cytochrome-c-t and 2--7--dichlorofluorescein

The aim of this work is to study the nature of reactive oxygen species, ROS, arisen from Chitosan/2-HP-β-Cyclodextrin/Chlorophyll a (CH/CD/Chla) blended biofilm under a photodynamic activity. Suitable molecules, called primary acceptors, able to react selectively with ROS, in turn generated by the photosensitizer (PS), herein Chla, are used to attempt this purpose. The changes of the absorption and the emission spectra of these acceptors after the irradiation of aqueous solution containing the active biofilm have provided the specific nature of ROS and thus the main pathway of reaction followed by PS, in our condition. The (1)O2 formation was unveiled using Uric Acid (UA) and 9,10-diphenilanthracene (DPA). On the other hand, 2,7- dichlorofluorescin and Ferricytochrome c (Cyt-c) were used to detect the formation of hydrogen peroxide and superoxide radical anion, respectively. Results suggest that among the possible pathways of reaction, namely Type I and Type II, potentially followed by PSs, in our condition the hybrid biofilm CH/CD/Chla follows mainly Type II mechanism with the formation of (1)O2. However, the latter is involved in subsequent pathway of reaction involving Chla inducing, in addition, the formation of O2(-) and H2O2.

Topics: Anthracenes; beta-Cyclodextrins; Chitosan; Chlorophyll; Chlorophyll A; Cytochromes c; Fluoresceins; Hydrogen Peroxide; Light; Membranes, Artificial; Photosensitizing Agents; Singlet Oxygen; Solutions; Superoxides; Uric Acid; Water

H(2)DCF-DA (dihydrodichlorofluorescein diacetate) is widely used to evaluate 'cellular oxidative stress'. After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-esterified to a hydrophilic alcohol [H(2)DCF (dihydrodichlorofluorescein)] that may be oxidized to fluorescent DCF (2',7'-dichlorofluorescein) by a process usually considered to involve ROS (reactive oxygen species). It is, however, not always recognized that, being a hydrophilic molecule, H(2)DCF does not cross membranes, except for the outer fenestrated mitochondrial ones. It is also not generally realized that oxidation of H(2)DCF is dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c, for neither superoxide, nor H(2)O(2), directly oxidizes H(2)DCF. Consequently, oxidation of H(2)DCF requires the presence of either cytochrome c or of both redox-active transition metals and H(2)O(2). Redox-active metals exist mainly within lysosomes, whereas cytochrome c resides bound to the outer side of the inner mitochondrial membrane. Following exposure to H(2)DCF-DA, weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the much more sensitive J774 cells. This fluorescence was only marginally enhanced following short exposure to H(2)O(2), showing that by itself it is unable to oxidize H(2)DCF. Cells that were either exposed to the lysosomotropic detergent MSDH (O-methylserine dodecylamide hydrochloride), exposed to prolonged oxidative stress, or spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence. The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.

Topics: Animals; Apoptosis; Cell Line; Cytochromes c; Cytosol; Fluoresceins; Fluorescence; Humans; Hydrogen Peroxide; Lysosomes; Mice; Mitochondria; Organometallic Compounds; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species

Hypericin is a photosensitizing pigment found in St. John's wort (Hypericum perforatum) displaying a high toxicity towards certain tumors. The fact that some non-tumor cells, especially monocytes and granulocytes, are resistant to its photocytotoxic effects, posed the question whether this insensitivity is due to their ability to accumulate vitamin C, an antioxidant which alleviates the deleterious work of free radicals. HL-60 promyelocytic tumor cells can be differentiated to neutrophilic granulocytes by treatment with dimethylsulfoxide and were used as cell model. In the differentiated cells, treatment with phorbol esters (PMA) stimulates vitamin C (ascorbate) transport. The uptake rates were unaltered by hypericin at concentrations below 1 microM and irradiation with visible light at a light dose of 6 J/cm2. Inhibition by higher concentrations of hypericin was most probably due to a combination of photocytotoxic properties of the dye and oxygen radicals generated during respiratory burst. Superoxide production by NADPH oxidase followed by reduction of ferricytochrome c was inhibited by hypericin. The degree of inhibition was dependent on the concentration of hypericin and light intensity: IC50-values were 1.7 and 0.7 microM under light doses of 3.6 and 10.8 J/cm2, respectively. Oxidative stress, monitored with 2',7'-dichlorofluorescein (DCF) was only slightly decreased by ascorbate even at higher concentrations of hypericin. In contrast to its effect on the ferricytochrome c-reduction, irradiation had no significant influence on DCF-fluorescence. However, the viability of the cells was strongly decreased after photosensitization and no significant improvement was obtained by ascorbate. Results from this work indicate that ascorbate transport per se is not altered during photodynamic therapy and vitamin C does not interfere with hypericin-induced photodamage of cellular targets.

Topics: Anthracenes; Antineoplastic Agents; Antioxidants; Ascorbic Acid; Biological Transport; Cell Differentiation; Cytochromes c; Fluoresceins; HL-60 Cells; Humans; Light; NADPH Oxidases; Oxidation-Reduction; Oxidative Stress; Perylene; Phorbol Esters; Photochemotherapy; Protein Kinase C; Reactive Oxygen Species; Respiratory Burst; Superoxides