in HeLa cells after 1 or 18 h of incubation with thiazines followed by red-light irradiation for 15 min [leading to surviving fractions (SF) of about 65% (SF65) or 1% (SF1), respectively]. Untreated control cells showed the normal distribution of interphase microtubules, whereas considerable or severe disorganization of the microtubule network was observed after SF65 or SF1 photodynamic treatments, respectively. A great amount of blebs showing homogeneous fluorescence was also found on the cell surface after SF1 treatments. Possible mechanisms responsible for the photodamage to microtubules induced by thiazine dyes are briefly discussed.

Photokilling mechanisms induced by zinc(II)-phthalocyanine on cultured tumor cells.

The photosensitizing effects of liposomal zinc(II)-phthalocyanine (ZnPc) on HeLa cells, with emphasis on morphological changes and mechanisms for cell death, have been studied. No dark toxicity for ZnPc alone was found. Incubation for 1 h with ZnPc followed by red light irradiation induced a variable decrease in the surviving of cells, which was related to both drug concentration and irradiation time. A lethal photodynamic effect (100% of the cells are killed: LD100) was induced by 5 x 10-6 M ZnPc and 5-min irradiation, whereas a sublethal effect (60% of the cells are killed: LD60) was detected with 10 7 M ZnPc and 3 min of red light. Toluidine blue and Hoechst 33258 staining showed characteristic alterations of cell morphology. Numerous bubbles on the plasma membrane were found immediately after an LD100 treatment, and a necrotic morphology appeared 24 h later. On the contrary, severe cell shrinkage with nuclear fragmentation. characteristic of apoptosis. was observed 8 and 24 h after LD60 treatments. In this case, propidium iodide-acridine orange labeling and the TUNEL assay confirmed the occurrence of apoptosis. The highest amount of apoptotic cells appeared 24 h after LD60 treatments, particularly in detached cells, as revealed by cell counting and DNA electrophoresis. Both apoptotic and necrotic mechanisms for cell death occur in HeLa cells in dependence on the experimental protocol of ZnPc photodynamic treatments.

Photodamage induced by Zinc(II)-phthalocyanine to microtubules, actin, alpha-actinin and keratin of HeLa cells.

We have studied the photosensitizing effects of zinc(II)-phthalocyanine (ZnPc) on the cytoskeleton of HeLa cells using sublethal (10(-7) M, followed by 1 or 3 min of red light to induce 20%, LD20, or 60%, LD60, cell death, respectively) or lethal (5 x 10(-6) M and 15 min of irradiation, LD100) experimental conditions. The immunofluorescent analysis of the cytoskeleton showed a variable photodamage to microtubules (MT), actin microfilaments (AF) and intermediate filaments of keratin (KF), as well as on alpha-actinin, which was dependent on treatment conditions. Both sublethal treatments induced deep alterations on interphase and mitotic MT. The mitotic index increased with time with the maximum at 18 h (12%) or 24 h (14%) after LD20 or LD60, respectively. The alterations on AF and alpha-actinin were much more severe than those observed on KF at any evaluated time. With the exception of the KF, which remained partially organized, the MT and AF network was severely damaged by the lethal treatment. Western blot analysis for alpha-tubulin, G-actin and alpha-actinin from soluble and insoluble fractions confirmed the results observed by immunofluorescence, thus indicating that these cytoskeletal components are involved in cell damage and death by ZnPc photosensitization.

Voltammetric behavior of berenil.

Berenil is reduced on mercury drops electrode in buffered aqueous media. The reduction of -N=N- group is controlled by diffusion. Polarographic waves are of analytical usefulness. The hydrogen discharge is favored by the basic centers of the molecule in Co(II)/ammonia-buffered media.

Effect of copper coordination complexes on sister-chromatid exchanges in plant cells.

The effectiveness of several copper coordination complexes in the induction of sister-chromatid exchanges (SCE) in meristematic cells of Allium cepa roots was studied. A significant increase in the frequencies of SCE was observed when cells were exposed to different concentrations of copper complexes for 3 h. This effect, which is concentration dependent, seems to be related to the possibility of intercalative binding modes between the planar copper complexes and DNA.

Analytical SEM of cell polyanions stained with tris(2,2'-bipyridine)ruthenium(II).

Peritoneal mast cells and lymphocytes from mice were placed on graphite supports, fixed in methanol, stained with the new fluorochrome tris(2,2'-bipyridine)ruthenium(II) and microanalysed using scanning electron microscopy (SEM). X-ray microanalysis showed the expected signal of P and S (K alpha lines) in emission spectra of lymphocytes and mast cells. The signal of Ru (L alpha 1 and L beta 1) overlapped with that of Cl, although the peak in the corresponding region was about 7 times higher than that from unstained cells. X-ray images showed the topographic localization of P, S and Ru in mast cells and lymphocytes and confirmed the accumulation of the Ru complex in heparin- and DNA-containing structures. These results indicate that, by using suitable marker elements and detection methods, analytical SEM is a useful complement in cytochemical studies.

Image processing and analysis of fluorescent labelled cytoskeleton.

Cytoskeleton elements play an important role in cellular processes including normal and pathological conditions or even cell death. Although immunofluorescence microscopy techniques have proved useful to study the cytoskeleton, description and comparative results commonly rely on the direct visual analysis of the targeted cells. We report here an objective and quantitative method to analyze the cytoskeletal pattern of cultured HeLa cells based on image processing and analysis (IPA) of fluorescent labelled cytoskeleton components. Different structural quantifiable characteristics using IPA facilities (densitometric profile, number of branched points, Euler number, convexity, orientation value) allow accurate characterization and differentiation of the three types of fibre nets studied (microtubules, actin microfilament, and cytokeratin intermediate filaments). The application of the analytical methods presented here provides information concerning the organization and modification of cytoskeleton components, and could aid in the further understanding of structural and functional aspects of the cytoskeleton under normal and pathological conditions.

Chronic intoxication by heroin; histopathological effects on seminiferous tubules.

Seminiferous tubules from heroin abusers and from rats chronically intoxicated by heroin samples presented a striking reduction in the thickness of the germinal epithelium. Light and electron microscopical studies showed a considerable increase of lipids and phagosomes in Sertoli cells, disorganization of their junction complexes, detachment of immature germ cells which appeared free in the tubular lumen, and formation of giant multinucleate spermatids. These alterations point out that Sertoli cells could be the target element for the toxic effect of heroin samples on the seminiferous epithelium.

Monomerizing effect of caffeine, o-phenanthroline, and tannin on cationic dyes: a model system to analyze spectral characteristics of the intercalative binding to nucleic acids.

If used as co-solutes in concentrated solutions of cationic planar dyes, caffeine, o-phenanthroline, and tannin induce striking hyperchromic and bathochromic shifts in their absorption spectra. Likewise, the fluorescence of acridine orange at high concentration greatly increases in the presence of caffeine, the emission peak appearing at a shorter wave-length. These spectral changes, which are similar to those produced by organic solvents, detergents, and alpha-cyclodextrin, reflect the disaggregating (monomerizing) capacity of the co-solutes on stacked chromophores. After washing with saturated solutions of caffeine or o-phenanthroline, the chromatin fluorescence by intercalating fluorochromes is reduced or abolished, which suggests competition effects for intercalative binding modes. These results support the use of caffeine, o-phenanthroline, and tannin in spectroscopic and histochemical studies of dye-stuff interactions with DNA and chromatin.