Reduced cellular glutathione levels do not affect the cytotoxicity or photocytotoxicity of the cationic photosensitiser Victoria Blue BO

Victoria Blue BO (VBBO) is thought to exert its photocytotoxic effects via free radical generation. Glutathione and related enzymes are associated with the protection of normal tissues against free-radical damage and have also been implicated in multiple drug resistance. It might, therefore, be expected that cells containing higher levels of glutathione would be resistant to the cytotoxic effects of VBBO. The total glutathione content for a murine mammary tumour cell line, EMT6-S, was found to be lower than in a multi-drug resistant cell line, EMT6-R, 21.84+/-2.54 microg (mg protein)(-1) and 18.79+/-2.7 microg (mg protein)(-1), respectively; however, this was not found to be a significant difference (p > 0.05, Student t-test). Buthionine sulfoximine, a potent inhibitor of gamma-glutamyl cysteine synthetase, brought about a reduction in glutathione levels in both EMT6-S and EMT6-R cell lines in a concentration-dependent manner. Buthionine sulfoximine administration was effective in reducing intracellular glutathione levels by up to 90% in both types of cells. Interestingly, glutathione depletion of EMT6-S and EMT6-R cells did not enhance the photocytotoxic effect of VBBO, suggesting that the primary site of action of VBBO may be at an intracellular site not protected by glutathione or that the mechanism of action is not via the in situ generation of free radical species.     

Intracellular localisation studies of doxorubicin and Victoria Blue BO in EMT6-S and EMT6-R cells using confocal microscopy

The subcellular localisation of doxorubicin and Victoria Blue BO (VBBO) in a murine mammary tumour cell line EMT6-S, and the resistant sub-lineEMT6-R was studied, using confocal microscopy, in order to investigate their sites of action. In cells treated with doxorubicin (10 μ M) for 90 min, the pattern of intracellular drug distribution differed between the two cell lines. Doxorubicin was found to localise mainly in the nucleus of the sensitive cell line, whereas weak fluorescence was observed in the cytoplasm of the resistant cells, in a punctuate pattern, with no nuclear involvement. The drug also appeared to be effluxed more rapidly by the resistant cell line. The accumulation of doxorubicin at various time intervals over 1h in EMT6-S cells showed that the drug clearly interacted with both the plasma membrane and the nucleus. In contrast to doxorubicin, the intracellular distribution of VBBO in both EMT6-S and EMT6-R was similar, VBBO was clearly localised throughout the cytoplasm, in a punctuate pattern, which may be consistent with the widespread distribution of mitochondria. A more apical pattern of accumulation was noted in the EMT6-R cell line. No interaction with the plasma membrane was evident. These results indicate that the main modes of action for the two drugs differ markedly, suggesting involvement of both the membrane and the nucleus in the case of doxorubicin, but mitochondrial involvement for VBBO. This revised version was published online in August 2006 with corrections to the Cover Date.     

Retinal pigment epithelium pigment granules stimulate the photo-oxidation of unsaturated fatty acids

The cellular pigments of the retinal pigment epithelium (RPE) have been shown to catalyze free radical activity, especially when illuminated with visible or ultraviolet light. This activity is sufficient to cause photooxidation of several major cellular components. The present investigation determined the relative ability of melanin, lipofuscin, and melanolipofuscin granules isolated from human and bovine eyes to oxidize polyunsaturated fatty acids, specifically linoleic and docosahexaenoic acids. The dark reactivity as well as the light-stimulated reactions were determined. The production of hydroperoxide derivatives of the linoleic and docosahexaenoic acids were determined by NADPH oxidation coupled to the activity of glutathione peroxidase, and also by production of thiobarbituric acid reactive substances. All RPE pigment granules stimulated fatty acid oxidation when irradiated with short wavelength (< 550 nm) visible light, with the melanosomes exhibiting the greatest light-induced activity. Only lipofuscin granules, however, caused peroxidation of fatty acids in the dark. These findings provide additional support for the role of RPE pigments in "blue light toxicity" as well as indicating that accumulation of lipofuscin may contribute to increased photooxidation in the aging RPE.     

Regulation of Protein Synthesis during Photomorphogenesis of Gametophytes of the Fern Onoclea sensibilis

Gametophytes of the fern Onoclea sensibilis grow as filaments in the dark and in red light and become planar in blue light. Pulse-labeling 4-day-old gametophytes with [³⁵S]methionine at different times after transfer to dark, red, and blue light environments revealed higher rates of amino acid uptake and protein synthesis in blue light than in red light or in the dark. Characterization of the extant and newly synthesized soluble proteins by one- and two-dimensional gel electrophoresis showed that the patterns of protein accumulation and synthesis in gametophytes exposed to short periods of red or blue light were qualitatively indistinguishable from those of gametophytes maintained in the dark. However, some striking increases and decreases in the levels of certain polypeptides were noted and these changes were accentuated during continued growth of gametophytes in the different environments. The results show that photomorphogenesis of gametophytes of O. sensibilis is associated with quantitative rather than qualitative changes in the population of mRNAs available for translation.     

Blue-light-control of the uptake of amino acids and of ammonia in Chlorella mutants

In non-photosynthetic, yellow or colourless mutant cells of Chlorella kessleri , grown with nitrate as sole nitrogen source, blue light inhibited the uptake of the amino acids glycine, proline and arginine and of ammonia in growing cells, while it enhanced the uptake of these amino acids in resting cells. On the other hand, in cells grown with ammonia as the only nitrogen source without nitrate reductase activity, blue light did not influence the uptake of amino acids and of ammonia in growing cells, while it enhanced the uptake of amino acids in resting cells. Addition of methionine sulphoximine, a potent inhibitor of glutamine synthetase, to growing cells, resulted in intracellular ammonia-accumulation and inhibition of uptake of glycine and of ammonia. For the colourless mutant, blue light was shown to activate purified nitrate reductase. These results indicate that in the mutant cells of Chlorella examined, uptake of ammonia seems to be influenced by nitrate reductase and the uptake of amino acids was influenced by both nitrate reductase and an unknown blue-light-receptor(s). The uptake of urea in mutant cells is not influenced by the irradiation with blue light. Uptake of glycine was also increased after addition of glucose (hexose) in the dark. Because blue light is known to enhance the breakdown of starch, a reaction producing glucose for oxidative degradation in the algae used, the role of glucose (hexose) in the blue light-affected uptake of amino acids is discussed.     

Effcts of Blue Light and Ammonia on Nitrogen Metabolism in a Colorless Mutant of Chlorella

Illumination of a colorless mutant of Chlorella vulgaris 1lh(M125) with blue light enhanced both the uptake of nitrate andthe release of ammonia. These effects were not observed underillumination with red light. The release of ammonia was alsoenhanced by the addition of methionine sulphoximine (MSX), aninhibitor of glutamine synthetase (GS). Addition of MSX to culturesin the dark increased the rate of breakdown of starch. Algal cells grown in nitrate-containing medium did not showthe aminating activity of glutamate dehydrogenase (GDH). Additionof large (millimolar) amounts of ammonia in the dark resultedin the induction of NADPH-GDH activity and, in addition, a decreasein GS activity. From these results it appears that GS catalyzesthe primary step in the assimilation of ammonia in algal cellsgrown in nitrate-containing medium. Two isoforms (GS1 and GS2)of GS have been separated by ion exchange chromatography. Theactivities of both isoforms were decreased upon the additionof ammonia. Illumination of the alga with blue light at intensities up to10,000 mW m^–2 enhanced the measurable activity of GS invitro, while higher intensities were ineffective. In red lightno such effect was observed. The effects of blue light and ammonia on nitrogen metabolismin algal cells are discussed. (Received November 25, 1988; Accepted March 6, 1989)     

Nitrate uptake and extracellular alkalinization by the green alga Hydrodictyon reticulatum in blue and red light

Nitrate uptake and the medium alkalinization related to it were studied with nets of the coenocytic, giant cell, green alga Hydrodictyon reticulatum. A comparison of red, blue and white light irradiation showed no special control of nitrate uptake and of the corresponding alkalinization of the external medium by light quality, but rather a response as expected for the photosynthetic apparatus. In the dark, nitrate uptake rates amounted to one-fifth of those in saturating white light. This is in contrast to the chlorococcal microalga Monoraphidium braunii, where blue light specifically switched on nitrate uptake-dependent alkalinization and where uptake and reduction of nitrate strongly depended on blue light; the rates in pure red light and in the dark being very low. The stoichiometric ratio between nitrate taken up and extracellular alkalinization was close to 1 (0.86) in air with CO2 but close to 2 (1.84) in N2 for nitrate pre-loaded cells. In the absence of any carbon source, a high proportion of the absorbed and reduced nitrogen is released, most of it as ammonium which causes the excess alkalinization and some as nitrite, which lowers the ratio. Nitrite and ammonium release rates under anaerobic, CO2-free conditions were also independent of red or blue light and continued for several hours when the medium was buffered at pH 6. The data indicate that nitrate uptake, but less its reduction, is regulated differently in vacuolate, coenocytic algae from microalgae. In Hydrodictyon, nitrate uptake and reduction seems to be controlled by energy supply; in various microalgae, in addition, it is controlled specifically by blue light.     

Use of Victoria blue in the detection of intrahepatocyte HBs Ag: comparison with other methods

The authors tested Victoria blue staining in intrahepatocytic HBs Ag detection and compared the results with those obtained by the following methods: direct immunofluorescence, orcein and peroxidase-antiperoxidase (PAP). Although PAP appears to be the best technique for diagnostic research, Victoria blue is a sound method for routine diagnosis in hepatology.     

Callose deposition in leaves of cowpea (Vigna unguiculata [L.] Walp.) as a sensitive response to high Mn supply

First macroscopic visible symptoms of Mn toxicity in cowpea (Vegna unguiculata [L.] Walp.) plants grown in solution culture were dark brown spots on the older leaves. Close to these spots, large quantities of substances which fluorescence with aniline blue were deposited, indicating formation of (1,3)-β-glucan (callose). Callose formation in the leaf epidermis was a more sensitive indicator of Mn toxicity than the appearance of macroscopic symptoms, or the Mn concentration in the leaf.     

The effect of photosensitizing dyes on the 3H-thymidine incorporation of cells grown in vitro

The photodynamic inactivation of ³H-thymidine incorporation in mouse embryo (ME) and mouse L cells by acridine orange (AO), methylene blue (MB) or neutral red (NR) has been studied by estimating the number of nuclei capable of incorporating ³H-thymidine during a 24 h period following light exposure. In the dark NR and AO reduced the number of ME-nuclei incorporating ³H-thymidine but MB caused an increase in non-scheduled DNA synthesis. The dark effect on L cells was less but the photoinactivation of thymidine uptake was proportionally greater in these cells. Polyoma virus was shown to be capable of growing in cells whose thymidine uptake was reduced or completely stopped by photoinactivation with NR. However, if the NR damage was very great, or when AO was used to photosensitize cells, the synthesis of viral DNA was interfered with.     

A Technique for Demonstrating Glands in Miracidia of Some Fasciolidae (Trematoda)

A technique (periodic acid-Schiff—Victoria blue) is described which selectively stains glands in miracidia of some fasciolide trematodes. In this method, sections of miracidia are first stained with periodic acid-Schiff and then with an iron-rescorcin lake of Victoria blue. Following this treatment, the semetory granules of the apical gland are blue and the anterior glands are red.     

The Control of Cadmium Uptake in the Lichen Genus Peltigera

Intra- and extracellular Cd uptake in the lichen genus Peltigerawere investigated, and intracellular uptake found to displayMichaelis-Menten kinetics. Compared with values reported forZn uptake in free-living algae and fungi, Peltigera had lowaffinities for Cd and low maximum uptake rates. Intra-, andto a lesser extent extracellular uptake rates were temperaturedependent. When lichens were incubated concurrently with Cdand equimolar concentrations of a range of other cations, mostwere found to reduce both extra- and intracellular Cd uptake,implying that the Cd uptake systems had low specificities. Mg,though not a strong competitor for extracellular Cd uptake,inhibited intracellular Cd uptake to a similar extent to borderlineelements, and it is postulated that intracellular Cd uptakeoccurs by a system which normally transports Mg. Although concurrently-suppliedcations reduced Cd-induced inhibitions of photosynthesis, thereductions were not proportional to the effect of the cationson intracellular Cd uptake. This indicated that other cationsaffected the toxicity of Cd to photosynthesis by some meansin addition to reducing Cd uptake. Intracellular Cd uptake waslight-stimulated, suggesting that a close relationship existedbetween metal uptake and metabolism. The rate of intracellularCd uptake in the dark was probably not directly linked to thesupply of respirable reserves, as it was unaffected by prolongedstorage in the dark, and was not increased by adding glucose.It is hypothesized that light-stimulated Cd uptake representsactive entry into algal cells, but with uptake in the dark itis not clear which symbiont is involved, and whether energyis required. Key words: Lichen, Cadmium uptake, Temperature, Light, Cation competition     

Phytotoxicity of Air Pollutants: Evidence for the Photodetoxification of SO(2) but Not O(3)

Pisum sativum L. cv Alsweet (garden pea) and Lycopersicon esculentum flacca Mill. (tomato) were used to evaluate the phytotoxicity of SO₂ and O₃ in the light and dark. Plants were grown in controlled environment chambers and exposed to SO₂ or O₃ in the light or dark at the same environmental conditions at which they were grown. The pea plants were treated with fusicoccin to ensure open stomata in the dark; the stomata of the tomato mutant remained open in the dark. Both species exhibited 64% to 80% less foliar necrosis following exposure to SO₂ (0.5 to 1.0 microliter per liter for 2 hours) in the light than in the dark. The decrease in SO₂ injury for light versus dark exposed plants was greater in fully expanded than expanding leaves. Both species exhibited 30% greater foliar necrosis following exposure to O₃ (0.2 microliter per liter for 2 hours) in the light than dark. The increase in O₃ injury in the light versus dark was similar for leaves at all stages of expansion. Leaf conductance to water vapor was 7% to 11% and 23% higher in the light than dark for fusicoccin-treated peas and tomato plants, respectively, indicating greater foliar uptake of both pollutants in the light than dark. Thus, the decreased SO₂ toxicity in the light was not associated with pollutant uptake, but rather the metabolism of SO₂. In contrast, the increased toxicity of O₃ in the light was at least in part associated with increased uptake or could not be separated from it.     

;Dark-Lethality' of Certain Chlamydomonas reinhardtii Strains Is Prevented by Dim Blue Light

Cells of certain Chlamydomonas reinhardtii strains dramatically swell and ultimately disintegrate when grown in the dark but not in the light. This effect of darkness is delayed: cells undergo about two divisions before swelling. Measurements on O₂ uptake and ATP content indicate that the cells grown in the dark are not deficient in energy supply. Dim blue light not effective in photosynthesis prevents `dark-lethality'. Inhibition of protein synthesis during the latent period with cycloheximide also prevents dark-lethality. We propose that blue light stimulates synthesis of one or more proteins which are part of or are important in assembly of an osmoregulatory complex.     

Synthesis and photobiological study of a novel chlorin photosensitizer BCPD-18MA for photodynamic therapy

This paper reports synthesis and photobiological properties of a novel chlorin photosensitizer BCPD-18MA. Cytotoxicity, cellular uptake, subcellular location, biodistribution, photodynamic therapy (PDT) efficiency, cell apoptosis as well as histological analysis of the liposomal-delivered BCPD-18MA (L-BCPD-18MA) was studied using mammary adenocarcinoma MDA-MB-231 cells and Lewis lung carcinoma (LLC) implanted in C57BL/6 mice as experimental models. The results showed that L-BCPD-18 was incorporated rapidly into MDA-MB-231 cells and localized partially in mitochondria. L-BCPD-18 induced cell apoptosis by PDT. In addition, biodistribution of L-BCPD-18MA in LLC-bearing mice demonstrated a fast clearance rate of the drug and good skin-related tumor selectivity. Finally, entrapment of BCPD-18 into liposomes resulted in a dramatic impairment of dark toxicity and a notable improvement of PDT antitumor efficacy in vitro. Compared with liposomal-delivered BPDMA (L-BPDMA), L-BCPD-18MA exhibited low dark toxicity and high PDT efficiency on MDA-MB-231 cells. The photodynamic efficacy of L-BCPD-18MA on LLC-bearing mice is comparable to that of L-BPDMA, implying that L-BCPD-18MA is a potential antitumor candidate for PDT.     

8-Hydroxy-2-(1H-1,2,3-triazol-1-yl)-1,4-naphtoquinone derivatives inhibited P2X7 Receptor-Induced dye uptake into murine Macrophages

Extracellular adenosine 5′-triphosphate (ATP) triggers the P2X7 receptor (P2X7R) ionic channel to stimulate the release of the interleukin-IL-1β cytokine into macrophages. The current study explored the reaction of six structurally diverse triazole derivatives on P2X7-mediated dye uptake into murine peritoneal macrophages. P2X7R activity determined by ATP-evoked fluorescent dye uptake. Triazole derivatives toxicity measured using dextran rhodamine exclusion based colorimetric assay. A740004 and BBG, both P2X7R antagonist, inhibited ATP-induced dye uptake. In contrast, the derivatives 5a, 5b, 5e, and 5f did not diminish P2X7R activity in concentrations until 100?µM. 5c and 5d analogs caused a potent inhibitory activity on P2X7-induced dye uptake. Dextran Rhodamine exclusion measurements after 24?h of continuous treatment with triazole derivatives indicated a moderated toxicity for all molecules. In conclusion, this study showed that a series of new hybrid 1,2,3-triazolic naphthoquinones reduces P2X7R-induced dye uptake into murine macrophages. In silico analysis indicates a good pharmacokinetic profile and molecular docking results of these analogs indicate the potential to bind into an allosteric site located into the P2X7R pore and juxtaposed with the ATP binding pocket. In this manner, the compounds 5c and 5d may be used as a scaffold for new P2X7R inhibitors with reduced toxicity, and good anti-inflammatory activity.     

Interactions of green or red light with blue light on the dark closure of Albizzia pinnules

The interactions of green or red light with blue light on the dark closing of Albizzia julibrissin Durazz. pinnules have been investigated. Irradiations at 430, 450 and 470 nm progressively delay dark closing with increasing photon fluence rates. Red or green light alone has no effect. However, when the blue fluence rate is low, both red and green light interact with it and increase the delaying effect of the blue light. When the blue fluence rate is high, green light interacts with it to negate some of the effectiveness of the blue light, while red light has no effect. This is similar to results obtained previously with far-red light. It is suggested that the same unidentified photoreceptor is operating in both the far-red and blue regions. The results also indicate the presence of a blue-only absorbing photoreceptor whose action is increased by phytochrome.     

Identification of naturally occurring spirostenols preventing beta-amyloid-induced neurotoxicity

22R-Hydroxycholesterol is an intermediate in the steroid biosynthesis pathway shown to exhibit a neuroprotective property against beta-amyloid (1-42) (Abeta) toxicity in rat PCl2 and human NT2N neuronal cells by binding and inactivating Abeta. In search of potent 22R-hydroxycholesterol derivatives, we assessed the ability of a series of naturally occurring entities containing the 22R-hydroxycholesterol structure to protect PC12 cells against Abeta-induced neurotoxicity, determined by measuring changes in membrane potential, mitochondrial diaphorase activity, ATP levels and trypan blue uptake. 22R-Hydroxycholesterol derivatives sharing a common spirost-5-en-3-ol or a furost-5-en-3-ol structure were tested. Although some of these compounds were neuroprotective against 0.1 microM Abeta, only three protected against the 1-10 microM Abeta-induced toxicity and, in contrast to 22R-hydroxycholesterol, all were devoid of steroidogenic activity. These entities shared a common structural feature, a long chain ester in position 3 and common stereochemistry. The neuroprotective property of these compounds was coupled to their ability to displace radiolabeled 22R-hydroxycholesterol from Abeta, suggesting that the Abeta-22R-hydroxycholesterol physicochemical interaction contributes to their beneficial effect. In addition, a 22R-hydroxycholesterol derivative inhibited the formation of neurotoxic amyloid-derived diffusible ligands. Computational docking simulations of 22R-hydroxycholesterol and its derivatives on Abeta identified two binding sites. Chemical entities, as 22R-hydroxycholesterol, seem to bind preferentially only to one site. In contrast, the presence of the ester chain seems to confer the ability to bind to both sites on Abeta, leading to neuroprotection against high concentrations of Abeta. In conclusion, these results suggest that spirost-5-en-3-ol naturally occurring derivatives of 22R-hydroxycholesterol might offer a new approach for Alzheimer's disease therapy.     

Ca uptake and plasma membrane depolarization associated with blue light-sensitive exogenous NADH oxidation by Cuscuta protoplasts

Protoplasts isolated from the apical segments of Cuscuta reflexa exhibited blue light-sensitive PM-linked NADH oxidase activity and increased rate of Ca²⁺-uptake in presence of NADH in dark, which was also stimulated by blue light. Contrary to marginal inhibition by Con A treatment, the ATPase inhibitors significantly inhibited the Ca²⁺ uptake by the protoplasts both in dark and under blue light. The Ca²⁺-calmodulin antagonists, W-7 and calmidazolium, also inhibited Ca²⁺-uptake by protoplasts under similar conditions. The state of PM polarization was monitored by the fluorescent dye 9-amino acridine. It was observed that PM-linked NADH oxidation caused hyperpolarization of the membrane, the exposure of which to blue light resulted in membrane depolarization. The presence of Ca²⁺-calmodulin antagonists or Con A treatment completely abolished the blue light-induced membrane depolarization. It is argued that these actities at the PM, having some glycoproteic components, are functionally closely involved in blue light-induced signal transduction in Cuscuta     

The effect on sister-chromatid exchanges of drugs and dyes by intercalation and photoactivation.

Intercalating dyes (acridine orange, proflavin and methylene blue) and drugs (chlorpromazine, promazine and chlorprothixene) were tested for their ability to induce sister-chromatid exchanges (SCEs) with and without photoactivation by visible light. Whereas in the dark all substances tested increased the frequency of SCEs, a superimposed effect of visible light on SCE formation was observed for the acridines proflavin and acridine orange, but not for the pheneothiazine derivatives methylene blue and chlorpromazine. These results are discussed in connection with the known mutagenic effects of these substances and with the factors that may be involved in SCE formation induced by intercalating molecules in the absence and presence of visible light.