Detection and identification of 1-methylethyl and methyl radicals generated by irradiating tea tree (Melaleuca alternifolia) oil with visible light (436 nm) in the presence of flavin mononucleotide and ferrous ion

Abstract

Here, we determined the electron spin resonance (ESR) spectra of standard reaction mixtures (I) containing 25 μM flavin mononucleotide (FMN), 0.018% tea tree (Melaleuca alternifolia) oil, 1.9 M acetonitrile, 20 mM phosphate buffer (pH 7.4), 0.1 M α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN), and 1.0 mM FeSO₄(NH₄)₂SO₄ irradiated with 436 nm visible light (7.8 J/cm²). Prominent ESR signals (α^N = 1.58 mT and α^Hβ = 0.26 mT) were detected, suggesting that free radicals form in the standard reaction. In order to know whether singlet oxygen (¹O₂) is involved in the radical formation or not, ESR measurement was performed for the standard D₂O reaction mixture (I) which contained 25 μM FMN, 0.0036% tea tree oil, 1.9 M acetonitrile-d3, 20 mM phosphate buffer (pH 7.4), 0.1 M 4-POBN and 1.0 mM FeSO₄ in D₂O. The ESR peak height of the standard D₂O reaction increased to 169 ± 24% of the control. Thus, ¹O₂ seems to be involved in the formation of the radicals because D₂O increases the lifetime of singlet oxygen. High-performance liquid chromatography-ESR-mass spectrometry analyses detected 1-methylethyl and methyl radicals in the standard reaction. The radicals appear to form through the reaction of ferrous ion with α-terpinene endoperoxide (ascaridole), which generated from the reaction of α-terpinene with ¹O₂. The 1-methylethyl and methyl radicals may exert a pro-oxidant effect under these conditions.     

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response¹. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)².The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.     

Ultra-violet mediated cytotoxic activity of β-carboline alkaloids

Six β-carboline alkaloids were screened for phototoxicity using yeast and bacterial bioassay systems. Five of the compounds tested showed various degrees of activity. The remaining compound, 6-methoxy-tetrahydroharman, was devoid of activity.     

Iterative tomography with digital adaptive optics permits hour-long intravital observation of 3D subcellular dynamics at millisecond scale

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UV-mediated antibiotic activity of some compositae species

Roots, leaves, and flowers of 80 species of Compositae were tested for phototoxic activity against Candida albicans. Many genera showed activity, especially in the roots. No active genera were found in the tribe Cichorieae. Phototoxic compounds were isolated from Chrysanthemum leucanthemum florets and Cirsium arvense roots. Chemotaxonomic evidence plus preliminary chemical data suggests that the compounds are polyacetylenic in nature. Unlike other phototoxic compounds, these are inactive against human skin.     

PDT effects of m-THPC and ALA, phototoxicity and apoptosis

The purpose of this study was to estimate the efficacy of an endogenous sensitizer (-aminolevulinic acid (or ALA) induced protoporphyrin IX (or PpIX)) and an exogenous sensitizer (meta(tetrahydroxyphenyl)chlorin or m-THPC) on two different cell lines, rat colon adenocarcinoma PROb cells and murine melanoma B16A45 (B16) cells, in apoptosis production. After sensitizer incubation, cells were irradiated with an argon dye laser. LD₅₀ with m-THPC was 2.8 g/ml and 4.7 g/ml under irradiation of 25 J/cm² respectively for PROb and B16 cells. With ALA, LD₅₀ was 150 g/ml and 175 g/ml under 25 J/cm² respectively for PROb and B16 cells. Apoptosis induction by m-THPC or ALA-PDT was detected by DNA gel electrophoresis and quantified using an ELISA assay 24 h after PDT. The maximal apoptosis enrichment factor (MAEF) was reached for 6 g/ml m-THPC at 10 J/cm² for PROb and B16 cells and for 50 g/ml ALA at 25 J/cm² for PROb or B16 cells. Both m-THPC and PpIX are efficient photosensitizers and apoptosis inducers. However, MAEF is obtained by sensitizer or laser doses inducing very different phototoxic effects: MAEF was obtained after m-THPC-PDT with LD₇₈ for PROb cells and LD₃₀ for B16 cells and after ALA-PDT with LD₂₂ for PROb cells and LD₁₈ for B16 cells. However the overall m-THPC/PDT apoptotic induction (under the curve surface analysis) was not different whatever the cell line for 10 and 25 J/cm². On the contrary, ALA-PpIX/PDT apoptotic induction was twice for 25 J/cm² as compared to 50 J/cm² (p < 0.01) for both the PROb and B16 cells. These results indicate that the apoptosis rate in PDT cell killing varies considerably according to cell type and sensitizer.     

Development of a highly sensitive in vitro phototoxicity assay using the SkinEthicTM reconstructed human epidermis

The reconstituted human epidermis model SkinEthic^TM was used to evaluate the phototoxicity of topically applied chemicals. For comparison with published data, we first tested a library of 13 nonphototoxic (NPT) and phototoxic (PT) compounds, applied onto SkinEthic^TM reconstituted human epidermal tissues, in a protocol as close as possible to the one described by Liebsch using another skin tissue model. The results showed that, under these nonoptimized conditions, the SkinEthic^TM model was already able to fully discriminate between known NPT and PT compounds. Furthermore, these epidermal tissues being highly resistant to UVA irradiation, it was possible to increase irradiation by (at least) 3-fold without decrease in tissue viability. In such conditions, the phototoxicity assay is much more sensitive, so that the model is expected to be of great interest for the detection not only of strong but also of weak phototoxic compounds. This revised version was published online in July 2006 with corrections to the Cover Date.     

光活化多炔类化合物对蚊幼虫的毒力

采用自制的光活化实验装置,测定了11个合成的多炔类化合物对致倦库蚊Culex quinquefasciatus 4龄幼虫的光活化毒力,发现部分化合物在近紫外光照射条件下,能明显地提高光活化毒杀效应,测得化合物5(1-苯基-4-(3,4-亚甲基二氧)苯基-丁二炔)光照与未光照处理LC₅₀分别是0.35 μg/mL和8.89 μg/mL。实验中发现蚊虫先接触药后,再进行光照处理,才能较好地发挥毒效,而且毒杀效应与光照时间呈正相关。模拟田间试验表明,太阳光能显著提高化合物5毒杀蚊幼虫的药效。利用抗氧化剂进行猝灭作用试验,间接地证明化合物5的光活毒杀机理是与过氧化作用有关。分析结构与活性关系,发现二苯基-丁二炔衍生物比二烷基取代丁二炔活性高,苯基上不同取代基也影响光活毒杀效果,它们的活性顺序是：亚甲基二氧基>甲氧基>邻硝基>间硝基>甲基酯。