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.

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.

Two combined photosensitizers: a goal for more effective photodynamic therapy of cancer.

Photodynamic therapy (PDT) is a clinically approved therapeutic modality for the treatment of diseases characterized by uncontrolled cell proliferation, mainly cancer. It involves the selective uptake of a photosensitizer (PS) by neoplastic tissue, which is able to produce reactive oxygen species upon irradiation with light, leading to tumor regression. Here a synergistic cell photoinactivation is reported based on the simultaneous administration of two PSs, zinc(II)-phthalocyanine (ZnPc) and the cationic porphyrin meso-tetrakis(4-N-methylpyridyl)porphine (TMPyP) in three cell lines (HeLa, HaCaT and MCF-7), using very low doses of PDT. We detected changes from predominant apoptosis (without cell detachment) to predominant necrosis, depending on the light dose used (2.4 and 3.6 J/cm(2), respectively). Analysis of changes in cytoskeleton components (microtubules and F-actin), FAK protein, as well as time-lapse video microscopy evidenced that HeLa cells were induced to undergo apoptosis, without losing adhesion to the substrate. Moreover, 24 h after intravenous injection into tumor-bearing mice, ZnPc and TMPyP were preferentially accumulated in the tumor area. PDT with combined treatment produced significant retardation of tumor growth. We believe that this combined and highly efficient strategy (two PSs) may provide synergistic curative rates regarding conventional photodynamic treatments (with one PS alone).

Replacing xylene with n-heptane for paraffin embedding.

In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.

An artificial neural network model for predicting the subcellular localization of photosensitisers for photodynamic therapy of solid tumours.

Photodynamic therapy (PDT) is a promising modality for the treatment of tumours based on the combined action of a photosensitiser (PS), visible light and molecular oxygen, which generates a local oxidative damage that leads to cell death. The site where the primary photodynamic effect takes place depends on the subcellular localization of the PS and affects the mode of action and efficacy of PDT. It is therefore of prime interest to develop structure-subcellular localization prediction models for a PS from its molecular structure and physicochemical properties. Here we describe such a prediction method for the localization of macrocyclic PSs into cell organelles based on a wide set of physicochemical properties and processed through an artificial neural network (ANN). 128 2D-molecular descriptors related to lipophilicity/hydrophilicity, charge and structural features were calculated, then reduced to 76 by using Pearson's correlation coefficient, and finally to 5 using Guyon and Elisseeff's algorithm. The localization of 61 PSs was compiled from literature and distributed into 3 possible cell structures (mitochondria, lysosomes and "other organelles"). A non-linear ANN algorithm was used to process the information as a decision tree in order to solve PS-organelle assignment: first to identify PSs with mitochondrial and/or lysosomal localization from the rest, and to classify them in a second stage. This sequential ANN classification method has permitted to distinguish PSs located into two of the most important cell targets: lysosomes and mitochondria. The absence of false negatives in this assignation, combined with the rate of success in predicting PS localization in these organelles, permits the use of this ANN method to perform virtual screenings of drug candidates for PDT.

A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents.

Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H(2)O(2). After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H(2)O(2) concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.

Uptake and localization mechanisms of fluorescent and colored lipid probes. 1. Physicochemistry of probe uptake and localization, and the use of QSAR models for selectivity prediction.

We outline the factors involved in precise targeting of lipids and membranes by probes, namely, lipid and probe chemistry, geometry/topography of probe delivery, and probe uptake kinetics. The special case of probe orientation within membranes also is considered. The varieties of commercially available fluorophores are described, and an overview of probe physicochemical properties (amphiphilicity, conjugated system size, electrical properties, head group size, lipophilicity and solubility) is provided together with notes on their parameterization. Probe-lipid physicochemical interactions, and their relations to parameterization, then are discussed including the nature and derivation of decision-rule QSAR models, partitioning and insertion of probes into bulk lipids and complications of this, partitioning and insertion of probes into membranes, and flip-flop of probes across membrane leaflets. A general QSAR algorithm for understanding lipid probe application then is set out. Problems and limitations are outlined. Biological issues include varied biomembrane composition, cell line effects and toxicity of fluorescent probes. Methodological issues include difficulties of estimating certain numerical structure parameters, the impure character of many fluorochromes and dyes, and the perturbation of biomembrane structure by fluorescent probes.

Inaccurate chemical structures of dyes and fluorochromes found in the literature can be problematic for teaching and research.

Abstract Representations of the chemical structures of dyes and fluorochromes often are used to illustrate staining mechanisms and histochemical reactions. Unfortunately, inaccurate chemical structures sometimes are used, which results in problems for teaching and research in histochemistry. We comment here on published examples of inadequate chemical drawing and modeling. In particular, omission of hydrogen atoms can lead to misleading hydrogen-bonding interactions, and inaccurate drawing and modeling procedures result in a variety of implausible molecular structures. The examples and arguments given here are easily intelligible for non-chemists and could be used as part of a training approach to help avoid publication of misleading or puzzling dye structures and molecular models for illustrating biological staining and histochemical studies.

Binding of cationic dyes to DNA: distinguishing intercalation and groove binding mechanisms using simple experimental and numerical models.

Simple methods for predicting intercalation or groove binding of dyes and analogous compounds with double stranded DNA are described. The methods are based on a quantitative assessment of the aspect (width to length) ratio of the dyes. The procedures were validated using a set of 38 cationic dyes of varied chemical structures binding to well oriented DNA fibers and assessing binding orientation by linear dichroism and polarized fluorescence. We demonstrated that low aspect ratio dyes bound by intercalation, whereas more rod-like dyes were groove binders. Some problems that result and possible applications are discussed briefly.

Selective labeling of lipid droplets in aldehyde fixed cell monolayers by lipophilic fluorochromes.

We evaluated a number of lipophilic dyes and fluorochromes, including oxazone and thiazone derivatives of oxazine and thiazine dyes, scintillator agents, a carotenoid and a metal-porphyrin complex for visualization of lipid droplets within aldehyde fixed cultured HeLa and BGC-1 cells. Observation under ultraviolet, blue or green exciting light revealed selective fluorescence of lipid droplets, particularly after treatment with aqueous solutions of Nile blue and brilliant cresyl blue oxazones, toluidine blue thiazone, or propylene glycol solutions of canthaxanthin, ethyl-BAO, and ZnTPyP. Mounting in water was required to maintain the fluorescence of lipids; the use of glycerol, Mowiol or Vectashield was not adequate. The effect of dye structure on staining intensity was assessed with the aid of numerical structure parameters modeling lipophilicity (HI and log P), overall size (MW) and the size of the conjugated system (conjugated bond number; CBN). The best stains for lipid droplets were relatively lipophilic (HI > 4.0, log P > 5.0), of small size overall (MW < 370), with small conjugated systems (CBN < 24), and not significantly amphiphilic. The two hydrophobic-hydrophilic parameters (the classic log P and the hydrophobic index, HI; values calculated by molecular modeling software) were highly correlated; however, HI was a more suitable hydrophobicity index for the dyes studied here.

Improving images of fluorescent cell labeling by background signal subtraction.

The uptake and selective accumulation of fluorescent labels and drugs into organelles of cultured cells currently are widely investigated in biomedical research. In such studies, co-localization procedures are frequently used to identify the accumulation sites of compounds with biological activity. A drawback with fluorescent labeling is the autofluorescence of some cell organelles, which can hinder the precise assessment of co-localization. We report here labeling of the Golgi apparatus of A-549 cells using the photosensitizer zinc(II)-phthalocyanine (ZnPc) and co-localization with the Golgi probe NBD C6-ceramide. The blue autofluorescence signal of mitochondria can be subtracted easily from the original picture by image processing, after which the co-localization of the isolated red ZnPc signal with the green signal from the Golgi probe is considerably improved.

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.

Photodynamic Therapy of the Murine LM3 Tumor Using Meso-Tetra (4-N,N,N-Trimethylanilinium) Porphine.

Photodynamic therapy (PDT) of cancer is based on the cytotoxicity induced by a photosensitizer in the presence of oxygen and visible light, resulting in cell death and tumor regression. This work describes the response of the murine LM3 tumor to PDT using meso-tetra (4-N,N,N-trimethylanilinium) porphine (TMAP). BALB/c mice with intradermal LM3 tumors were subjected to intravenous injection of TMAP (4 mg/kg) followed 24 h later by blue-red light irradiation (λmax: 419, 457, 650 nm) for 60 min (total dose: 290 J/cm(2)) on depilated and glycerol-covered skin over the tumor of anesthetized animals. Control (drug alone, light alone) and PDT treatments (drug + light) were performed once and repeated 48 h later. No significant differences were found between untreated tumors and tumors only treated with TMAP or light. PDT-treated tumors showed almost total but transitory tumor regression (from 3 mm to less than 1 mm) in 8/9 animals, whereas no regression was found in 1/9. PDT response was heterogeneous and each tumor showed different regression and growth delay. The survival of PDT-treated animals was significantly higher than that of TMAP and light controls, showing a lower number of lung metastasis but increased tumor-draining lymph node metastasis. Repeated treatment and reduction of tissue light scattering by glycerol could be useful approaches in studies on PDT of cancer.

Morphological criteria to distinguish cell death induced by apoptotic and necrotic treatments.

We present a comparative study of apoptotic and necrotic morphology (light and scanning electron microscopy), induced by well known experimental conditions (photodynamic treatments, etoposide, hydrogen peroxide, freezing-thawing and serum deprivation) on cell cultures. Our results indicate that morphological criteria (apoptotic cell rounding and shrinkage, and appearance of membrane bubbles in early necrosis) allow to distinguish these cell death mechanisms, and also show that, independently of the damaging agents, the necrotic process occurs in a characteristic sequence (coalescence of membrane bubbles in a single big one that detaches from cells remaining on the substrate).

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.

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.

Recycling cultured cells for immunofluorescent labeling.

A method to use sequential rounds of immunofluorescent labeling in cell cultures is presented. The method is based on the utilization of a non-liquid reducing agent, sodium dithionite, in conjunction with ionic or non-ionic detergents (SDS or TX100, respectively) at room temperature. This method preserves cell morphology and substrate antigenicity, and operates through the complete extraction of most primary and secondary antibodies. Using this protocol, the sequential immunolocalization of different proteins is possible, without signal interference with previous immunolabeling rounds. In addition, the method is also useful to recycle blotted membranes in immunoblots.

Fixation and permanent mounting of fluorescent probes after vital labelling of cultured cells.

The use of fluorescent probes for the visualization of organelles in living cells and assessment of live/dead cells has an increasing importance in cell biology. However, rapid and irreversible morphological changes of labelled cells (due to the photosensitizing effect of most fluorescent probes) make prolonged observation and detailed analysis of living cells under continuous excitation difficult. In this study, we describe a method for fixing and mounting cultured HeLa and 3T3 cells labelled with acridine orange (lysosomes), rhodamine 123 (mitochondria), Hoechst 33342 (nuclei), and propidium iodide/acridine orange (live/dead HeLa cells subjected to nutrient deprivation). Fixation is performed with vapours of a commercially available formaldehyde solution for 0.5-1 min followed by air drying and permanent mounting in DPX. After this procedure, both the general morphology and selective fluorescent labelling of cells are well preserved. The method of vapour fixation and DPX mounting is simple, rapid and reproducible, allowing definitive preservation of the fluorescence pattern observed in unfixed cell cultures.

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.