【作者】 李维维；

【导师】 郑江； 彭缨；

【作者基本信息】 沈阳药科大学 ， 药物化学， 2017， 博士

【摘要】 吡咯里西啶生物碱(PAs)是一类广为人知的毒性化合物,分布于多种植物。暴露于PAs类化合物能引起肝毒性、肺毒性、致癌性、致畸性。据文献报道,在世界各地有大量暴露于PAs导致毒性的案例。PAs是威胁人类健康的一类有害物质,因此,对PAs导致毒性的机制的探究是必要的。野百合碱(Monocrotaline)是PAs中的有毒成员,研究表明其在代谢、毒性等方面具有代表性,所以我们以野百合碱为研究对象,对PAs的致毒机制进行探究。呋喃类化合物广泛分布在各种水果、食物、中药材及饮料中。据文献报道,许多呋喃类化合物具有严重的毒性反应。随着人们对养生的兴趣逐渐加深,各种中药材、水果、药膳等天然产物的摄入量也随之增加,对呋喃类化合物致毒机制的探究也愈发重要。黄药子为中药薯蓣科植物黄独的块根,临床报道了很多关于黄药子导致肝毒性的案例,严重时甚至引起死亡。黄独素B是黄药子中主要的呋喃二萜类化合物,简称DIOB,有文献报道DIOB能够引起严重的肝毒性,我们以DIOB为研究对象,探究其致毒机制。具体的研究包括以下几个部分:1.野百合碱要经历代谢活化过程生成反应性中间体脱氢野百合碱(DHM),才会产生毒性反应。脱氢PAs与蛋白质的共价结合是引发毒性的关键步骤,已有文献报道脱氢PAs能与蛋白质L-半胱氨酸巯基共价结合,使蛋白质失活,从而产生细胞毒性。本研究首次报道脱氢PAs能修饰蛋白质L-赖氨酸残基。我们检测到DHM与L-赖氨酸形成的结合物,分别为A1’-A3’。A1’是DHM与双分子赖氨酸形成的7位与9位均结合赖氨酸的双分子赖氨酸结合物,即7,9-di-Lys-DHP。DHM与单分子赖氨酸形成2个结合物A2’和A3’,分别是7-Lys-DHP和9-Lys-DHP,7-Lys-DHP为主要结合物。我们以小肽(八肽)为模型,检测DHM是否与含有赖氨酸的小肽反应。通过二级质谱分析,检测到未被修饰的y5和b2片段,同时检测到被修饰的b3*和y6*片段,提示DHM与小肽的反应发生在赖氨酸的ε-氨基。而且,DHM能与具有三维结构的模型蛋白的的赖氨酸残基反应,蛋白质的赖氨酸残基主要结合在DHM母核千里光次碱的7位碳原子上。最后,给小鼠腹腔注射野百合碱和倒千里光碱(retrorsine),在小鼠肝脏水解液中也检测到7-Lys-DHP,说明小鼠肝脏蛋白质的赖氨酸残基也被反应性代谢物修饰,这种修饰随着剂量的升高不断增加,可以作为PAs生物碱修饰体内蛋白质的生物标记物。这可能是野百合碱导致肝脏毒性的重要原因之一。2.PAs是一个具有双亲电基团的分子,有可能一侧与蛋白质结合一侧与DNA结合,形成DNA-蛋白质交联产物,阻碍DNA的复制,翻译等功能。脱氢PAs能与蛋白质的半胱氨酸的疏基和赖氨酸的ε-氨基反应,还能与DNA的2’-脱氧鸟苷的2位氨基和2’-脱氧腺苷的6位氨基结合。DHM与它们的混合液分别进行反应,都检测到交联结合物。我们选取小牛胸腺DNA(ctDNA)作为模型DNA,提供碱基,然后分别加入小分子赖氨酸以及半胱氨酸,再对DNA进行彻底水解,只检测到Cys-DHP-dG结合物。我们选取牛血清白蛋白(BSA)作为模型蛋白,提供赖氨酸和半胱氨酸,然后分别加入小分子2’-脱氧鸟苷以及2’-脱氧腺苷,再对蛋白质进行彻底水解,也只检测到Cys-DHP-dG结合物。然后,我们采用ctDNA提供碱基,BSA提供氨基酸,与DHM孵育,分别对蛋白质和DNA进行彻底水解,同样只检测到Cys-DHP-dG结合物,说明DHM确实与具有空间结构的DNA和蛋白质形成交联产物。小鼠腹腔注射野百合碱,8.0 h获取肝脏组织,制备肝脏匀浆液,采用糜蛋白酶和链霉蛋白酶对蛋白质进行彻底水解,采用DNA酶Ⅰ,磷酸二酯酶Ⅰ和碱性磷酸酶对DNA进行彻底水解,水解产物采用LC-MS/MS方法进行分析。我们在小鼠肝脏内也检测到DHM与2’-脱氧鸟苷和半胱氨酸反应形成的结合物,其结构为:7(R)-Cys-DHP-dG,说明给予野百合碱后,在小鼠肝脏中经代谢生成的DHM与DNA和蛋白质发生交联,DHM母核千里光次碱的7位碳与蛋白质的半胱氨酸巯基结合,9位碳与DNA的2’-脱氧鸟苷结合。在腹腔注射倒千里光碱(retrorsine)的肝脏中也能检测到7(R)-Cys-DHP-dG。反应性代谢物与DNA和蛋白质发生交联结合,这可能是PAs导致肝癌的重要原因之一。3.我们对DIOB进行了详细的毒性研究,DIOB引起的肝毒性呈现明显的时间-依赖性和剂量-依赖性,DIOB给药200 mg/kg时,小鼠血清ALT活性升高20倍,肝脏病理组织切片结果显示肝细胞出现灶状坏死。我们进一步探究了 DIOB代谢与肝脏毒性之间的相关性,KTC(75 mg/kg)连续预处理再给予DIOB(200 mg/kg,KTC+DIOB组)与只给予DIOB(200 mg/kg)相比,小鼠表现出的肝脏损伤大大降低,GSH消耗变得相对较少,AUC0-t值升高(2.9倍),尿液中DIOB-GSH结合物的排泄量降低,原型药物的排泄量大大增加。我们对小鼠用BSO预处理后再给予DIOB,相较于单独给予DIOB,引起小鼠血清中ALT值剧烈升高,病理组织切片也观察到大量的细胞坏死。为了探究呋喃环在DIOB诱导的肝毒性中所起的作用,我们对DIOB进行选择性还原,把呋喃环还原成四氢呋喃,即tetrahydro-DIOB。腹腔注射给予相同剂量的tetrahydro-DIOB(200 mg/kg),小鼠未表现出任何肝脏毒性,无论是ALT水平,还是肝脏病理组织切片,都与正常组无任何差异,说明呋喃环在DIOB引起的肝脏毒性中起着至关重要的作用。本实验的结果表明,顺烯二醛反应性代谢中间体在DIOB诱导产生的肝脏毒性中起着非常重要的作用。小鼠腹腔注射给予DIOB后,肝脏中GSH水平的下降趋势并不明显,提示反应性代谢产物与关键蛋白的共价结合可能是DIOB诱导产生肝脏毒性的原因。动物体内实验结果表明,细胞色素P450 3A家族是参与DIOB代谢生成反应性代谢产物的主要酶。更多还原

【Abstract】 Pyrrolizidine alkaloids(PAs)are widespread hepatotoxins,occur in varieties of plant species.Exposure to PAs may cause severe hepatic injury,pulmonary toxicity,carcinogenesis,and genotoxicity.Numerous PAs poisoning cases have been documented in many countries.PAs have become a potential public health issue to humans and livestock.Monocrotaline is a retronecine-type PAs found in the plant family leguminosae(genus Crotalaria).Monocrotaline was selected to explore the mechanisms of PAs-induced hepatotoxicitiesFuran-containing compounds refer to a class of compounds containing furan ring(s),which are abundant in fruits,herbs,foods,and beverages.Many furan-containing compounds have been reported to be toxicities.The risks for intake of toxic furans have been rising,due to the rapid growth of globe-wide consumption of medical remedies,dietary supplements,and"natural" foods.Diosbulbin B(DIOB),a furanoid,is a major constituent of herbal medicine Dioscorea bulbifera L.Exposure to DIOB caused liver injury in humans and experimental animals.The mechanisms of DIOB-induced hepatotoxicities remain unknown.The studies performed are summarized as below.1.The execution of the toxicities of the alkaloids requires metabolic activation.Protein modificcation by reactive metabolites of PAs has been suggested to be an important mechanism of the toxic actions of PAs.Studies showed that dehydro-PAs modified cysteine residues of hepatic proteins in animals administered with PAs.The objectives of the present study were to define the interactions of dehydromonocrotaline(DHM)with lysine,lysine derivatives.The reaction of DHM with L-lysine produced three DHM-derived adducts,namely A1’-A3’,The observed molecular ion of A1’(m/z 410),along with its mass spectrum,indicates that the adduct contained two molecules of Lys,named as 7,9-di-Lys-DHP.The adducts A2’ and A3’observed were most likely the two regioisomers resulting from alkylating at C7 and C9 of the necine base,i.e.7-Lys-DHP and 9-Lys-DHP respectively.7-Lys-DHP was the major mono-Lys-DHP adduct.A lysine-containing peptide(8 amino acids)was employed for the investigation of the interaction of Lys residues with DHM.The observation of b3*and y6*,along with the retaining of b2 and y5,indicates the modification of Lys residue of the peptide by the necine base.A model protein,bovine serum albumin was used to explore the lysine modification of proteins by DHM.DHM was found to react with ε-amino group of all model compounds tested after incubation with DHM,and the modification reaction preferentially occurred at C7 of the necine base.The lysine residue modification with the same regioselectivity was also observed in hepatic proteins of mice treated with monocrotaline.The observed modification increased with the increasing in the doses administered in the animals.This work allowed us to better understand the mechanisms of hepatotoxicity of monocrotaline.2.Dehydro-PAs are well-known bidentate alkylating agents with two electrophilic benzylic-like carbons at C7 and C9,thus it can react with DNA and protein to form DNA-protein cross-links.DPCs strongly disrupt normal DNA-protein interactions and interfere with DNA replication,transcription,and repair,which ultimately threatens genomic integrity and cell viability.DHM can react with lysine/cysteine,dG(2’-Deoxyguanosine)/dA(2’-Deoxyadenosine),respectively.As an initial step,we incubated DHM with lysine/cysteine and dG(2’-Deoxyguanosine)/dA(2’-Deoxyadenosine),and cross-link adducts were monitored by the AB Sciex 4000 Q-Trap MS.A dG(2’-Deoxyguanosine)/dA(2’-Deoxyadenosine)containing DNA(ctDNA)was employed for the investigation of the interaction of DHM with dG/dA and lysine/cysteine,the cross-linked DNA samples were enzymatically hydrolyzed to nucleosides with DNAase I,phosphodiesterase I,and alkaloid Phosphatase.Five analytes showed identical chromatographic and mass spectrometric behaviors as those of authentic Cys-DHP-dG adducts were detected in the mixture,and no such adduct was found in the digestion mixture without DHM.A lysine/cysteine containing protein(BSA)was employed for the investigation of the interaction of DHM with dG/dA and lysine/cysteine,the cross-linked protein samples were enzymatically hydrolyzed to amino acids with α-chymotrypsin and pronase E.Two Cys-DHP-dG adducts were detected in the mixture,and no such adduct was found in the digestion mixture without DHM.In addition,DHM reacts with BSA and ctDNA to form DNA-protein cross-links in vitro.Only 7(R)-Cys-DHP-dG was observed in hepatic homogenates of mice treated with monocrotaline.This work allowed us to better understand the mechanisms of hepatotumorigenicity of monocrotaline.3.The hepatotoxicities of DIOB were evaluated in detail.Intraperitoneal administration of DIOB caused elevations of serum ALT activities in a time-dependent and dose-dependent manner.A single dose of 200 mg/kg produced over twenty-fold elevations in serum ALT activities.Histopathologic evaluation showed focal necrosis in the liver of mice given DIOB at 200 mg/kg.Pretreatment with KTC protected the animals from hepatotoxicities and hepatic GSH depletion induced by DIOB,increased area under the concentration-time curve of blood DIOB,decreased urinary excretion of GSH conjugates derived from DIOB,and increased urinary excretion of parent drug.Pretreatment with BSO exacerbated DIOB-induced hepatotoxicities.In order to define the role of furan moiety in DIOB-induced liver toxicities,we replaced the furan of DIOB with a tetrahydrofuran group by chemical hydrogenation of the furan ring of DIOB.No liver injury was observed in the animals given the same doses of tetrahydro-DIOB.The furan moiety was essential for DIOB-induced hepatotoxicities.The results implicate the cis-enedial reactive metabolite of DIOB was responsible for the observed toxicities.The observed modest depletion of hepatic GSH in DIOB-treated animals suggests the actions of one or more reactive metabolites,and the hepatic injury observed could be due at least in part to reactions of these metabolites with crucial biomolecules.Cytochrome P450 3A enzymes are implicated in DIOB-induced hepatotoxicities by catalyzing the formation of the reactive metabolite of DIOB.更多还原