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题名: 含巯基药物对氯代醌及铜诱导的毒性保护机制研究
作者: 刘玉祥
学位类别: 博士
答辩日期: 2015-05
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 朱本占
关键词: 卤代醌 ; ; 含巯基的药物 ; 2 ; 3-二巯基丁二酸 ; 细胞毒性 ; DNA双链断裂,Halogenated quinones ; Copper ; Thiol-containing drugs ; 2 ; 3-Dimercaptosuccinic acid ; Cytotoxicity ; DNA double-strand breaks
其他题名: Mechanism of Protection by Thiol-containing Drugs against Chlorinated Quinone and Copper Induced Toxicty
学位专业: 环境科学
中文摘要:    
    卤代醌是一类能够在体内产生有害效应如急性肝毒性、肾毒性以及致癌性的有毒物质。我们最近的研究发现卤代醌可与H2O2通过不依赖于金属的有机类Fenton反应产生羟基自由基(?OH)。铜是一种具有氧化还原活性的过渡金属,它构成了多种金属酶的活性中心。尽管铜是人体必需的微量元素,但当铜过量时,它也可通过类Fenton反应产生活性氧物种(ROS)从而导致生物毒性。众所周知,低分子量含巯基药物如青霉胺、2,3-二巯基丁二酸等都具有一定的抗氧化性能。然而,目前还不清楚这些含巯基的药物是否能对卤代醌或铜诱导的DNA损伤起到保护作用?如果是,那么其具体的保护作用机制为何?针对上述问题,我们开展了本项研究,研究主要分为以下三个部分:

    1、卤代醌导致HepG2细胞毒性的结构-效应研究
     四氯-1,4-苯醌 (TCBQ)是研究最为广泛的卤代醌,其对多种生物系统均显示有毒性作用。然而,迄今为止尚无报道针对所有卤代醌进行系统性的结构-效应研究。本研究中,我们以HepG2细胞作为模型细胞,对卤代醌导致的细胞毒性进行了详细深入探究。我们发现3种四卤代醌(四氟-1,4-苯醌(TFBQ)、四氯-1,4-苯醌 (TCBQ)、四溴-1,4-苯醌 (TBBQ)),5种不同的氯代醌(2-氯-1,4-苯醌(2-CBQ)、2,5-二氯-1,4-苯醌(2,5-DCBQ)、2,6-二氯-1,4-苯醌(2,6-DCBQ)、三氯-1,4-苯醌(TriCBQ)和TCBQ),TCBQ及其两种水解产物(三氯羟基-1,4-苯醌(TrCHQ)和2,5-二氯-3,6-二羟基-1,4-苯醌(DDBQ))的细胞毒性的强弱顺序依次为:TBBQ > TCBQ > TFBQ; TriCBQ > TCBQ > 2-CBQ > 2,5-DCBQ > 2,6-DCBQ; TCBQ > TrCHQ > DDBQ。进一步研究发现,这些卤代醌的细胞毒性由它们产生ROS的能力(即氧化还原能力)以及与谷胱甘肽直接结合的能力所决定。

    2、新型氯化消毒副产物三氯苯醌与H2O2导致DNA双链断裂的分子机制研究
     最近,三氯-1,4-苯醌(TriCBQ)作为一种新型氯化消毒副产物,在饮用水和游泳池水中均被检测到。我们前期的研究发现卤代醌可与H2O2通过不依赖于金属的途径产生高活性且对生物体有重要作用的?OH。然而,目前还不清楚TriCBQ和H2O2是否能导致DNA双链断裂?如果能,那么含巯基的药物是否能对其起到保护作用。本研究发现:TriCBQ与H2O2联合暴露可导致pBR322 DNA产生双链断裂;而单独暴露TriCBQ、H2O2则不能。典型的?OH清除剂如二甲基亚砜和乙醇能显著抑制TriCBQ/H2O2导致的DNA双链断裂;通过电子自旋共振-自旋捕获研究发现TriCBQ/H2O2体系产生的?OH量与DNA的双链断裂具有较好相关性。多种含巯基的小分子药物如卡普托利、青霉胺、N-乙酰-半胱氨酸、2,3-二巯基丁二酸可很好地保护TriCBQ/H2O2导致的DNA双链断裂,其保护作用机制主要是这些含巯基的药物可直接与TriCBQ反应形成活性较低的醌-巯基偶联物。
 
    3、2,3-二巯基丁二酸保护铜所致DNA断裂损伤的作用机理:
    与铜形成不具氧化还原活性的铜络合物 2,3-二巯基丁二酸(DMSA)是一种重金属络合剂,其具有毒性低,安全且给药方便等特点,因此在临床上被广泛用于治疗铅、砷等重金属中毒。然而,目前尚不清楚DMSA是否能对铜介导的氧化损伤产生保护作用?如果能,其分子作用机制又为何?本研究中,我们深入探讨了DMSA对铜/抗坏血酸体系介导的DNA氧化损伤的影响。以DNA链断裂作为DNA氧化损伤的标志。研究结果发现:DMSA能显著保护铜/抗坏血酸介导的DNA氧化损伤,且这种保护效应呈浓度依赖方式(0.1-1.0 mM)。与之相对的是,典型的?OH清除剂二甲基亚砜即使浓度高达500 mM,对铜/抗坏血酸介导的DNA损伤也只提供微弱的保护作用。进一步利用紫外-可见和低温电子自旋共振等分析技术发现,DMSA能够显著抑制铜催化的抗坏血酸的氧化过程,并能有效地与组氨酸以及1,10-邻菲罗啉竞争结合一价铜离子Cu(I)。综上可知,DMSA抑制铜介导的DNA氧化损伤应归因于DMSA与铜形成了不具氧化还原活性的DMSA-Cu(I)络合物。
英文摘要:     Halogenated  quinones  represent  a  class  of  toxicological  intermediates  that  can create   a   variety   of   hazardous   effects   in   vivo,   including   acute   hepatoxicity, nephrotoxicity, and carcinogenesis. We found recently that hydroxyl radical (•OH) can be produced by  halogenated quinones in the  presence of H2O2 via  metal-independent organic Fenton-like reaction.  Copper is a  redox-active transition metal comprising  of the active center of a wide variety of metalloenzymes. Although copper is  an essential trace element for humans, it is also toxic  when in excess, possibly due to formation of reactive  oxygen  species   (ROS)  via  Fenton-like   reaction.  Low  molecular   weight thiol-containing  drugs  such  as  penicillamime  and  2,3-dimercaptosuccinic  acid  are known  for  general  antioxidant  properties.  However,  it  is  not  clear  whether  DNA damage  induced   by  halogenated   quinone  or  copper   can  be  protected   by  these thiol-containing drugs;  and  if so,  what are  their  respective underlying  mechanisms.Our research includes the following three parts:
      1.   The   cytotoxicity  induced   by   halogenated  quinones   on   HepG2   cells:  A structure-activity study
    Although   tetrachloro-1,  4-benzoquinone   (TCBQ),   one   of   the  most   studied halogenated quinones, has been shown to be toxic  in various biological systems, there was   no  systematic   structure-activity   study  on   the   cytotoxicity  induced   by   all halogenated quinones so far. In this study, we  investigated the cytotoxicity induced by halogenated quinones  using HepG2 cell as  model cell culture  system. We found  that the cytotoxicity induced by the three tetrahalogenated quinones (tetrafluoro-1,4-benzoquinone   (TFBQ),   TCBQ   and   tetrabromo-1,4-benzoquinone (TBBQ)),   the    five   different    chlorinated   quinones    (2-chloro-1,4-benzoquinone (2-CBQ), 2,5-dichloro-1,4-benzoquinone (2,5-DCBQ),2,6-dichloro-1,4-benzoquinone   (2,6-DCBQ),  trichloro-1,4-benzoquinone   (TriCBQ)and TCBQ), TCBQ and its two hydroxylation products (trichlorohydroxy-1,4-benzoquinone   (TrCHQ)   and  2,5-dichloro-3,   6-dihydroxy-1,4-benzoquonine (DDBQ)) decrease in  the following order: TBBQ  > TCBQ > TFBQ;TriCBQ >  TCBQ >  2-CBQ >  2,5-DCBQ > 2,6-DCBQ;  TCBQ >  TrCHQ >  DDBQ.The cytotoxicity was  found to be  determined by both their  redox activity to generate reactive oxygen species (ROS) and their direct binding ability with GSH.
     2.Mechanism   of   DNA   double-strand   cleavage   by    a   new   chlorination disinfection byproduct trichloro-1,4-benzoquinone and H2O2
     Trichloro-1,4-benzoquinone  (TriCBQ)  has   been  recently  identified  as   a  new chlorination disinfection  byproduct in drinking  and swimming pool  water. We found previously that the highly reactive and biologically important •OH can be produced by halogenated quinones  and H2O2  independent of transition  metal ions.  However, it is not clear  whether DNA  double-strand breaks  (DSBs) can  be induced by  TriCBQ in the presence  of H2O2;  and if  so, whether TriCBQ/H2O2  induced DNA  DSBs can be protected by thiol-containing drugs?  Here we show that DNA DSBs can be  generated only when pBR322 DNA was treated with the combination of both TriCBQ and H2O2,while either TriCBQ or  H2O2 alone cannot. Typical  •OH scavengers such as dimethyl sulfoxide and ethanol  can markedly inhibit TriCBQ/H2O2-induced  DSBs, and a good correlation was  observed between DNA DSBs  and •OH  generation by TriCBQ/H2O2
as measured  by ESR  spin-trapping method. DNA  damage induced by  TriCBQ/H2O2 could be inhibited by  several thiol-containing drugs such  as captopril, penicillamime,N-acetyl-cysteine  and 2,3-dimercaptosuccinic  acid. The  protection  was found  to  be due to  their direct  interactions  with TriCBQ  to form  the less  reactive quinone-thiol conjugates.
    3.Mechanism of protection by 2,3-dimercaptosuccinic acid against copper-induced DNA cleavage via forming a redox-inactive copper complex
    2,3-Dimercaptosuccinic acid  (DMSA) has  been widely  used in  the treatment  of lead, arsenic and other heavy metal poisoning in clinic because of its low toxicity, safe and convenient  administration. However,  it is  not clear whether  DMSA can provide protection against copper-induced  oxidative damage; and if so,  what is its underlying molecular   mechanism.   In   this  study,   we   investigated   the   role   of   DMSA  in
copper-induced   oxidative    damage   to    DNA,    using   copper-containing    system Cu(II)/ascorbate. Oxidative damage to DNA was  measured as stand breakage. DMSA (0.1-1.0  mM)  provided strong  and  dose-dependent  protection  against  oxidation  of DNA by  Cu(II)/ascorbate system.  In contrast,  only limited  protection  was observed
with the typical  •OH scavenger  dimethyl sulfoxide, even at  concentrations as high as 500 mM.  DMSA also  ignificantly inhibited  copper-catalyzed oxidation of  ascorbate and  competed   effectively  with  histidine  and   1,10-phenanthroline  for  binding   of cuprous   copper,   but   not   cupric   copper,   as   demonstrated   by   UV-visible   and low-temperature electron spin  resonance techniques. We  conclude that the  protection by  DMSA   against  copper-mediated   oxidative   damage  to   DNA  is   through   the formation of a redox-inactive DMSA-cuprous complex.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34505
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Recommended Citation:
刘玉祥. 含巯基药物对氯代醌及铜诱导的毒性保护机制研究[D]. 北京. 中国科学院研究生院. 2015.
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