Melatonin defends mouse oocyte quality from benzo[ghi]perylene-induced deterioration.

Benzo[ghi]perylene (B[ghi]P) is a polycyclic aromatic hydrocarbon widely found in haze. Long-term exposure to humans or animals can cause serious damage to the respiratory system. Melatonin is an endogenous natural hormone synthesized and released by the pineal gland. In this study, we investigated the effects of melatonin on in vitro cultured B[ghi]P-exposed mouse oocytes and the protective roles of melatonin. Our data indicate that B[ghi]P exposure leads to meiotic maturation arrest and reduced ability of sperm binding and parthenogenetic activation. Also, B[ghi]P exposure disrupts actin filament dynamics, spindle assembly, and kinetochore-microtubule attachment stability, which results in oocyte aneuploidy. Simultaneously, B[ghi]P exposure disturbs the distribution of mitochondria, increases the level of oxidative stress, and induces apoptosis of oocytes. Whereas all of these toxic effects of B[ghi]P can be restored after melatonin supplement. In conclusion, our findings validate that melatonin has a certain protective effect on preventing the reduced oocyte quality caused by B[ghi]P exposure during meiotic maturation in mouse oocytes.

In vitro screening of calli of mungbean to cercosporin, a photoactivated toxin.

Mungbean or Green gram [Vigna radiata (L.) R. Wilczek] is an arid/semiarid pulse crop, native to India, grown mostly as a rotational crop with cereals like wheat, rice, maize, sorghum, etc. It is an affordable source of protein, carbohydrate, vitamins and minerals preferred for its nutrient digestibility, food processing properties and bioavailability. India accounts for 65% of mungbean's world acreage and 54% of its world production. Various pests, diseases and environmental stresses have kept mungbean yield quite unstable over decades and researcher's worldover are looking for resistant varieties to overcome these challenges. Cercospora leaf spot (CLS) caused by Cercospora canescens is one of the most destructive diseases of mungbean and the key polyketide toxin cercosporin plays an important role in pathogenesis. Such toxins as selective agents in the tissue culture medium can help in selecting genotype with suitable levels of resistance to the toxin and/or to the pathogen among the available germplasm. Here, we standardized the dose of cercosporin for in vitro selection of resistant mungbean genotypes and variable expression of peroxidase, catalase and superoxide dismutase. Murashige and Skoog (MS) medium supplemented with 1.0 mg L-1 NAA and 1.0 mg L-1 BAP was standardized for the development of callus from mungbean using hypocotyls as an explant. The calli from six cultivars of mungbean were tested in medium amended with cercosporin (0-40 µg mL-1) and calli survived up to 20 µg mL-1 of cercosporin. The calli from resistant cultivars survived 83.33-93.00%, and showed lower reduction in fresh weight (25.97-28.83%). Calli from the susceptible cultivars survived 50-60% and showed higher reduction in fresh weight. Callus showed browning, exposure to cercosporin (5-20 µg mL-1). Enzymes assay from survived calli of different cultivars showed higher peroxidase activity (7.90-8.91 ∆OD min-1 mg­1 callus), superoxide dismutase (0.96-1.03 ∆OD min-1 mg-1 callus) and a lower catalase (0.35-0.43 µ moles of H2O2 utilized min-1 mg­1 callus) in resistant, followed by moderately resistance and susceptible cultivars. The necrosis in leaves was recorded with 200 µg mL-1 of cercosporin, and no visible necrosis was observed below this concentration. Enzyme assayed from the controlled and cercosporin-treated (100-200 µg mL-1) leaves of mungbean genotypes showed variable activity of peroxidase, catalase and superoxide dismutase.

Liposomal hypocrellin B as a potential photosensitizer for age-related macular degeneration: pharmacokinetics, photodynamic efficacy, and skin phototoxicity in vivo.

Photodynamic therapy (PDT) has been successfully implemented as a treatment for wet age-related macular degeneration (AMD), but very few photosensitizers have been developed for clinical use. Herein, we describe a novel formulation of liposomal hypocrellin B (LHB) that was prepared by high-pressure homogenization. The encapsulation efficiency and PDT efficacy in vitro of this new preparation were found to remain nearly constant over 1 year. Moreover, LHB is rapidly cleared from the blood, with a half-life of 2.319 ± 0.462 h and a very low serum concentration at 24 h after injection. Testing in a rat model of choroidal neovascularization (CNV) showed that leakage of blood vessels in CNV lesions was significantly reduced when LHB PDT was given at a dose of 1 mg kg(-1) along with yellow laser irradiation; the damage to the collateral retina and the retinal pigment epithelium was minimal. Skin phototoxicity assays showed that only two of the 200 mice given a 4 mg per kg dose of LHB experienced an inflammatory reaction in the auricle irradiated at 24 h after dosing. These data collectively indicate that LHB may be a safe and effective photosensitizer for vascular-targeted PDT of AMD.

Hypericin phototoxicity induces different modes of cell death in melanoma and human skin cells.

Hypericin, the major component of St. John's Wort, absorbs light in the UV and visible ranges whereupon it becomes phototoxic through the production of reactive oxygen species. Although photodynamic mechanisms (i.e. through endogenous photosensitizers) play a role in UVA phototherapy for the treatment of skin disorders such as eczema and psoriasis, photodynamic therapy employing exogenous photosensitizers are currently being used only for the treatment of certain forms of non-melanoma skin cancers and actinic keratoses. There are few reports however on its use in treating melanomas. This in vitro study analyses the phototoxic effect of UVA (400-315 nm) - activated hypericin in human pigmented and unpigmented melanomas and immortalised keratinocytes and melanocytes. We show that neither hypericin exposure nor UV irradiation alone reduces cell viability. We show that an exposure to 1 microM UVA-activated hypericin does not bring about cell death, while 3 microM activated hypericin induces a necrotic mode of cell death in pigmented melanoma cells and melanocytes and an apoptotic mode of cell death in non-pigmented melanoma cells and keratinocytes. We hypothesis that the necrotic mode of cell death in the pigmented cells is possibly related to the presence of melanin-containing melanosomes in these cells and that the hypericin-induced increase in reactive oxygen species leads to an increase in permeability of melanosomes. This would result in toxic melanin precursors (of an indolic and phenolic nature) leaking into the cytoplasm which in turn leads to cell death. Hypericin localisation in the endoplasmic reticulum in these cells shown by fluorescent microscopy, further support a disruption in cellular processing and induction of cell death. In contrast, this study shows that cells that do not contain melanosomes (non-pigmented melanoma cells and keratinocytes) die by apoptosis. Further, using a mitochondrial-specific fluorescent dye, we show that intracellular accumulation of hypericin induces a mitochondrial-associated caspase-dependent apoptotic mode of cell death. This work suggests that UVA is effective in activating hypericin and that this phototoxicity may be considered as treatment option in some cases of lentigo maligna or lentigo maligna melanoma that are too large for surgical resection.

Reduction in hypericin-induced phototoxicity by Hypericum perforatum extracts and pure compounds.

Clinical evidence suggests that administration of Hypericum perforatum (Hp) extracts containing the photo-activated hypericin compounds may cause fewer skin photosensitization reactions than administration of pure hypericin. This study was conducted to determine whether the phototoxicity of hypericin in HaCaT keratinocytes could be attenuated by H. perforatum extracts and constituents. Two extracts, when supplemented with 20 microM hypericin: (1) an ethanol re-extraction of residue following a chloroform extraction (denoted ethanol(-chloroform)) (3.35 microM hypericin and 124.0 microM total flavonoids); and (2) a chloroform extract (hypericin and flavonoids not detected), showed 25% and 50% (p<0.0001) less phototoxicity than 20 microM hypericin alone. Two H. perforatum constituents, when supplemented with 20 microM hypericin: (1) 10 microM chlorogenic acid; and (2) 0.25 microM pyropheophorbide, exhibited 24% (p<0.05) and 40% (p<0.05) less phototoxicity than 20 microM hypericin alone. The peroxidation of arachidonic acid was assessed as a measure of oxidative damage by photo-activated hypericin, but this parameter of lipid peroxidation was not influenced by the extracts or constituents. However alpha-tocopherol, a known antioxidant also did not influence the amount of lipid peroxidation induced in this system. These observations indicate that hypericin combined with H. perforatum extracts or constituents may exert less phototoxicity than pure hypericin, but possibly not through a reduction in arachidonic acid peroxidation.

Photogenotoxicity of hypericin in HaCaT keratinocytes: implications for St. John's Wort supplements and high dose UVA-1 therapy.

Extract of St. John's Wort (Hypericum perforatum) is commonly used as natural remedy for treatment of mild to moderate depression. However, it contains a powerful photoactive component, hypericin, which can cause a severe photodermatitis when eaten by grazing animals (hypericism). In humans, there is evidence that supplementation with St. John's Wort can reduce the minimal erythemal dose (MED) in patients undergoing high dose UVA-1 phototherapy. This is a recent development in phototherapy where the most erythemogenic parts of the UVA spectrum are filtered out, allowing delivery of higher doses of the longer wavelengths of UVA. Although current published evidence suggests that the plasma levels of hypericin are unlikely to cause clinical phototoxicity, it has been established that photoactive compounds can cause DNA damage at sub-toxic and sub-erythemal doses, the effects of which might not be apparent for many years after the event. The present study used HaCaT keratinocytes to investigate the photoclastogenic ability of hypericin on irradiation with UVA. The results show that although the combination of hypericin and UVA light increased the genotoxic burden, when all factors are taken into account, the risk of significant photogenotoxic damage incurred by the combination of Hypericum extracts and UVA phototherapy may be low in the majority of individuals.

Stability of different formulations and ion pairs of hypericin.

Hypericin, solubilized in an instillation fluid consisting of an aqueous buffer supplemented with 1% plasma proteins, is currently used as a clinical diagnostic tool for the detection of superficial TCC (transitional cell carcinoma) tumors. However, the development of a sterile and stable hypericin stock formulation, excluding the presence of plasma constituents, would be an important factor in a more general clinical application of the method. Therefore, we investigated the stability of several heat sterilized hypericin formulations and ion pairs. Besides sodium hypericinate (in distilled water, in phosphate buffer, in polyethyleneglycol (PEG) 400), several other hypericinate salts (potassium, lysine, TRIS or hexylamine) were investigated. As to that, the physical appearance of different hypericin concentrates stored at 4 and 37 degrees C was investigated. Besides, after dilution into cell culture medium, the ability of hypericin remaining to accumulate in tumor cells and demonstrating photocytotoxic effects upon light irradiation was assessed. These findings suggest that PEG 400 is an excellent hypericin formulation, since it maintained the stability of the compound for at least 120 d when stored at either 4 or 37 degrees C. PEG 400 therefore is a suitable vehicle for the storage of hypericin prior to preparation of the bladder instillation solution.

Photooxidation of lens alpha-crystallin by hypericin (active ingredient in St. John's Wort).

Hypericin is the active ingredient in the over the counter antidepressant medication St. John's Wort. Hypericin produces singlet oxygen and other excited state intermediates that indicate it should be a very efficient phototoxic agent in the eye. Furthermore it absorbs in the UV and visible range, which means it can potentially damage both the lens and the retina. Lens alpha-crystallin, isolated from calf lenses, was irradiated in the presence of hypericin (5 x 10(-5) M, 10 mM ammonium bicarbonate, pH 7.0) and in the presence and absence of light (> 300 nm, 24 mW/cm2). Hypericin-induced photosensitized photopolymerization as assessed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Further analysis of the oxidative changes occurring in alpha-crystallin using mass spectrometry showed specific oxidation of methionine, tryptophan and histidine residues, which increased with irradiation time. Hypericin did not damage the lens protein in the dark. Damage to alpha-crystallin could undermine the integrity of the lens directly by protein denaturation and indirectly by disturbing chaperone function. Therefore, in the presence of light, hypericin can induce changes in lens protein that could lead to the formation of cataracts. Appropriate precautions should be taken to protect the eye from intense sunlight while on this antidepressant medication.

Photocytotoxicity of protohypericin after photoconversion to hypericin.

In the present study, protohypericin was synthesised in order to compare its intrinsic photocytotoxicity with that of hypericin. The experimental work was performed in specific filtered light conditions that prevented both an unintended photoconversion of protohypericin and photosensitization of the cells. Assessing the photocytotoxicity as a function of irradiation time, it was found that the photocytotoxicity of both compounds converged after a long irradiation time (i.e., 15 min), while the difference between the photocytotoxicities was maximal after a short irradiation time (i.e., 1 min). Since this could not be accounted for by a redistribution of protohypericin during irradiation, and the different irradiation times corresponded to different degrees of photoconversion of protohypericin into hypericin, the results clearly suggest that protohypericin exhibits intrinsically a dramatically lower photoactivity as compared to hypericin.

Hypericin: a new laser phototargeting agent for human cancer cells.

Laser activation of anthracycline-related drugs combines chemotherapy with photoablation for improved treatment. Hypericin, a structurally related anthraquinone, was tested for laser activation and cytotoxicity in human cancer cells. Viability of P3 squamous cell carcinoma cells incubated with 1 to 20 microgram/mL hypericin was reduced by more than 95% after 1 minute exposure at 4 degrees C to an argon laser (514 nm, 5 W), a KTP-532 laser (532 nm, 5 W), or a 20-A xenon lamp. Viability was reduced over 90% in six human carcinoma, sarcoma, and melanoma cell lines by this combined treatment, but only trace toxicity was seen after separate exposure to hypericin or light alone. These results show that hypericin is a sensitive agent for phototherapy of human cancer cells in vitro and indicate that this drug may be useful for tumor targeting via minimally invasive imaging-guided laser fiber optics.

[Genotoxicity of a standardized Hypericum extract]. / Genotoxizität eines standardisierten Hypericum-Extraktes.

St. John's wort (Hypercum perforatum) contains hypericin and hypericin-like substances as well as flavonoids, of which particularly Quercetin has generated a wide-spread controversial discussion with respect to mutagenic action. The genotoxicity of a standardized aqueous ethanolic Hypericum extract (Hypericum extract Steigerwald, Psychotonin M) was verified in different in-vivo and in-vitro testsystems with mammalian cells. The in-vitro investigations were performed with the HGPRT (hypoxanthine guanidine phosphoribosyl transferase)-test, UDS (unscheduled DNA synthesis)-test and with the cell transformation test using Syrian hamster embryo cells. Both the in-vitro tests as well as the in-vivo tests--fur spot test of the mouse and the chromosome aberration test with the bone marrow cells of the chinese hamster--were negative, giving completely no indication of a mutagenic potential of Hypericum extract. These investigations lend support to the view that results from bacterial short-term tests are of very limited transferability to human.

Petroleum distillates suppress in vitro metabolic activation: higher [S-9] required in the Salmonella/microsome mutagenicity assay.

To determine if standard conditions used in the Salmonella/mammalian microsome mutagenicity assay could reliably screen complex petroleum samples, two high-boiling (700-1,070 degrees F) distillates and their separated aromatic fractions were tested. The initial mutagenic activities were inconsistent with the samples' known polyaromatic hydrocarbon (PAH) contents and observed potencies in a dermal carcinogenesis bioassay. A significant mutagenic response was observed only at S-9 concentrations 5 to 10 times higher than those used in the standard assay, supporting the use of elevated levels of S-9 in the Salmonella/microsome assay to assess the carcinogenic potential of petroleum-derived materials. All four samples masked the expected mutagenic activity of added PAHs (benzo[a]pyrene and perylene). Data suggested that petroleum distillates suppress the functional efficacy of the S-9; possible mechanisms are discussed.