Modulation of the phototoxic effect of hypericin in human leukemia CEM cell line by N-ethylmaleimide,amiloride and omeprazole

Hypericin is a photosensitizing plant pigment from Hypericum perforatum with multiple modes of light-induced biological activities due to production of singlet oxygen and/or excited-state proton transfer with consequent pH drop in the hypericin environment. In the present work, we studied the effects of three inhibitors of crucial mechanisms responsible for intracellular pH (pHi) regulation on hypericin phototoxicity: N-ethylmaleimide (NEM), an inhibitor of H+-ATPase, 5'-(N,N-dimethyl)-amiloride (DMA), an inhibitor of Na+/H+ exchanger, and omeprazole (OME), an inhibitor of H+K+-ATPase. Our experiments show that the effect of hypericin at 1 x 10(-5) and 1 x 10(-6) mol x l(-1) was significantly potentiated by NEM (1 x 10(-7)-1 x 10(--9) mol x l(-1)) and DMA (1 x 10(-6) and 1 x 10(-7) mol x l(-1)) in leukemic CEM cell line. On the other hand, OME had no significant effect on hypericin cytotoxicity. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of hypericin environment could play a role in the biological activity of hypericin.     

Mitochondria are a primary target of hypericin phototoxicity: synergy of intracellular calcium mobilisation in cell killing

Hypericin, a naturally occurring anthraquinone synthesised by hypericum, upon light activation exhibits photodynamic cytotoxicity attributed mainly to the production of reactive oxygen species. This study aimed to elucidate the primary subcellular targets and mechanistic aspects of hypericin photosensitization in human prostate carcinoma cells. Depletion of intracellular glutathione (>85%) via inhibition of gamma-glutamyl-cysteine synthase had no effect on hypericin (5 microM) phototoxicity, thus precluding any direct oxidative involvement of H2O2. There was no change in intracellular SOD activity immediately after hypericin irradiation (1.5-5 J cm(-2)). Evaluation of the lysosomal enzyme hexosaminidase activity showed: (a) 60% cell loss 22 h following irradiation (1.5 J cm(-2)) and (b) a steady rate of lysosomal leakage to the cytosol (25%), at the same time and irradiation. However, lysosomal damage appears to be a slower process compared to the rapid loss of mitochondrial function, as reflected from parallel tetrazolium to formazan assays. The activity of cytosolic and mitochondrial aconitase, an enzyme exquisitely sensitive to oxidation, revealed a dose correlated loss of activity in the mitochondria immediately following hypericin photoactivation. The use of ionomycin, which modulates both internal Ca2+ stores and external Ca2+ transport during hypericin photosensitization, profoundly enhanced photocytotoxicity. Our data supports a direct mitochondrial hypericin phototoxicity that does not involve glutathione/H2O2 homeostasis. Further a potential synergistic treatment combining mitochondrial targeting of photosensitisers and Ca2+ mobilisation was identified.     

Photosensitization of aqueous model systems by hypericin.

Absorption and fluorescence measurements of purified hypericin (HY) were made in various media. Photosensitization of two aqueous systems was investigated: resealed red blood cell membranes (ghosts) and hen lysozyme (Lys). Solubilization of HY by ghost membranes was shown by means of diffuse reflectance spectroscopy. Visible light irradiation of the ghosts incorporating HY led to lipid peroxidation with evidence of singlet oxygen involvement. A binding model applicable for insoluble ligands is indicative of strong HY binding to HSA. The HY-HSA complex photosensitized inactivation of Lys. The pseudo-first-order reaction kinetics with protection by azide ion are consistent with a Type II mechanism mediated by singlet oxygen. The results are discussed in the context of the HY photodynamic and antiretroviral activities.     

On the mechanism of Candida spp. photoinactivation by hypericin

The photoprocesses involved in hypericin photoinactivation of three different Candida species (C. albicans, C. parapsilosis and C. krusei) have been examined. Production of singlet oxygen from the triplet state and of superoxide from both the triplet state and the semiquinone radical anion are demonstrated. Hydrogen peroxide is formed downstream of these early events. The outcome of the photodynamic treatments is dictated by the intracellular distribution of hypericin, which is different in the three species and affects the ability of hypericin to produce the different reactive oxygen species and trigger cell-death pathways. The results are in line with the previously-observed different susceptibilities of the three Candida species to hypericin photodynamic treatments.     

Potentiation of lymphocyte activation by colchicine.

Proliferation of human lymphocytes induced by IO4- is potentiated by 30 min exposure to colchicine (10(-6)M), whereas the response to Con A is inhibited. Treatment with colchicine before or after IO4- modification has similar enhancing effects. Lumicolchicine does not alter proliferative responses. In addition to the proliferation of IO4--oxidized cells, irradiated IO4- modified lymphocytes induce proliferation when mixed with untreated lymphocytes. Enhancement occurs in both these conditions only when IO4--modified cells are treated with colchicine. Preliminary data indicate that proliferation in mixed lymphocyte cultures is also potentiated when either stimulating or responding cells are pretreated with colchicine. These findings suggest a selective stimulatory effect of colchicine on lymphocyte responses induced by cell-cell contact. Agents that modify microtubular assemblies might regulate the induction of immune responses that involve cellular interactions.     

Potentiation of histamine release by sodium cromoglycate.

Human lung slices passively sensitized with allergic serum released histamine when incubated with specific antigen and anti-IgE but anti-IgG had no effect. Sodium cromoglycate (SCG) inhibited antigen induced histamine release but the dose-response curve was bell-shaped. Inhibition of anti-IgE induced release was linearly related to dose, whereas that induced by anti-IgG was potentiated by increasing doses of SCG. After sensitization with allergic serum in which IgE had been inactivated by heating, specific antigen released little or no histamine but this was potentiated by SCG. It is concluded that SCG inhibits IgE mediated but potentiates IgG mediated allregic reactions thus explaining its characteristic dose-response curve $\text{in vitro}$.     

Antiretroviral activity of synthetic hypericin and related analogs.

Hypericin and pseudohypericin are naturally occurring polycyclic quinones which have recently been shown to inhibit the infectivity of several retroviruses, including human immunodeficiency virus. To better understand the antiviral mechanisms of these compounds, hypericin and a series of analogous quinones were synthesized and tested for anti-retroviral activity against equine infectious anemia virus (EIAV). Treatment of EIAV-infected cells with hypericin reduced the production of infectious virus by 99.99%. None of the analogs were found to inhibit virus replication. These results suggest that the complete ring structure of hypericin is required, but not sufficient, for antiviral activity.     

Assay of omeprazole and omeprazole sulfone by semi-microcolumn liquid chromatography with mixed-function precolumn

A column-switching system based on semi-microcolumns was used for direct analysis of omeprazole and omeprazole sulfone in human plasma samples. Plasma samples were injected into a mixed-function (MF Ph-1) column (35 mm×4.6 mm I.D.) to remove proteins and other non-specific peak producing substances from the analyte-containing time zone. The analyte-containing fraction was thereafter transferred to a C-18 semi-microcolumn (250 mm×1.5 mm I.D.) after concentration at the C-18 intermediate column. The absorbance at 302 nm in a ultraviolet (UV) detector was recorded to measure the concentration. The detection limit for omeprazole and omeprazole sulfone in the present method was 10 ng/ml. Interbatch variation (coefficient of variation) of the QC samples spanned less than 10% and intra-batch variation less than 2%. The recovery ratios of omeprazole and omeprazole sulfone were over 98%. The current method can be used as a simpler procedure with similar sensitivity and reproducibility as previously reported methods.     

Potentiation of infection by epinephrine

A subinfective dose of Staphylococcus aureus (6 X 10(6)) does not produce wound infection in the rat model. Reducing the dosage (concentration) of epinephrine results in decreased numbers of viable bacteria in tissue and a lower incidence of wound infection in this model.     

Transient uptake of hypericin by chrysomelids is regulated by feeding behaviour

Abstract. The uptake of hypericin from the foliage of Hypericum perforatum by four species of adult chrysomelid beetles, Chrysolina brunsvicensis , C. geminata, C. hyperici and C. varians , was investigated under laboratory conditions. In no species were detectable levels of hypericin passed from the larval to the pupal or adult stages. The total amount of hypericin found in the adult beetles was positively related to the rate of ingestion of foliage. When feeding activity lessened or ceased, body levels of hypericin declined. In adults of C. brunsvicensis, C. hyperici and C. varians maximal body levels of hypericin occurred after 1–10 days of feeding. In C. brunsvicensis a maximal average level of c. 0.96 μg/beetle was obtained. After 20 days body levels in C. brunsvicensis declined to nearly zero. Adults of C. brunsvicensis contained more hypericin than the other species of beetles, but in no case was enough hypericin sequestered to match the original values (c. 13.3 μg/beetle) published by Rees (1969). Our results caste doubt on the role of hypericin as a sequestered defence against predators.
Analysis of body distribution of hypericin indicated the majority was in the gut lumen (80%). An input-output analysis of hypericin ingested by adults of C. brunsvicensis showed that the greatest majority of hypericin was eliminated in the faeces (up to 85%). Between 15% and 35% of the ingested hypericin was unaccounted for. This loss is the result of a post-defecational degradation of hypericin (perhaps bacterial).