Ultrastructural changes induced in HeLa cells after phototoxic treatment with harmine.

In the present work, we have continued our studies on harmine phototoxicity in human tumour cells. The toxicity of harmine in the dark was analysed by a quantitative neutral red uptake assay, and subcellular sensitive targets following harmine photosensitization were de fi ned by electron microscopic analysis of HeLa cells. The results obtained indicated that this compound shows a clear dose-dependent cytotoxic effect in the dark. The combined treatment with suitable doses of harmine and UV radiation was very effective at an early stage, although maximal cell killing appeared 48 h after photodynamic activation. Ultrastructural examination of HeLa cells immediately after the photodynamic treatment revealed lysosomal destabilization and profound cytoplasmic vacuolization that evolved to cytolysis, which is typical of necrotic cell death. It is concluded that harmine could be a valuable photosensitizer whose biological applications merit further evaluation.

Ecotoxicological evaluation of carbamazepine using six different model systems with eighteen endpoints.

The occurrence of pharmaceutically active compounds in the aquatic environment has been recognized as one of the emerging issues in environmental chemistry. However, the ecotoxicological effects of pharmaceuticals have still not been researched adequately. Carbamazepine, an anticonvulsant commonly present in surface and groundwater, was studied, using six ecotoxicological model systems with eighteen endpoints evaluated at different exposure time periods. The battery included the immobilization of Daphnia magna, bioluminescence inhibition in the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris, and micronuclei induction and root growth inhibition in the plant Allium cepa. Cell morphology, neutral red uptake, total protein content, MTS metabolization, lactate dehydrogenase leakage and activity and glucose-6-phosphate dehydrogenase activity were studied in the salmonid fish cell line RTG-2. The total protein content, LDH activity, neutral red uptake and MTT metabolization in Vero monkey kidney cells were also investigated. The most sensitive system to carbamazepine was the Vero cell line, followed by Chlorella vulgaris, Vibrio fischeri, Daphnia magna, Allium cepa, and RTG-2 cells. EC50 values from 19 microM in Vero cells at 72 h to more than 1200 microM in other systems, were obtained. Comparing the concentrations in water and the toxicity quantified in our assay systems, carbamazepine is not expected to produce acute toxic effects in the aquatic biota under these circumstances, but chronic and synergistic effects with other chemicals cannot be excluded.

An in vitro study on the kinetics, subcellular distribution and phototoxicity of harmine in human tumor cells.

The beta-carboline alkaloid harmine, which was found to possess interesting phototoxic properties in different biological systems, was investigated for photokilling of human tumor cells in vitro. Harmine was readily accumulated by HeLa cells, localized preferably in cytoplasm, being a non toxic compound when used at low concentrations. The photoactivation of harmine-loaded cells with UV radiation showed lysosomal damage, reflected by altered localization of the fluorescent probe acridine orange, as well as an evident cell killing which increase with time up to a maximal value 48 h after the photodynamic treatment.

A test battery for the ecotoxicological evaluation of pentachlorophenol.

Experimental bioassays are currently used in ecotoxicology and environmental toxicology to provide information for risk assessment evaluation of new chemicals and to investigate their effects and mechanisms of action; in addition, ecotoxicological models are used for the detection, control and monitoring of the presence of pollutants in the environment. As a single bioassay will never provide a full picture of the quality of the environment, a representative, cost-effective and quantitative test battery should be developed. The effects of pentachlorophenol were studied using a battery of ecotoxicological model systems, including immobilization of Daphnia magna, bioluminiscence inhibition in the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris, and micronuclei induction in the plant Allium cepa. The inhibition of cell proliferation and MTT reduction were investigated in Vero cells. Neutral red uptake, cell growth, MTT reduction, lactate dehydrogenase leakage and activity were studied in the salmonid fish cell line RTG-2, derived from the gonad of rainbow trout. Pentachlorophenol was very toxic for all biota and cells. The system most sensitive to pentachlorophenol, was micronuclei induction in A. cepa, followed by D. magna immobilization, bioluminescence inhibition in V. fischeri bacteria at 60 min and cell proliferation inhibition of RTG-2 cells at 72 h. Inhibition of cell proliferation and MTT reduction on Vero monkey cells showed intermediate sensitivity.

Simple beta-carboline alkaloids as nucleic acids fluorochromes.

Treatment of cell smears (chicken blood, Trypanosoma cruzi epimastigotes, Ehrlich ascites tumor cells and mouse spleen) with simple beta-carboline alkaloids induced a strong bluish white fluorescence emission of condensed chromatin and basophilic cytoplasm under ultraviolet excitation. The compounds used were harmine, harmol, harman, norharman, harmalol and harmaline (25-50 micrograms/ml aqueous solutions for 2-3 min). Spectrofluorometric studies on harmine solutions in vitro (emission peak at 420 nm) showed fluorescence quenching at high concentrations as well as in the presence of DNA. Microscopic fluorescence features of these new fluorochromes and possible binding modes to nucleic acids are discussed.

S-adenosyl-L-homocysteine: a non-cytotoxic hypomethylating agent.

The cytotoxic effect caused by the hypomethylating agent S-adenosyl-L- homocysteine (SAH) was compared with that of two drugs commonly used to induce DNA hypomethylation, 5-azacytidine and 5-aza-2'-deoxycytidine. Two in vitro cytotoxicity tests, the tetrazolium MTT assay and the intracellular lactate dehydrogenase (LDH) activity test, suggest that SAH induces hypomethylation without causing any cytotoxic effect. We propose the use of SAH as a non-cytotoxic agent which may be more suitable for inducing experimental DNA hypomethylation.

Fluorescence reactions of orcein: new applications for an old stain.

The fluorescence properties of orcein are described in this paper. Under suitable exciting light, acidic solutions of orcein showed emission peaks at 587 nm (low concentration) and at 620 nm (high concentration). In fluorescence microscopy an intense orange-red emission was found in the cytoplasm and nucleoli from acetic orcein squashes of meristematic cells, as well as in the cytoplasm of Ehrlich tumour cells and in chicken erythrocyte nuclei. When semithin sections of Epon-embedded tissues were treated with orcein, a strong fluorescence reaction (orange-red) appeared in starch granules, cell walls, elastin, collagen, reticulin, keratohyalin, chromatin and mucin. Cytofluorometric measurements of orcein-stained chromatin revealed an emission peak at 585 nm with a shoulder at 620 nm. These spectroscopic and microscopical results suggest the possibility of employing orcein as a fluorochrome.

Autofluorescence and induced fluorescence in Epon embedded tissue sections.

The autofluorescence of tissue components in Epon semithin sections depends on the exciting wavelength, section thickness, and the structure under observation. The degree of autofluorescence emission of a given component affects the fluorescence reaction induced by fluorochromes, a feature which must be taken into account when fluorescence microscopy is applied to the study of Epon sections.

[Visualization of specific leukocyte granules using morin and other fluorochromes]. / Visualización de gránulos específicos de leucocitos por medio de morina y otros fluorocromos.

The visualization of the specific granulation of leukocytes is useful in hematology to identify de cell type in leukemic diseases. The authors employed the acidic fluorochrome morin after testing its high affinity for the fluorescent reaction with morin in human and chicken peripheral blood smears, and in streak smears from rabbit spleen. The best results were obtained with a solution prepared either with 0.10 mg morin per ml of 50 per 100 ethanol, or with a saturated solution of morin in distilled water diluted to one fourth in 25 per 100 ethanol. When observed under blue-violet excited light (436 nm) the acidophilic granules of leukocytes produce a bright yellow fluorescence, while the cell nuclei show a faint greenish fluorescence. As for the mechanism of the reaction, it consists possibly in an interaction between morin and the basic components of the granules. Other acidic fluorochromes, as primulin and hematoxylin, produce similar fluorescent reactions with the acidophilic granules.

Fluorescence patterns of chromatin and cytoplasm by hematoxylin solutions.

Treatments of chicken blood and Ehrlich ascites tumor smears with hematoxylin solutions give a fluorescence reaction in chromatin, basophilic cytoplasm and leukocyte granules. In these structures the fluorescence emission increases upon dye aging and prolonged staining times. We present a preliminary spectral analysis suggesting the possibility to employ hematoxylin as a fluorochrome.

Citoquimica de la union de colorantes y fluorocromos con DNA y cromatina. / Cytochemistry of the binding of dyes and flurochromes with DNA and chromatin

El estudio de la interaccion entre colorantes y fluorocromos basicos y acidos nucleicos ha llegado a adquirir una gran importancia en la biologia molecular actual, asi como indudables repercusiones sobre las ciencias biomedicas. El analisis citoquimico de la tincion y/o fluorescencia de la cromatina por colorantes y fluorocromos derivados del xanteno, azina, tiazina, isoquinolina, bezotiazol y ftalocianina, permite obtener indicaciones sobre especificidades y mecanismos de union con el DNA, asi como correlacionar los resultados microscopicos con las caracteristicas fisicoquimicas de los colorantes y de su interaccion con acidos nucleicos

Citoquimica de la union de colorantes y fluorocromos con DNA y cromatina. / Cytochemistry of the binding of dyes and flurochromes with DNA and chromatin

El estudio de la interaccion entre colorantes y fluorocromos basicos y acidos nucleicos ha llegado a adquirir una gran importancia en la biologia molecular actual, asi como indudables repercusiones sobre las ciencias biomedicas. El analisis citoquimico de la tincion y/o fluorescencia de la cromatina por colorantes y fluorocromos derivados del xanteno, azina, tiazina, isoquinolina, bezotiazol y ftalocianina, permite obtener indicaciones sobre especificidades y mecanismos de union con el DNA, asi como correlacionar los resultados microscopicos con las caracteristicas fisicoquimicas de los colorantes y de su interaccion con acidos nucleicos