Isolation and indentification of a new toxin,altenuene, from the fungus Alternaria tenuis

A previously unknown metabolite was isolated from four week old cultured of the fungus, Alternaria tenuis. The compound shows cytotoxic activity against five bacteria tested by the paper-disc agar-plate method. After purification by column chromatography on silica gel, the metabolite crystallized from acetone: hexane mixtures as colorless neddles neddles that melted at 190–191° (corr.). High resolution mass spectrometry indicated a parent ion with mol. wt. 292.0938, which identified the empirical formula as C₁₅H₁₆O₆. Structural assignments based on infrared, proton magnetic resonance, and ultraviolet spectra established the structure of the compound as 2′,3′,4′,5′-tetrahydro-3,3′,4′-trihydroxy-5-methoxy-6′-mehyl-dibenzo-α-pyrone to which we have given the trival name, altenuene. Altenuene is structurally related to two previously characterized A. tenius metabolites, alternariol and alternariol monomethyl ether.     

Aflatoxins in sunflower seeds: Influence of Alternaria alternata on aflatoxin production by Aspergillus parasiticus

The aim of the present work was to determine the influence of Alternaria alternata upon aflatoxin production by Aspergillus parasiticus.A mixture of spores of both strains was inoculated in sunflower seeds at 0,90 a_w, and incubated for 42 days at 28 °C ±1.The cultures were observed and analyzed every 7 days to determine the infection level of the seeds and the production of aflatoxins. Results showed that when the seeds were inoculated only with Aspergillus parasiticus, 100% were infected from the 7th day.When Aspergillus parasiticus and Alternaria alternata were simultaneously inoculated the infection level of the seeds was 100% for Aspergillus parasiticus following 7 days of inoculation and 0% for Alternaria alternata. After the 14th day of inoculation there was no significant difference in the infection percentage of both strains (approximately 80% of each one). As far as toxin production is concerned a remarkable decrease was observed when seeds were inoculated with both strains simultaneously.In accordance to the results, Alternaria alternata would not compete with Aspergillus parasiticus in colonization of seeds but would either degrade the aflatoxins by Aspergillus parasiticus or compete for aflatoxin biosynthesis precursors. Alternaria alternata could also secrete some substance that specifically inhibits aflatoxin synthesis.     

Occurrence of alternariol, alternariol monomethyl ether and tenuazonic acid in Argentinean tomato puree

The occurrence ofAlternaria mycotoxins was investigated in 80 samples of tomato puree processed and sold in Argentina. Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) were searched for by liquid chromatography. Thirty-nine of the 80 samples showed mycotoxin contamination. TA was found in 23 samples (39-4021 μg/kg), AOH in 5 samples (187-8756 μg/kg), and AME in 21 samples (84-1734 μg/kg). Co-occurrence of two of these toxins was detected in 10 samples. This is the first report of natural occurrence of AOH, AME and TA in tomato products in Argentina.     

Effect of heat treatments on stability of altemariol, alternariol monomethyl ether and tenuazonic acid in sunflower flour

A study was carried out to evaluate the effect of heat treatment on the stability of alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) in sunflower flour and the effectiveness of this treatment by a biological assay in rats. The concentrations of AOH and AME remained constant during heating at 100°C for up to 90 minutes while TeA concentration decreased with time to 50% after 90 minutes. The most effective treatment in reducing AOH and AME levels was heating at 121°C for 60 minutes. Histopathological evaluation in the biological assay in rats fed withAlternaria toxins showed marked atrophy and fusion of villi in the intestines and liver cell damage; these lesions were less severe in rats fed heat-treated sunflower flour in line with the reduced toxin content. However, a lower weight gain and a noticeable renal damage in rats were produced when they fed decontaminated flour.     

荧光探针在光动力疗法亚细胞损伤位点研究中的应用

目的：应用荧光探针在光动力疗法研究中检测亚细胞损伤位点。方法：传代培养鼠肺毛细血管内皮细胞,将血卟啉单甲醚(HMME)与内皮细胞共同孵育24小时后,加入线粒体探针Bhodamine-123、内质网探针DioC6(3)和溶酶体探针Lucifer yellow分别对细胞器染色。首先采用激光共聚焦显微镜对光敏剂进行亚细胞定位。应用荧光显微镜汞灯照射激发光敏剂的光动力效应,加入ROS探针H2DCF-DA检测产生的单线态氧。分别在激发前后采集Pdloclamine-123、Lucifer yellow和DioC6(3)的荧光图像。结果：线粒体探针Phodamine-123的荧光图像在光动力损伤前后差异显著,原有形态特点发生明显改变;Phodamine-123在光动力损伤后再分布于细胞核区。结论：血卟啉单甲醚介导的光动力效应导致亚细胞水平多位点损伤,线粒体和核膜可能是PDT敏感位点;荧光探针标记检测光动力损伤亚细胞位点方法简便可靠。     

Activities of human recombinant cytochrome P450 isoforms and human hepatic microsomes for the hydroxylation ofAlternaria toxins

TheAlternaria toxins alternariol (AOH), alternariol-9-methyl ether (AME), altenuene (ALT) and isoaltenuene (iALT) undergo extensive oxidative metabolism, but the cytochrome P450 (CYP) isoforms responsible for the reported hydroxylation reactions are yet unknown. In the present study, the activities of twelve human CYP isoforms for the hydroxylation of AOH, AME, ALT and iALT at different positions have been determined. The most active monooxygenase for AOH and AME was CYP1A1, and lower activities were observed for CYP1A2, 2C19 and 3A4. Hydroxylation at C-2 of AOH and AME was the preferred reaction of most isoforms. For ALT and iALT, CYP2C19 had the highest activity, followed by 2C9 and 2D6. The dominating metabolite of all active isoforms was the 8-hydroxylated ALT and iALT. The activities of the CYP isoforms are consistent with the pattern of metabolites of theAlternaria toxins obtained with pooled human hepatic microsomes. Based on the activities of the CYP isoforms, a significant extrahepatic hydroxylation must be expectede.g. in the lung and esophagus for AOH and AME, and in the intestine and ovaries for ALT and iALT. As all major hydroxylation products are catechols, the extrahepatic metabolism ofAlternaria toxins may be of toxicological relevance. Presented at the 30^th Mycotoxin Workshop, Utrecht, Netheriands, April 28–30. 2008. Financial support: State of Baden-Württemberg (Research Program “Mycotoxins” as part of the Research Initiative “Food and Health”)     

Phototoxicity of Hemoporfin to ovarian cancer

Hematoporphyrin monomethyl ether (Hemoporfin) is a novel porphyrin-related photosensitizer. Photocytotoxic effect of Hemoporfin to ovarian cancer is still unclear. We used human epithelial ovarian carcinoma cell line SKOV3 and its xenograft model in nude mice to investigate the Hemoporfin-based photodynamic therapy (PDT) for ovarian cancer. The growth rates of SKOV3 cells were determined by MTT assays. Flow cytometry combined with dual Annexin V/PI staining was used to identify the death mode of the cells following PDT. We demonstrated that Hemoprofin-based PDT induced significant cell death via direct necrosis induction, and the photocytotoxity to SKOV3 cells is dose related. With SKOV3 xenograft model in nude mouse, we further demonstrated that Hemoporfin-based PDT is effective for controlling the tumor growth. Our results suggest that Hemoporfin is a promising novel photosensitizer for the treatment of ovarian cancer and merit further evaluation in the clinical practice.     

光动力疗法防治损伤性血管内膜增生的实验研究

目的 :观察光动力疗法 (PDT)防治损伤性血管内膜增生的近期疗效 ,并筛选PDT参数。方法 :首先筛选PDT参数。兔 2 4只 ,按L4 ( 2 3)正交实验方案分为四组 ,处理因素为血啉甲醚 5或 10mg/kg、功率密度 30或 90mw/cm2 、照射时间 15或 30min。球囊法损伤髂总动脉内膜 ,随后进行PDT处理 ,2 1天后取材 ,以中膜与内膜面积的比值为指标分析各处理因素的作用。其次观察PDT疗效。兔 2 4只 ,随机分成 4组 :( 1)单纯损伤组 ;( 2 )PDT对照组 ,内膜损伤后先激光照射 ,后注射血啉甲醚 ;( 3)低PDT剂量组 ,内膜损伤后先注射血啉甲醚 5.0mg/kg ,然后以 30mw/cm2 照射 15min ;( 4 )高PDT剂量组 ,以 90mw/cm2 照射 ,其余同低剂量组。以内膜与中膜面积的比值为指标分析PDT疗效。结果 :预选参数的最优搭配为 :功率密度为 30mw/cm2 ,照射时间为 15min ,血啉甲醚剂量为 5mg/kg。低剂量组与高剂量组的S1/S2 比值分别比单纯损伤组低 71.9% (P <0 .0 1)和 59.4 % (P <0 .0 5) ,比PDT对照组低 63.1% (P <0 .0 1)和 4 6.5% (P <0 .0 5)。结论 :经皮血管内光动力疗法可能成为防治损伤性血管内膜增生的有效方法。     

<Emphasis Type="Italic">Alternaria</Emphasis> toxins: DNA strand-breaking activity in mammalian cells<Emphasis Type="Italic">in vitro</Emphasis>

Treatment, for 1 h, of cultured Chinese hamster V79 cells, human liver HepG2 cells, and human colon HT-29 cells with theAlternaria toxins alternariol (AOH) and alternariol methyl ether (AME) caused a concentration-dependent induction of DNA strand breaks at concentrations ranging from 5 to 50 micromolar. After treatment for 24 h, DNA strand breaks were observed in HepG2 but not HT-29 cells. Analysis of the 24 h-incubation media of HT-29 cells showed that both toxins were completely conjugated, whereas 75% were still present as unconjugated compounds in the 24 h-media of HepG2 cells. Lysates of both cell types fortified with UDPGA were found to convert both toxins into two glucuronides each, but HT-29 cells exhibited a much high activity than HepG2 cells and gave rise to a different ratio of glucuronides. It is concluded that glucuronidation eliminates the DNA strandbreaking potential of AOH and AME, and that the two glucuronides of eachAlternaria toxin are generated by different UGT isoforms. Presented at the 29^th Mykotoxin-Workshop, Fellbach, Germany, May 14–16, 2007 Financial support: State of Baden-Württemberg (Research Program “Mycotoxins” as part of the Research Initiative “Food and Health”)     

血啉甲醚体外光敏效应观察

本文用化学发光法检测ＨＭＭＥ的’Ｏ２和ＲＯＳ产量,并与ＨｐＤ相比较,同时观察ＨＭＭＥ光敏效应与浓度、激光照射功率密度、照射时间、激光波长的关系。结果显示：ＨＭＭＥ浓度在２．５－４０μｇ／ｍｌ范围内,其光敏效应随浓度的增加而呈线形上升,以后便略下降;５７８．２ｎｍ激光以１０ｍＷ／ｃｍ＾２照射２．５μｇ／ｍｌ,ＨＭＭＥ的’Ｏ２和ＲＯＳ产量是ＨｐＤ的８倍;ＨＭＭＥ光敏效应与激光照射的功率密度密切相关。各     

光动力疗法诱导增生滑膜细胞凋亡的初步研究

目的：观察光动力疗法（PDT）诱导类风湿性关节炎（RA）动物模型的增生滑膜细胞的凋亡情况。方法：兔AIA模型在关节类出现第七天进行PDT治疗,随机治疗一侧膝关节,另一侧作自身对照,免耳静脉注入HMME10mg/Kg,20分钟后,膝关节用金蒸气激光照射,激光波长627.8nm,功率密度100mW/cm^2,能量密度100J/cm^2,24小时后取膝关节滑膜作病理检查,并用脱氧核苷酸末端转移酶介导的缺口末端标记法（TUNEL）原位检测凋亡结论。结果：PDT治疗组凋亡阳性细胞较对照组明显增多,图像分析结果单位视野内阳性细胞数和面密度PDT治疗组高于对照组,统计学检验差异有显著性,凋亡细胞核直径PDT治疗组较小,与对照组相比,统计学检验差异有显著性。结论：PDT有可能通过诱发滑膜细胞的凋亡,使增生的滑膜细胞减少。     

血啉甲醚在鼻咽癌细胞中的浓度分布

利用激光诱导荧光方法实验测定了新型光敏剂血啉甲醚（Hematoporphyrin Monomethyl Ether, HMME）在鼻咽癌细胞株CNE中的时间分布和浓度变化规律.实验结果表明,鼻咽癌细胞株CNE的荧光光谱中的两个特征峰615 nm和675 nm证实了鼻咽癌细胞对HMME的选择性特异吸收.HMME在鼻咽癌细胞中的潴留浓度在混合培养后的140分钟达到最大值.当用于培养鼻咽癌细胞的HMME药物浓度低于32 μg/ml时,测量得到的相对荧光光谱强度与HMME浓度呈现出很好的线性关系,通过拟合方程可以间接确定在给定注射剂量条件下鼻咽癌细胞中所潴留HMME的浓度.结果可以为HMME在光动力学诊断与治疗中的临床应用提供理论依据和剂量参考.     

血卟啉单甲醚在A549肺癌细胞内亚细胞分布的动态变化

目的:探讨血卟啉单甲醚(Hematoporphyrin monomethyl ether,HMME)在A549肺癌细胞内亚细胞分布的动态变化。方法:传代培养A549肺癌细胞,分别与光敏剂HMME孵育2 h和12 h。应用由荧光显微镜及电感耦合器材(Charge-coupled device,CCD)组成的高分辨率荧光显微成像系统,结合荧光探针标记技术,采用细胞器-细胞荧光强度比值法研究HMME在不同时间的亚细胞分布情况。结果:在2 h和12 h两个孵育时间点高尔基体的平均荧光强度比值(J1/J2)值都最高;随着孵育时间延长,A549细胞的四种细胞器J1/J2值都升高且溶酶体幅度最大。结论:孵育时间是影响HMME亚细胞分布的一个重要因素。随着孵育时间的延长,A549肺癌细胞各细胞器吸收HMME能力逐渐增强,尤以溶酶体显著。     

Alternaria metabolites in sunflower seeds

The alternariol and alternariol monomethyl ether contamination in sunflower seeds was determined. The levels of alternariol found ranged between 35 to 792 µg/kg and 90 to 630 µg/kg for alternariol monomethyl ether. The fungicides showed different effect on the mycotoxin production depending on the substrate, strains and toxin analysed. Mercury phenyl acetate, Octave, Metiram, Thiram and Orthene inhibited alternariol monomethyl ether production while for alternariol production only Thiram, Metiram and Octave were effective.     

Lignans of Araucaria angustifolia and 13C NMR analysis of some phenyltetralin lignans

Secoisolariciresinol monomethyl ether and lariciresinol-4-methyl ether were isolated from the knots of dead trees of Araucaria angustifolia. On the basis of spectral evidence, the position of the OH group was located in these compounds. The ¹³C NMR spectra of the phenyltetralin lignans galbulin, galcatin, isogalcatin and cyclogalgravin have also been recorded and the signals assigned, based on the methyl shifts of cyclogalgravin.     

原子力显微镜观测血卟啉单甲醚对细菌光动力杀伤作用

[目的]探讨血卟啉单甲醚(Hematoporyrin monomethyl Ether,HMME)对革兰氏阳性(G )、阴性(G-)菌的光动力杀伤作用.[方法]通过平板菌落计数法和原子力显微镜(AFM),观察细菌与HMME作用前后形貌的变化.[结论]当HMME浓度为50 μg/mL,可见光(光功密度为200 mW/cm2)光照30min时90%以上的金黄色葡萄球菌(Staphyrlococcus aureus)能被杀死,无光照时对S.aureus杀灭效果显著.同等条件下,无论光照还是无光照,HMME对大肠杆菌(E.coli)无明显的杀伤作用.AFM图像显示,S.aureus细菌表面破坏严重,完全碎裂成鱼鳞状的片状堆积.对HMME作用后的E.coli扫描可见,菌体原来光滑的表面变成网格状的裂纹排列.[讨论]HMME对G 有明显的光失活效应,而对G-效果不明显.AFM的超微图像显示HMME对细菌细胞的攻击位点主要在细胞膜上.AFM为我们研究光敏剂对细菌的光动力损伤作用机制的可视化提供了依据.