致命鹅膏菌毒素对DMBA/巴豆油诱导小鼠皮肤肿瘤的治疗作用

研究了致命鹅膏中的毒素在安全剂量下抑制小鼠皮肤肿瘤的效果,并对此毒素的药用安全性进行初步评价。采用经典的DMBA/巴豆油诱导小鼠皮肤乳头状瘤形成的二阶段致癌模型,观察和评价致命鹅膏毒素在不同剂量下抑制肿瘤的效果,并且通过测定血清中氨基己糖的含量来判断对肝损伤的情况。结果表明,致命鹅膏毒素在某一低剂量下对DMBA/巴豆油诱导小鼠皮肤乳头状瘤有较好的治愈效果,且对小鼠肝脏无损伤,证明了致命鹅膏毒素可以较好地治疗皮肤癌。     

甘草黄酮体抗促癌,抗致突和抗氧化作用的研究

G9315是从胀果甘草[Glycyrrhizae inflata Bat(Ⅲ))中提取的含有6个黄酮体的复合物。2mg剂量明显抑制二甲基苯蒽(DMBA)合并巴豆油诱发的小鼠皮肤乳头瘤的生成,抑瘤作用主要在促癌阶段。1mg剂量显著抑制巴豆油诱发的小鼠耳部水肿。小鼠口服G_(9315)(0.5g/kg/d×7)明显对抗环磷酰胺诱发的骨髓微核细胞增加。20—40μg/ml显著抑制TPA促进的~(32)pi参入HeLa细胞的磷脂部分。20μg/ml显著抑制巴豆油诱发的Wistar大鼠中性粒细胞(PMN)和Balb/c新生小鼠皮肤表皮细胞的化学发光以及肝线粒体的脂质过氧化。G(9315)(10—20μg/ml)有效对抗CCl_4诱发的小鼠肝微粒体脂质过氧化。     

转化生长因子β mRNA在佛波酯刺激的小鼠表皮和化学性诱发的小鼠皮肤肿瘤中的高水平表达

转化生长因子β是一种细胞多功能调节肽,目前研究认为其和促癌过程有密切关系。本文利用Northern印迹分析技术研究了TGF-β mRNA在促癌物佛波酯刺激的小鼠表皮和化学性诱发的小鼠二阶段皮肤肿瘤中的表达情况。结果证明,巴豆油2—20 μL和TPA 5—40nmol一次局部涂用于小鼠皮肤,均可明显增加小鼠表皮TGF-β mRNA,呈较好的浓度依赖性。TGF-β mRNA表达在二甲基苯蒽(DMBA)和巴豆油诱发的二阶段小鼠皮肤乳头瘤和癌中均高于正常和瘤旁表皮。所检测到的TGF-βmRNA为2.5kb和1.9kb二种条带,但以2.5kb为主。     

二甲基苯蒽/佛波酯两步化学法诱导小鼠皮肤癌的模型优化

目的对两步化学诱导法建立BALB/c小鼠皮肤癌模型进行方法优化。方法将BALB/c小鼠背部剃毛暴露出约2 cm×2 cm的皮肤,小鼠随机分为空白对照组和4个模型组,4个模型组第1周分别涂1、2、4、7次100μg/100μL DMBA/丙酮液,后续均1周涂两次4μg/100μL TPA/丙酮液,记录小鼠体重、成瘤时间、成瘤数量,并在12周后处死小鼠取肿瘤组织及瘤旁组织做HE染色分析。结果 1周涂1次DMBA成瘤周期长;1周两次成瘤周期短,质量高;1周4次会损伤皮肤但不影响肿瘤的形成;1周7次对皮肤伤害较大并导致小鼠死亡。结论采用第1周涂两次DMBA启动,后续用TPA连续诱导建立BALB/c小鼠皮肤癌模型操作简便,成瘤期短,癌变率高,与人类皮肤癌发生发展相似,可用于建立研究皮肤癌较为理想的实验动物模型。     

我国28种鹅膏菌主要肽类毒素的检测分析*

利用高效液相色谱(HPLC)技术对产于我国的28种鹅膏菌的主要肽类毒素(鹅膏毒肽和鬼笔毒肽)进行了检测分析,并和采于欧洲(德国)的毒鹅膏Amanita phalloides作对照,结果表明,3种东亚所特有的鹅膏菌(灰花纹鹅膏、致命鹅膏和黄盖鹅膏白色变种)和欧洲毒鹅膏所含毒素种类多、含量高,其子实体菌盖部位主要毒素总量分别达到12583.7μg/g、8152.6μg/g、1058.2μg/g、7456.2μg/g干重子实体,这4种鹅膏菌可称之为剧毒鹅膏菌。其它25种鹅膏菌中有10种检测出含有微量鹅膏毒肽,含量在19.5μg/g-151.2μg/g之间。在4种剧毒鹅膏菌中,子实体组织部位不同,毒素含量以及鹅膏毒肽和鬼笔毒肽在其中的分布也不一样,菌盖中的毒素含量最高,菌柄的毒素含量次之,菌托中的毒素含量最低;对于灰花纹鹅膏、致命鹅膏和黄盖鹅膏白色变种,无论在菌盖、菌柄和菌托中,鹅膏毒肽类毒素的含量都高于鬼笔毒肽类毒素,尤其以α-amanitin的相对含量最高;而在欧洲毒鹅膏中,菌盖、菌柄和菌托中都以鬼笔毒肽为主,尤其以phallacidin的相对含量最高,并且从菌盖至菌柄到菌托,鬼笔毒肽的相对含量依次增加。     

我国鹅膏菌新发现种——致命鹅膏(Amanita exitialis)的肽类毒素分析

采用高效液相色谱（HPLC）技术对在广州发现的鹅膏菌新种——致命鹅膏(Amanita exitialis)不同组织部位的肽类毒素(鹅膏毒肽和鬼笔毒肽)的含量进行了分析,结果表明,致命鹅膏是一种剧毒蘑菇,其毒素含量相当高,子实体中组织部位不同,毒素含量以及鹅膏毒肽和鬼笔毒肽在其中的分布也不一样,菌盖中的毒素含量最高,达8152.6μg/g干重,菌柄的毒素含量次之,为3742.3μg/g干重,菌托中的毒素含量最低,只有1142.5μg/g干重;在菌盖、菌柄和菌托中都以鹅膏毒肽为主,尤其以αamanitin的相对含量最高,但从菌盖至菌柄到菌托,鬼笔毒肽尤其是Phallacidin的相对含量依次增加。     

促癌物TPA诱导小鼠表皮鸟氨酸脱羧酶mRNA表达和维甲类化合物对该表达的影响

強皮肤促癌物十四烷酰佛波醋酸酯(TPA)局部应用时可触发一系列的生物化学改变,其中最明显的事件之一就是对ODC活性的短暂而急到的诱导,而这种诱导作用与其促癌作用密切相关。利用Northern印迹分析和条带(Slot)印迹分析证明,10nmol/L TPA一次局部处理小鼠背部皮肤可刺激ODC mRNA(2.0kb大小)表达,在4h左右最为明显,随后逐渐降低。10nmol/L TPA多次处理小鼠皮肤(每2天一次,共4次)也有类似的促进作用,但却在6h左右最为明显。在二甲基苯蒽和巴豆油诱发的二阶段小鼠皮肤乳头瘤和癌组织中也观察到了相同大小的ODC mRNA的高水平表达,尤以癌组织最高。新维甲类化合物R8605虽能明显抑制巴豆油诱导的ODC活性,但却未见对TPA诱导的ODC mRNA增加有明显抑制作用。     

我国28种鹅膏菌主要肽类毒素的检测分析

利用高效液相色谱(HPLC)技术对产于我国的28种鹅膏菌的主要肽类毒素(鹅膏毒肽和鬼笔毒肽)进行了检测分析,并和采于欧洲(德国)的毒鹅膏。Amanita phalloides作对照,结果表明,3种东亚所特有的鹅膏菌(灰花纹鹅膏、致命鹅膏和黄盖鹅膏白色变种)和欧洲毒鹅膏所含毒素种类多、含量高,其子实体菌盖部位主要毒素总量分别达到12583．7μg／g、8152．6μg／g、1058．2μg／g、7456．2μg／g干重子实体,这4种鹅膏菌可称之为剧毒鹅膏菌。其它25种鹅膏菌中有10种检测出含有微量鹅膏毒肽,含量在19．5μg／g—151．2μg/g之间。在4种剧毒鹅膏菌中,子实体组织部位不同,毒素含量以及鹅膏毒肽和鬼笔毒肽在其中的分布也不一样,菌盖中的毒素含量最高,菌柄的毒素含量次之,菌托中的毒素含量最低;对于灰花纹鹅膏、致命鹅膏和黄盖鹅膏白色变种,无论在菌盖、菌柄和菌托中,鹅膏毒肽类毒素的含量都高于鬼笔毒肽类毒素,尤其以α-amanitin的相对含量最高;而在欧洲毒鹅膏中,菌盖、菌柄和菌托中都以鬼笔毒肽为主,尤其以phallacidin的相对含量最高,并且从菌盖至菌柄到菌托,鬼笔毒肽的相对含量依次增加。     

云南楚雄致命鹅膏中环肽毒素的检测与分析

采用反相高效液相色谱法对采自云南楚雄双柏县的致命鹅膏在3个不同生长期中不同部位的6种环肽毒素含量进行了检测和分析。结果表明,致命鹅膏含有α-, β-鹅膏毒肽、羧基三羟鬼笔毒肽和羧基二羟鬼笔毒肽,未检出γ-鹅膏毒肽和二羟鬼笔毒肽。生长期毒素总量最高（9.3mg/g）、从成熟期（7.5mg/g）到衰老期（6.5mg/g）逐渐降低,但鬼笔毒肽的相对含量随着年龄增长而逐渐增加,鹅膏毒肽与鬼笔毒肽比值从生长期、成熟期到衰老期分别为2.6、1.4和0.9。在3个不同发育阶段中,4种毒素含量从菌盖、菌柄到菌托逐渐降低,而鬼笔毒肽的相对含量逐渐增加。α-鹅膏毒肽和β-鹅膏毒肽在生长期菌盖中含量最高,分别为7.4mg/g和3.1mg/g,而羧基三羟鬼笔毒肽和羧基二羟鬼笔毒肽在衰老期的菌盖中含量最高,分别为2.8mg/g和2.1mg/g。     

皮肤光损伤肿瘤模型的建立

目的模拟人皮肤肿瘤形成的自然环境因素,建立光损伤小鼠皮肤肿瘤模型。方法采用完全随机的两因素析因设计。选择对光线敏感的BALB／c小鼠,背部皮肤脱毛后UVC照射（56mJ／cm2）,隔天1次,8周结束;二甲基苯蒽／丙酮液（100μg／200μL）外涂,每周1次,共7周,12周后处死。连续测量并记录小鼠背部肿瘤数量和直径,描绘时间一荷瘤数动态变化图;皮肤组织病理学观察肿瘤形成的组织细胞形态学改变。结果单纯UVC照射组无肿瘤出现;UVC＋DMBA模型组小鼠6周后背部开始出现肉眼可见的瘤体,第9周荷瘤率达到100％,11、12周荷瘤数渐趋稳定,肿瘤体积有增大趋势,平均荷瘤数为（4．57±3．0）个,肿瘤平均体积为（44．91±4．6）mm。。HE染色、瑞氏染色、基底膜带染色均显示为早期皮肤鳞状细胞癌。而单纯DMBA组荷瘤数第8．5周达高峰,后呈下降趋势,肿瘤自然消退率高,数量不稳定。结论DMBA是皮肤肿瘤建模的主要因素,但停止该处理因素后荷瘤数量呈下降趋势;UVC作为一个独立因素,在12周内未能诱导出皮肤肿瘤,但UVC与DMBA联合应用具有交互协同作用,可较快速地建立皮肤肿瘤模型。该模型具有出瘤率高,出瘤整齐,荷瘤量多,除去处理因素后瘤体数量依旧稳定,且体积有增大趋势等特点,为皮肤肿瘤的防治研究提供有价值的实验工具。     

广东已知毒蘑菇种类

广东毒蘑菇种类有112种,分别隶属于子囊菌门(Ascomycota)的1科1属和担子菌门(Basidiomycota)的18科41属。其中种类最多的是鹅膏属(Amanita),有20种,占17.8%;其次为红菇属(Russula),有10种,占8.9%;第3是丝盖伞属(Inocybe),有8种,占7.1%。致命鹅膏(Amanitaexitialis)、铅绿褶菇(Chlorophyllum molybdites)和粗柄白鬼伞(Leucocoprinuscepaestipe)等是近几年在广州引起中毒的常见毒蘑菇种类,其中致命鹅膏的中毒死亡率最高。     

Peptide toxin components of Amanita exitialis basidiocarps

Eight peptide toxins were isolated and purified from basidiocarps of Amanita exitialis with high performance liquid chromatography and were subjected to ultraviolet, nuclear magnetic resonance and mass spectrometry. We identified seven peptide toxins, α-amanitin, β-amanitin, amaninamide, phallacin, phallacidin, phallisacin and desoxoviroidin. The molecular weight (729.5 Da) of the eighth compound did not match that of any reported Amanita toxins and, although the UV absorption spectrum indicated it to be a phallotoxin, further studies are required to identify this component. This is the first report of amaninamide, phallacin, phallisacin and desoxoviroidin in this lethal mushroom species.     

硒酸赖氨酸对四氧嘧啶诱发的小鼠肝损伤的防护作用

目的:研究硒酸赖氨酸对四氧嘧啶诱发的小鼠肝损伤的防护作用。方法:选取昆明小鼠50只,雌雄各半,随机分成五组,即对照组、模型组、低剂量组、中剂量组、高剂量组。采用四氧嘧啶致急性肝损伤模型,检测各组小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(AKP)活性,并对各组小鼠肝脏进行组织病理学观察。结果:硒酸赖氨酸能降低小鼠血清中ALT、AST、AKP活性(P<0.05或P<0.01),明显减轻四氧嘧啶致肝损伤小鼠肝细胞的病变及炎症反应。结论:硒酸赖氨酸具有对四氧嘧啶诱发小鼠肝损伤的保护作用。     

1-Chloromethylpyrene: a reference skin sensitizer and genotoxin

1-Chloromethylpyrene (1-CMP) has been evaluated as a model mutagen and toxin related to the ultimate electrophiles derived from benzo[a]pyrene and 1-nitropyrene. It was mutagenic to Salmonella (greater than 100 pg/plate) and exceptionally reactive to DNA when assessed by the 32P-postlabelling technique. 1-CMP was inactive in a mouse bone micronucleus assay when administered by gavage, probably due to hydrolysis, whose kinetics have been studied (t1/2 approximately 23 min at 37 degrees C). However, as expected, it was a potent skin toxin as determined by its activity as a mitogen to mouse skin and its contact allergenicity, as determined using the local lymph node proliferative assay. It is concluded that 1-CMP will probably be a potent human skin carcinogen and contact allergen.     

Production and characterization of Amanitin toxins from a pure culture of Amanita exitialis

Amanita exitialis Zhu L. Yang and T.H. Li is a lethal mushroom species recently isolated in Guangdong Province, China. In this report, a pure culture of this species was obtained for the first time. To confirm the identity of the pure culture, the internal transcribed spacer regions of the nuclear ribosomal DNA of the pure culture and of a typical fruiting body of the species were sequenced and compared. Further, amatoxins produced by pure cultures were analyzed and characterized by high-performance liquid chromatography and mass spectrometry analysis. The results showed that the pure cultures produced 728.3 +/- 43.8 microg g(-1) (dry matter) of alpha-Amanitin and 60.0 +/- 20.7 microg g(-1) (dry matter) of beta-Amanitin, respectively, a yield which is about 10% of that produced by fruiting bodies.     

An international collaborative study of the effect of active pertussis toxin on the modified Kendrick test for acellular pertussis vaccines

Speculation that the Japanese modified intra-cerebral challenge assay, which is used in several countries for control of acellular pertussis vaccines, depends on the presence of small amounts of active pertussis toxin led to an assumption that it may not be appropriate for highly toxoided or genetically detoxified vaccines. Consequently, at the recommendation of a World Health Organisation AD Hoc Working Group on mouse protection models for testing and control of acellular pertussis vaccine, the effect of pertussis toxin on the modified intra-cerebral challenge assay (modified Kendrick, MICA) was evaluated in an international collaborative study. Results of this study showed that for genetically detoxified vaccines both with and without active pertussis toxin the MICA clearly distinguished mice vaccinated with acellular vaccines from unvaccinated mice and gave a significant dose–response relationship. However, vaccine samples containing active pertussis toxin (5 or 50 ng/single human dose) appeared to be more potent than the equivalent sample without active pertussis toxin. Similar results were also given by two respiratory infection models (intranasal and aerosol) included in the study. The results also indicated that the effect of pertussis toxin may vary depending on mouse strain.     

Chemopreventive activities of etodolac and oxyphenbutazone against mouse skin carcinogenesis

Previous cancer chemoprevention studies have demonstrated that the non-steroidal anti-inflammatory drugs (NSAIDs) can be effective in suppressing the development of various human malignancies. Recently we identified the possible anti-tumor promoting potentials of 14 new NSAIDs in the Epstein–Barr virus early antigen activation assay induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). In this study we report the inhibition of 7,12-dimethylbenz (a) anthracene (DMBA) induced two-stage mouse skin carcinogenesis by etodolac (ETD), one of the most potent NSAIDs identified in our in vitro cancer chemopreventive screening of this group of drugs. Topical administration of ETD at a very low dose of 85 nmol showed a significant decrease in both tumor incidence and burden. This effect is also accompanied by a delay in the tumor latency period. Since ETD showed potent chemopreventive activity in both in vitro and in vivo studies, it warrants prompt consideration for trial in humans as a potential cancer chemopreventive agent. We also investigated oxyphenbutazone (OPB) another commonly used NSAID for its cancer chemopreventive effect on peroxynitrite (PN) induced-TPA promoted skin tumors in the mouse. Following tumor initiation with 390 nmol of PN, the skin tumor promotion with 1.7 nmol of TPA was significantly inhibited by oral administration of 0.0025% OPB. The results demonstrate that OPB is a potent cancer chemopreventive agent in the highly sensitive in vivo mouse test model we used.     

Distribution of the amatoxins and phallotoxins in Amanita phalloides. Influence of the tissues and the collection site

The toxin composition of 25 Amanita phalloides carpophores collected from three sites in Franche-Comté (France) differing in their geological and pedological characteristics was determined and the factors involved in the variations of the toxin concentration in the tissues were identified. The concentrations of the main amatoxins (beta-amanitin, alpha-amanitin, gamma-amanitin) and phallotoxins (phallacidin, phallisacin, phalloidin, phallisin, phalloin) in the six tissues constituting the carpophore, i.e. the cap (C), gills (G), ring (R), stipe (S), bulb (B) and volva (V) were evaluated by using high-performance liquid chromatography. The results analysed statistically showed that the toxin concentrations were tissue dependent, leading to classification of the tissues into two groups (B, V) and (C, G, R, S). The (B, V) group was distinguished by high amounts of phalloidin, phallisin and phallisacin, and the (C, G, R, S) group by the predominance of the amatoxins. The characteristics of the soil of the collection site also affected the toxin concentrations; however, this effect differed from one site to another and was not similar for all the tissues. Finally, the mean toxin profile in the carpophores from the three sites was evaluated. This study underscores the fact that environmental factors and mainly the soil type clearly have an effect on the toxin composition of A. phalloides carpophores.