喹诺酮类药物氧氟沙星致关节软骨细胞氧化损伤及其作用机制
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摘要
喹诺酮类药物(Quinolones,QNs)是一类化学合成的抗菌药物,具有4-喹诺酮的基本结构,其抗菌机理是对细菌DNA回旋酶具有选择性的抑制作用。其具有抗菌谱广、活性强、与其它抗菌药较少产生交叉耐药性等优点,在不到半个世纪的时间里先后研发了4代产品。第4代产品氟喹诺酮类药物以其对革兰氏阴性菌和革兰氏阳性菌的双重杀菌活性在临床上广泛应用。但其不良反应如光毒性、心脏毒性、软骨毒性等限制了在不同人群的应用,尤其是对软骨的毒性作用越来越受到人们的关注。QNs致关节软骨损伤的毒性作用机制尚未阐明。本课题以原代培养幼龄兔关节软骨细胞为试验模型,以氧氟沙星为代表药物,研究探讨氧化应激在QNs致软骨细胞毒性损伤中的作用,重点研究细胞内氧化与抗氧化系统状态,以及对生物大分子的氧化损伤评价。为阐明其不良反应的确切机制,评价其在儿童中应用的安全性,指导对此类药物的结构改造以及毒性不良效应的防治等方面提供实验依据。
1氧氟沙星致兔关节软骨细胞的氧化损伤作用
培养幼龄兔关节软骨细胞,应用不同浓度氧氟沙星对软骨细胞作用不同时间,观察细胞形态变化,检测软骨细胞存活率及细胞乳酸脱氢酶(LDH)的露出率,评价氧氟沙星致软骨细胞毒性损伤特征。应用荧光探针法检测细胞内活性氧(ROS)的含量,采用Griss法检测细胞内一氧化氮(NO)的含量,评价氧氟沙星致软骨细胞的氧化状态。应用TBA比色法、彗星实验以及蛋白免疫印迹法检测软骨细胞内脂质过氧化产物MDA的含量,DNA损伤程度和蛋白氧化损伤产物蛋白羰基的含量,评价氧氟沙星对软骨细胞生物大分子的氧化损伤作用。为了评价氧氟沙星对软骨细胞抗氧化系统的影响,检测细胞内抗氧化物还原型谷胱甘肽(GSH)含量以及细胞内抗氧化酶的活性。应用巯基化合物GSH、N-乙酰半胱氨酸(NAC)和二硫苏糖醇(DTT),观察其对氧氟沙星致软骨细胞毒性作用的影响,以进一步阐明氧化应激在氧氟沙星致细胞毒性中的作用。研究结果表明:①氧氟沙星可引起关节软骨明显的细胞毒性,具体表现在氧氟沙星致细胞培养上清中LDH露出率增加,可浓度和时间依赖性地降低软骨细胞存活率。②氧氟沙星引起细胞内ROS过量的产生,并且呈现一定剂量效应关系,同时引起细胞内NO含量的升高;氧氟沙星引起软骨细胞MDA含量增加,软骨细胞DNA尾长、尾DNA百分含量和尾距明显增加,以及蛋白羰基含量增加。表明氧氟沙星通过引起软骨细胞ROS及活性氮的增加,引起软骨细胞膜脂质过氧化损伤,DNA损伤及蛋白质氧化损伤。③氧氟沙星引起软骨细胞GSH含量减少,同时引起细胞内主要抗氧化物酶如谷胱甘肽过氧化物酶(GSH-px)、超氧化物歧化酶(SOD)以及过氧化氢酶(CAT)活性的降低。提示氧氟沙星削弱软骨细胞抗氧化能力。④GSH、NAC和DTT均能明显抑制氧氟沙星引起细胞ROS的过量产生,减轻SOD活性的降低以及提高细胞存活率。证实氧化应激参与了氧氟沙星引起的软骨细胞损伤,氧化应激在氧氟沙星引起软骨细胞毒性中具有重要的作用。
2氧化应激在氧氟沙星致软骨细胞线粒体损伤及细胞凋亡中的作用
氧化应激与线粒体功能及细胞凋亡的关系密不可分。本文进一步研究氧化应激在氧氟沙星引起软骨线粒体损伤及细胞凋亡中的作用。采用荧光探针法检测软骨细胞凋亡,评价氧氟沙星致软骨细胞死亡的特征。采用荧光探针Fluo-3/AM检测细胞内Ca~(2+)含量,评价氧氟沙星对软骨细胞Ca~(2+)稳态的影响。用线粒体膜电位及线粒体通透性转换孔(PTP)的变化,评价氧氟沙星对线粒体功能的影响,探讨其在细胞凋亡中的作用。应用巯基化合物GSH和NAC,观察其对氧氟沙星致软骨细胞Ca~(2+)稳态变化、线粒体膜电位及细胞凋亡的影响,探讨氧化应激在氧氟沙星致软骨细胞线粒体损伤及细胞凋亡中的作用。结果表明:①氧氟沙星以浓度时间依赖的方式引起软骨细胞凋亡。②氧氟沙星增加胞内Ca~(2+)浓度,改变细胞内Ca~(2+)稳态。③氧氟沙星引起软骨细胞线粒体损伤,具体体现在氧氟沙星以浓度和时间依赖的方式致软骨细胞线粒体膜电位降低。线粒体通透性转变抑制剂环孢菌素(CyA)能够抑制氧氟沙星引起的细胞存活率的降低,提示线粒体通透性转变在氧氟沙星的软骨细胞中具有重要的作用。④抗氧化剂GSH和NAC可以使软骨细胞凋亡减少、胞内Ca~(2+)浓度降低以及线粒体膜电位升高。表明抗氧化剂对氧氟沙星造成软骨细胞损伤有保护作用,提示氧化应激与氧氟沙星引起软骨细胞线粒体损伤及细胞凋亡有关。
本研究的创新点在于阐明了氧氟沙星对软骨细胞损伤的某些机制,发现氧氟沙星对软骨细胞损伤的机制是由于氧氟沙星使细胞内ROS增加,脂质过氧化产物增加,引起细胞膜性结构破坏,蛋白质损伤,DNA断裂,导致细胞凋亡增加。同时氧氟沙星引起细胞内Ca~(2+)超载,线粒体膜电位降低和PTP的开放,也导致细胞凋亡增加。证实抗氧化剂可以抑制氧氟沙星所引起的软骨细胞损伤,保护软骨细胞的结构与功能。具体得出以下结论:①氧化损伤是氧氟沙星致关节软骨毒性的重要机制之一;②氧氟沙星所致软骨细胞氧化应激可引起软骨细胞内Ca~(2+)超载、线粒体膜电位降低和PTP开放;③细胞凋亡是氧氟沙星致软骨细胞损伤的重要表现形式;④抗氧化剂可有效预防氧氟沙星的软骨细胞毒性。
Quinolones (QNs) belong to chemosynthesis antibacterials, which possess the elementary structure of 4-quinolone, and can selectively inhibit the DNA gyrase of bacterium. Due to its broad antibacterial spectrum, good antibacterial activity, low incidence of cross tolerance with other antibacterial agents, convenient administration and so on,there are 4 generation of QNs to have been developed in the near 50 years. Recently, FQNs is being widely used clinically because of its double bactericidal activity to G~-and G~+. However, all kinds of drug adverse reaction (ADR) has been observed in clinical therapy of QNs, such as ADR in photoxicity, heart toxicity, chondrotoxicity and so on. Especially, people gradually pay close attention to the chondrotoxicity of QNs since accumulating evidences have demonstrated that QNs can generate toxicity on articular cartilage of juvenile animals,which have suggested that QNs possess the same toxicity on people’s articular cartilage. In addition, lots of clinical data have shown that QNs can cause swelling and pain of joint after QNs has been administrated to children, and injuries of chondrocytes is observed in aborted fetus. So, QNs has been restricted to the application to the children, teenagers, pregnancy woman and lactation women. But the mechanism of ofloxacin-induced chondrotoxicity is not clear, especially, ofloxacin-induced oxidative damage. In the present study, we use the typical QNs, ofloxacin as the objective drug and the juvenile rabbit articular chondrocytes as experimental model to investigate the possible mechanism of QNs chondrotoxicity focusing on oxidative damage, DNA and protein damage, mitochondrion and the apoptosis.
1 Ofloxacin induced oxidative damage to joint chondrotoxicity of juvenile rabbit
Chondrocytes from juvenile rabbit joint are incubated with ofloxacin at concentrations of 0, 5, 10, 20, 40 and 80μg/ml, respectively. Cell viability assay was performed essentially by a modified MTT assay, and activity of lactate dehydrogenase (LDH) was determined. The results showed ofloxacin decreases the viability of chondrocytes in a time and concentration-dependent manner. And ofloxacin can increase LDH activity in culture medium, and injury cell membrane resulted in LDH leakage. The extent of oxidative stress is assessed by measuring reactive oxygen species (ROS), nitrogen oxide (NO) and antioxidant defense system including reduced glutathione (GSH) and avtivity of antioxidant enzymes. It is observed that ofloxacin induced a concentration-dependent increase in intracellular ROS production, which may be an early mediator of ofloxacin cytotoxicity. Furthermore, antioxidant enzyme activities, such as glutathione peroxidase (GSH-px), catalase (CAT) and superoxide dismutase (SOD), were rapid decreased after treatment with ofloxacin.
In addition, SOD decline and ROS production were strongly inhibited, and the loss in cell viability was partly abated by additional glutathione (GSH), N-acetylcysteine (NAC) and dithiothreitol (DTT). In conclusion, these results clearly demonstrate that oxidative stress plays an important role in ofloxacin-induced injuries of Juvenile rabbit joint chondrocytes.
It is well known that ROS may injure intracellular biomacromolecule, such as lipid, DNA and protein. Use chromatometry of TBA, comet assay and western blot to measure the content of MDA, the extent of DNA damage and the content of protein carbonylation. It was observed that ofloxacin resulted in a significant lipid peroxidation, revealed by a concentration-dependent increase in the level of thiobarbituric acid reactive substances (TBARS). The study indicated that ofloxacin can increase tail length, tail DNA% and tail moment resulted in DNA damage. Western blot analysis succeeded to detect specifically increased protein carbonylation by ofloxacin in chondrocytes.
2 Role of oxidative stress in ofloxacin-induced mitochondrion damage and apoptosis to chondrocytes
Chondrocytes from juvenile rabbit joint are incubated with ofloxacin at concentrations of 0, 5, 10, 20, 40 and 80μg/ml, respectively. Use different fluorescent probe to measure concentration of Ca~(2+), transmembrane potential of mitochondrion and cell apoptosis. The study showed ofloxacin increased concentration of Ca~(2+), decreased transmembrane potential of mitochondrion, and induced cell apoptosis to chondrocytes. And we also found scavenger of ROS could inhibit mitochondrial injury and suppress ofloxacin-induced apoptosis. In addition, cyclosporine (Cys), inhibitor of PTP, could inhibit cytotoxicity by ofloxacin to chondrocytes.
The results indicated ofloxacin could induce increase of ROS, increase the level of MDA and damage cell membranc, protein and DNA, and resulted in cell apoptosis. Ofloxacin also induce cell apoptosis by increasing concentration of Ca~(2+), decreasing transmembrane potential of mitochondrion and opening PTP. In addition, the study showed antioxidant may inhibit ofloxacin-induced damage to chondrocytes.
In conclusion, oxidative damage is one of the important mechanism in ofloxacin-induced damage to chongdrocytes. Oxidative stress increase concentration of Ca~(2+), decrease transmembrane potential of mitochondrion and open PTP. Lipid peroxidation damage induce chondrocytes apoptosis and antioxidants may prevent the damage of chondrocytes by ofloxacin.
1氧氟沙星致兔关节软骨细胞的氧化损伤作用
培养幼龄兔关节软骨细胞,应用不同浓度氧氟沙星对软骨细胞作用不同时间,观察细胞形态变化,检测软骨细胞存活率及细胞乳酸脱氢酶(LDH)的露出率,评价氧氟沙星致软骨细胞毒性损伤特征。应用荧光探针法检测细胞内活性氧(ROS)的含量,采用Griss法检测细胞内一氧化氮(NO)的含量,评价氧氟沙星致软骨细胞的氧化状态。应用TBA比色法、彗星实验以及蛋白免疫印迹法检测软骨细胞内脂质过氧化产物MDA的含量,DNA损伤程度和蛋白氧化损伤产物蛋白羰基的含量,评价氧氟沙星对软骨细胞生物大分子的氧化损伤作用。为了评价氧氟沙星对软骨细胞抗氧化系统的影响,检测细胞内抗氧化物还原型谷胱甘肽(GSH)含量以及细胞内抗氧化酶的活性。应用巯基化合物GSH、N-乙酰半胱氨酸(NAC)和二硫苏糖醇(DTT),观察其对氧氟沙星致软骨细胞毒性作用的影响,以进一步阐明氧化应激在氧氟沙星致细胞毒性中的作用。研究结果表明:①氧氟沙星可引起关节软骨明显的细胞毒性,具体表现在氧氟沙星致细胞培养上清中LDH露出率增加,可浓度和时间依赖性地降低软骨细胞存活率。②氧氟沙星引起细胞内ROS过量的产生,并且呈现一定剂量效应关系,同时引起细胞内NO含量的升高;氧氟沙星引起软骨细胞MDA含量增加,软骨细胞DNA尾长、尾DNA百分含量和尾距明显增加,以及蛋白羰基含量增加。表明氧氟沙星通过引起软骨细胞ROS及活性氮的增加,引起软骨细胞膜脂质过氧化损伤,DNA损伤及蛋白质氧化损伤。③氧氟沙星引起软骨细胞GSH含量减少,同时引起细胞内主要抗氧化物酶如谷胱甘肽过氧化物酶(GSH-px)、超氧化物歧化酶(SOD)以及过氧化氢酶(CAT)活性的降低。提示氧氟沙星削弱软骨细胞抗氧化能力。④GSH、NAC和DTT均能明显抑制氧氟沙星引起细胞ROS的过量产生,减轻SOD活性的降低以及提高细胞存活率。证实氧化应激参与了氧氟沙星引起的软骨细胞损伤,氧化应激在氧氟沙星引起软骨细胞毒性中具有重要的作用。
2氧化应激在氧氟沙星致软骨细胞线粒体损伤及细胞凋亡中的作用
氧化应激与线粒体功能及细胞凋亡的关系密不可分。本文进一步研究氧化应激在氧氟沙星引起软骨线粒体损伤及细胞凋亡中的作用。采用荧光探针法检测软骨细胞凋亡,评价氧氟沙星致软骨细胞死亡的特征。采用荧光探针Fluo-3/AM检测细胞内Ca~(2+)含量,评价氧氟沙星对软骨细胞Ca~(2+)稳态的影响。用线粒体膜电位及线粒体通透性转换孔(PTP)的变化,评价氧氟沙星对线粒体功能的影响,探讨其在细胞凋亡中的作用。应用巯基化合物GSH和NAC,观察其对氧氟沙星致软骨细胞Ca~(2+)稳态变化、线粒体膜电位及细胞凋亡的影响,探讨氧化应激在氧氟沙星致软骨细胞线粒体损伤及细胞凋亡中的作用。结果表明:①氧氟沙星以浓度时间依赖的方式引起软骨细胞凋亡。②氧氟沙星增加胞内Ca~(2+)浓度,改变细胞内Ca~(2+)稳态。③氧氟沙星引起软骨细胞线粒体损伤,具体体现在氧氟沙星以浓度和时间依赖的方式致软骨细胞线粒体膜电位降低。线粒体通透性转变抑制剂环孢菌素(CyA)能够抑制氧氟沙星引起的细胞存活率的降低,提示线粒体通透性转变在氧氟沙星的软骨细胞中具有重要的作用。④抗氧化剂GSH和NAC可以使软骨细胞凋亡减少、胞内Ca~(2+)浓度降低以及线粒体膜电位升高。表明抗氧化剂对氧氟沙星造成软骨细胞损伤有保护作用,提示氧化应激与氧氟沙星引起软骨细胞线粒体损伤及细胞凋亡有关。
本研究的创新点在于阐明了氧氟沙星对软骨细胞损伤的某些机制,发现氧氟沙星对软骨细胞损伤的机制是由于氧氟沙星使细胞内ROS增加,脂质过氧化产物增加,引起细胞膜性结构破坏,蛋白质损伤,DNA断裂,导致细胞凋亡增加。同时氧氟沙星引起细胞内Ca~(2+)超载,线粒体膜电位降低和PTP的开放,也导致细胞凋亡增加。证实抗氧化剂可以抑制氧氟沙星所引起的软骨细胞损伤,保护软骨细胞的结构与功能。具体得出以下结论:①氧化损伤是氧氟沙星致关节软骨毒性的重要机制之一;②氧氟沙星所致软骨细胞氧化应激可引起软骨细胞内Ca~(2+)超载、线粒体膜电位降低和PTP开放;③细胞凋亡是氧氟沙星致软骨细胞损伤的重要表现形式;④抗氧化剂可有效预防氧氟沙星的软骨细胞毒性。
Quinolones (QNs) belong to chemosynthesis antibacterials, which possess the elementary structure of 4-quinolone, and can selectively inhibit the DNA gyrase of bacterium. Due to its broad antibacterial spectrum, good antibacterial activity, low incidence of cross tolerance with other antibacterial agents, convenient administration and so on,there are 4 generation of QNs to have been developed in the near 50 years. Recently, FQNs is being widely used clinically because of its double bactericidal activity to G~-and G~+. However, all kinds of drug adverse reaction (ADR) has been observed in clinical therapy of QNs, such as ADR in photoxicity, heart toxicity, chondrotoxicity and so on. Especially, people gradually pay close attention to the chondrotoxicity of QNs since accumulating evidences have demonstrated that QNs can generate toxicity on articular cartilage of juvenile animals,which have suggested that QNs possess the same toxicity on people’s articular cartilage. In addition, lots of clinical data have shown that QNs can cause swelling and pain of joint after QNs has been administrated to children, and injuries of chondrocytes is observed in aborted fetus. So, QNs has been restricted to the application to the children, teenagers, pregnancy woman and lactation women. But the mechanism of ofloxacin-induced chondrotoxicity is not clear, especially, ofloxacin-induced oxidative damage. In the present study, we use the typical QNs, ofloxacin as the objective drug and the juvenile rabbit articular chondrocytes as experimental model to investigate the possible mechanism of QNs chondrotoxicity focusing on oxidative damage, DNA and protein damage, mitochondrion and the apoptosis.
1 Ofloxacin induced oxidative damage to joint chondrotoxicity of juvenile rabbit
Chondrocytes from juvenile rabbit joint are incubated with ofloxacin at concentrations of 0, 5, 10, 20, 40 and 80μg/ml, respectively. Cell viability assay was performed essentially by a modified MTT assay, and activity of lactate dehydrogenase (LDH) was determined. The results showed ofloxacin decreases the viability of chondrocytes in a time and concentration-dependent manner. And ofloxacin can increase LDH activity in culture medium, and injury cell membrane resulted in LDH leakage. The extent of oxidative stress is assessed by measuring reactive oxygen species (ROS), nitrogen oxide (NO) and antioxidant defense system including reduced glutathione (GSH) and avtivity of antioxidant enzymes. It is observed that ofloxacin induced a concentration-dependent increase in intracellular ROS production, which may be an early mediator of ofloxacin cytotoxicity. Furthermore, antioxidant enzyme activities, such as glutathione peroxidase (GSH-px), catalase (CAT) and superoxide dismutase (SOD), were rapid decreased after treatment with ofloxacin.
In addition, SOD decline and ROS production were strongly inhibited, and the loss in cell viability was partly abated by additional glutathione (GSH), N-acetylcysteine (NAC) and dithiothreitol (DTT). In conclusion, these results clearly demonstrate that oxidative stress plays an important role in ofloxacin-induced injuries of Juvenile rabbit joint chondrocytes.
It is well known that ROS may injure intracellular biomacromolecule, such as lipid, DNA and protein. Use chromatometry of TBA, comet assay and western blot to measure the content of MDA, the extent of DNA damage and the content of protein carbonylation. It was observed that ofloxacin resulted in a significant lipid peroxidation, revealed by a concentration-dependent increase in the level of thiobarbituric acid reactive substances (TBARS). The study indicated that ofloxacin can increase tail length, tail DNA% and tail moment resulted in DNA damage. Western blot analysis succeeded to detect specifically increased protein carbonylation by ofloxacin in chondrocytes.
2 Role of oxidative stress in ofloxacin-induced mitochondrion damage and apoptosis to chondrocytes
Chondrocytes from juvenile rabbit joint are incubated with ofloxacin at concentrations of 0, 5, 10, 20, 40 and 80μg/ml, respectively. Use different fluorescent probe to measure concentration of Ca~(2+), transmembrane potential of mitochondrion and cell apoptosis. The study showed ofloxacin increased concentration of Ca~(2+), decreased transmembrane potential of mitochondrion, and induced cell apoptosis to chondrocytes. And we also found scavenger of ROS could inhibit mitochondrial injury and suppress ofloxacin-induced apoptosis. In addition, cyclosporine (Cys), inhibitor of PTP, could inhibit cytotoxicity by ofloxacin to chondrocytes.
The results indicated ofloxacin could induce increase of ROS, increase the level of MDA and damage cell membranc, protein and DNA, and resulted in cell apoptosis. Ofloxacin also induce cell apoptosis by increasing concentration of Ca~(2+), decreasing transmembrane potential of mitochondrion and opening PTP. In addition, the study showed antioxidant may inhibit ofloxacin-induced damage to chondrocytes.
In conclusion, oxidative damage is one of the important mechanism in ofloxacin-induced damage to chongdrocytes. Oxidative stress increase concentration of Ca~(2+), decrease transmembrane potential of mitochondrion and open PTP. Lipid peroxidation damage induce chondrocytes apoptosis and antioxidants may prevent the damage of chondrocytes by ofloxacin.
引文
[1] Matsuda, A., Sugiyama M., Kitahara, S., Yoneyama, S., Kanoh, M. Safety evaluation of norfloxacin: subacute toxicity study in infant beagles. Pharmacometrics, 1991, 42: 1–19. (In Japanese.).
[2] Bouissou, H., Caujolle D., Caujolle, F., Milhaud, G. Cartilaginous tissue and nalidixicacide. C. R. Acad. Sci. Hebd Seances Acad. Sci, 1978, 286: 1743–1746.
[3] Bendele, A. M., Hulman, J. F., Harvey, A. K., Hrubey, P. S., Chandrasekhar S. Passive role of articular chondrocytes in quinolone-induced arthropathy in guinea pigs. Toxicol. Pathol, 1990, 18: 304–312.
[4] Gough, A. W., Barsoum, N. J., Mitchell, L., McGuire, E. J., De La Iglesia, F. A. Juvenile canine drug-induced arthropathy: clinicopathological studies on articular lesions caused by oxolinic and pipemidic acids. Toxicol. Appl. Pharmacol, 1979, 51: 177–187.
[5] Egerbacher M, Seiberl G, Wolfesberger B,et al. Ciprofloxacin causes cytoskeletal changes and detachment of human and rat chondrocytes in vitro. Arch Toxicol, 2000, 73(10-11): 557;
[6] Hayem, G., Domarle, O., Fay, M., Thuong-Guyot M., Pocidalo J. J., Carbon C. Lack of correlation between hydrogen peroxide production and nitric oxide production by cultured rabbit articular chondrocytes treated with fluoroquinolone antimicrobial agent. Toxicol. in vitro, 1996, 10: 515-555.
[7] Fridovich I. Fundamental aspects of reactive oxygen species, or what’s the matter with oxygen? Ann N Y Acad Sci, 1999, 893: 13-18.
[8] Yabe K., Satoh H., Ishii Y., Jindo T., Sugawara T., Furuhama K., Gorya M., Okada K. Early pathophysiologic feature of arthropathy in juvenile dogs induced by ofloxacin, a quinolone antimicrobial agent. Vet Pathol, 2004, 41:673–681.
[9] Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 1983, 5: 55–63.
[10] Wang Y. M., Peng S. Q., Zhou Q., Wang M. W., Yan C. H., Yang H. Y. , Wang G. Q. Depletion of intracellular glutathione mediates butenolide-induced cytotoxicity in HepG2 cells. Toxicol. Lett, 2006, 164: 231–238.
[11] Han SH, Espinoza LA, Liao HL, Boulares AH, Smulson ME. Protection by antioxidants against toxicity and apoptosis induced by the sulphur mustard analog 2-chloroethylethyl sulphide (CEES) in Jurkat T cells and normal human lymphocytes. Br J Pharmacol, 2004, 141: 795-802.
[12] Spitz D. R, Oberley LW. An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal. Biochem, 1989, 179: 8-18.
[13] Paglia, D.E., Valentine, W. N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med, 1967, 70: 158–70.
[14] Massod M. A. B., Abbas S. On the cytotoxicity and status of oxidative stress of two novel synthesized tri-aza macrocyclic diamides as studied in the V79 cell line. Bioorg. Med. Chem, 2007, 15: 3437–3444.
[15] Esterbauer, H., Cheeseman, K. H. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol, 1990, 186: 407–421.
[16] Singh, N.P., McKoy, M.T., Tice, R.R., Schneider, E.L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res, 1988, 175: 184–191.
[17] Liang-Jun Yan, William C. Orr, and Rajindar S. Sohal. Identification of Oxidized Proteins Based on Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis, Immunochemical Detection, Isoelectric Focusing, and Microsequencing. Analytical Biochemistry, 1998, 263: 67–71.
[18] Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem, 1951, 193: 265–275.
[19] Takizawa T, Hasimoto K, Itoh N, et al. Acomparativestudy of the repeat does toxicity of grepafloxacin and a number of other fluoroquinolones in rats. Hum Exp Toxicol, 1999, 18(1): 38.
[20] Simonin, M. A. S., Pascale, G. P., Minn A., Gillet P., Netter P., Terlain B. Proteoglyan and Collagen Biochemical Variations during Fluoroquinolone-Induced Chondrotoxicity in Mice,Dec. Antimicrob Agents Chemoth, 1999, 43: 2915–2921.
[21] Thuong-Guyot. M., Domarle O., Pocidalo J. J., and Hayem G. Effects of fluoroquinolones on cultured articular chondrocytes flow cytometric analysis of free radical production. J. Pharmacol. Exp. Ther, 1994, 271, 1544–1549.
[22] Bergamini, C.M., Gambetti, S., Dondi, A., Cervellati, C. Oxygen, reactive oxygen species and tissue damage. Curr. Pharm. Des, 2004, 10: 1611–1626.
[23] Petersen DR, Doorn JA. Reactions of 4-hydroxylnonenal with proteins and cellular targets. Free Radic Biol Med, 2004, 37 (7): 937-945.
[24] Uchida K, Kanematsu M, Sakai K, Matsuda T, Hattori N, Mizuno Y, Suzuki D, Miyata T, Noguchi N, Niki E, Osawa T. Protein-bound acrolein: potential markers for oxidative stress. Proc Natl Acad Sci USA, 1998, 95 (8): 4882-4887.
[25] Uchida K. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog Lipid Res, 2003, 42 (4): 318-343.
[26] Stark G. Functional consequences of oxidative membrane damage. J Membrane Biol, 2005, 205: 1-16.
[27] Del Rio D, Stewart AJ, Pellegrini N. A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr Metab Cardiovasc Dis, 2005, 15 (4): 316-328.
[28] 文镜, 李晶洁, 郭豫等. 用蛋白质羰基含量评价抗氧化保健食品的研究[J] . 中国食品卫生杂志, 2002 ,14 :13 - 17.
[29] T. Akasaka, S. Kurosaka, Y. Uchida, M. Tanaka, K. Sato, I. Hayakawa, Antibacterial activities and inhibitory effects of sitafloxacin (DU-6859a) and its optical isomers against type II topoisomerases, Antimicrob. Agents Chemother. 1998, 42: 1284–1287.
[30] Q. Liu, J.C. Wang, Similarity in the catalysis of DNA breakage and rejoining by type IA and IIA DNA topoisomerases, Proc. Natl. Acad. Sci. U.S.A. 1999, 96: 881–886.
[31] M. Fukuda, M. Inomata, K. Nishio, K. Fukuoka, F. Kanzawa, H. Arioka, T. Ishida, H. Fukumoto, H. Kurokawa,M. Oka, N. Saijo, A topoisomerase II inhibitor, NK109, induces DNA single- and double-strand breaks and apoptosis, Jpn. J. Cancer Res. 1996, 87: 1086–1091.
[32] Fridovich I. Superoxide radical and superoxide dismutases. Ann Rev Biochem, 1995, 64: 97-112.
[33] Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev, 1979, 59: 527-605.
[34] Masella R, Benedetto RD, Varì R, Filesi C, Giovannini C. Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione-related enzymes. J Nutr Biochem, 2005, 16: 577-586.
[35] Sies H. Glutathione and its role in cellular functions. Free Radic Biol Med, 1999, 27: 916-921.
[36] Armstrong, RN. Structure, catalytic mechanism, and evolution of the glutathione transferases. Chem Res Toxicol, 1997, 10: 2-18.
[37] van Bladeren PJ. Glutathione conjugation as a bioactivation reaction. Chem Biol Interact, 2000, 129: 61-76.
[38] V.L. Gabai, A.B. Meriin, J.A. Yaglom, V.Z. Volloch, M.Y. Sherman. Role of Hsp70 in regulation of stress-kinase JNK: implications in apoptosis and aging, FEBS Lett, 1998, 438: 1-4.
[39] Geske, F.J., Gerschenson, L.E. The biology of apoptosis. Hum. Pathol, 2001, 32: 1029–1038.
[40] 刘戎,宣昭林。活性氧、线粒体与细胞凋亡[J]。中国地方病防治杂志, 2005,15:285-288
[41] Ma L.X., Huang Y.H., Cheng L.M., Lei J. and Wang Q.R. Inducing effects of macrophagestimulating protein on the expansion of early hematopoietic progenitor cells in liquid culture, Chin Med J, 2007, 120(13):1192-1197.
[42] Lefeuvre M, Amjaad W, Goldberg M, Stanislawaski L. TEGDMA induces mitochondrial damage and oxidative stress in human gingival fibroblasts. Biomaterials, 2005, 26: 5130-5137.
[43] Ribeiro, C.; Vignes, M.; Brehelin, M. Xenorhabdus nematophila (enterobacteriacea) secretes a cation-selective calcium-independent porin which causes vacuolation of the rough endoplasmic reticulum and celllysis. J. Biol. Chem, 2003, 278:3030–3039.
[44] J Kslle DJ,Sarfian TA,Hahn H,et a1.Bcl一2 inhibition of neural death:decreased generation of reactive oxygen species.Science, 1993, 262(5137):1274—1277
[45] Batandier C, Fontaine E, Keriel C, Leverve XM. Determination of mitochondrial reactive oxygen species: methodological aspects. J Cell Mol Med, 2002, 6 (2): 175-187.
[46] Compton M, Virji S, Ward JM. Cyclophlin-D binds strongly to complexes of the voltage-dependent anion channel and the adeninine nucleotide translocase to form the permeability transition pore. Eur J Biochem, 1998, 258: 729-735.
[47] Zoratti M, Szabo I. The mitochondria permeability pore. Biochim Biophys Acta, 1995, 1241 (1): 139-176.
[48] Kowaltowski AJ, Castilho RF, Vercesi AE. Mitochondrial permeability transition and oxidative stress. FEBS Lett, 2001, 495 (1-2): 12-15.
[49] Jae SK, Lihua H, John JL. Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochem Biophys Res Commun, 2003, 304: 463-470.
[50] Halestrap AP. The mitochondrial permeability transition: a pore way to die. J Clin Basic Cardiol, 2002, 5: 29-41.
[51] Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, Crowe RA, Cascio WE, Bradham CA, Brenner DA, Herman B. The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta, 2002, 1366: 177-196.
[52] Kowaltowski AJ, Vercesi AE. Mitochondrial damage induced by conditions of oxidative stress. Free Radic Biol Med, 1999, 26 (3-4): 463-471.
[53] Ding WX, Shen HM, Ong CN. Critical role of reactive oxygen species and mitochondria permeability transition in microcystin-induced rapid apoptosis in rat hepatocytes. Hepatology, 2000, 32 (3): 547-555.
[54] Shirlee Tan,Tutaka Sash ,Yuanbin Liu,et a1. Regulation of reactive oxygen species production during programmed ceel death.J cell Biol, 1998, 141(6):1423 1432.
[55] MaconkeyDJ,Orrenius S. role of calcium in the regulation of apoptosis.Biolchem Biophys Res Commun,1997, 239: 357—366.
[2] Bouissou, H., Caujolle D., Caujolle, F., Milhaud, G. Cartilaginous tissue and nalidixicacide. C. R. Acad. Sci. Hebd Seances Acad. Sci, 1978, 286: 1743–1746.
[3] Bendele, A. M., Hulman, J. F., Harvey, A. K., Hrubey, P. S., Chandrasekhar S. Passive role of articular chondrocytes in quinolone-induced arthropathy in guinea pigs. Toxicol. Pathol, 1990, 18: 304–312.
[4] Gough, A. W., Barsoum, N. J., Mitchell, L., McGuire, E. J., De La Iglesia, F. A. Juvenile canine drug-induced arthropathy: clinicopathological studies on articular lesions caused by oxolinic and pipemidic acids. Toxicol. Appl. Pharmacol, 1979, 51: 177–187.
[5] Egerbacher M, Seiberl G, Wolfesberger B,et al. Ciprofloxacin causes cytoskeletal changes and detachment of human and rat chondrocytes in vitro. Arch Toxicol, 2000, 73(10-11): 557;
[6] Hayem, G., Domarle, O., Fay, M., Thuong-Guyot M., Pocidalo J. J., Carbon C. Lack of correlation between hydrogen peroxide production and nitric oxide production by cultured rabbit articular chondrocytes treated with fluoroquinolone antimicrobial agent. Toxicol. in vitro, 1996, 10: 515-555.
[7] Fridovich I. Fundamental aspects of reactive oxygen species, or what’s the matter with oxygen? Ann N Y Acad Sci, 1999, 893: 13-18.
[8] Yabe K., Satoh H., Ishii Y., Jindo T., Sugawara T., Furuhama K., Gorya M., Okada K. Early pathophysiologic feature of arthropathy in juvenile dogs induced by ofloxacin, a quinolone antimicrobial agent. Vet Pathol, 2004, 41:673–681.
[9] Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 1983, 5: 55–63.
[10] Wang Y. M., Peng S. Q., Zhou Q., Wang M. W., Yan C. H., Yang H. Y. , Wang G. Q. Depletion of intracellular glutathione mediates butenolide-induced cytotoxicity in HepG2 cells. Toxicol. Lett, 2006, 164: 231–238.
[11] Han SH, Espinoza LA, Liao HL, Boulares AH, Smulson ME. Protection by antioxidants against toxicity and apoptosis induced by the sulphur mustard analog 2-chloroethylethyl sulphide (CEES) in Jurkat T cells and normal human lymphocytes. Br J Pharmacol, 2004, 141: 795-802.
[12] Spitz D. R, Oberley LW. An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal. Biochem, 1989, 179: 8-18.
[13] Paglia, D.E., Valentine, W. N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med, 1967, 70: 158–70.
[14] Massod M. A. B., Abbas S. On the cytotoxicity and status of oxidative stress of two novel synthesized tri-aza macrocyclic diamides as studied in the V79 cell line. Bioorg. Med. Chem, 2007, 15: 3437–3444.
[15] Esterbauer, H., Cheeseman, K. H. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol, 1990, 186: 407–421.
[16] Singh, N.P., McKoy, M.T., Tice, R.R., Schneider, E.L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res, 1988, 175: 184–191.
[17] Liang-Jun Yan, William C. Orr, and Rajindar S. Sohal. Identification of Oxidized Proteins Based on Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis, Immunochemical Detection, Isoelectric Focusing, and Microsequencing. Analytical Biochemistry, 1998, 263: 67–71.
[18] Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem, 1951, 193: 265–275.
[19] Takizawa T, Hasimoto K, Itoh N, et al. Acomparativestudy of the repeat does toxicity of grepafloxacin and a number of other fluoroquinolones in rats. Hum Exp Toxicol, 1999, 18(1): 38.
[20] Simonin, M. A. S., Pascale, G. P., Minn A., Gillet P., Netter P., Terlain B. Proteoglyan and Collagen Biochemical Variations during Fluoroquinolone-Induced Chondrotoxicity in Mice,Dec. Antimicrob Agents Chemoth, 1999, 43: 2915–2921.
[21] Thuong-Guyot. M., Domarle O., Pocidalo J. J., and Hayem G. Effects of fluoroquinolones on cultured articular chondrocytes flow cytometric analysis of free radical production. J. Pharmacol. Exp. Ther, 1994, 271, 1544–1549.
[22] Bergamini, C.M., Gambetti, S., Dondi, A., Cervellati, C. Oxygen, reactive oxygen species and tissue damage. Curr. Pharm. Des, 2004, 10: 1611–1626.
[23] Petersen DR, Doorn JA. Reactions of 4-hydroxylnonenal with proteins and cellular targets. Free Radic Biol Med, 2004, 37 (7): 937-945.
[24] Uchida K, Kanematsu M, Sakai K, Matsuda T, Hattori N, Mizuno Y, Suzuki D, Miyata T, Noguchi N, Niki E, Osawa T. Protein-bound acrolein: potential markers for oxidative stress. Proc Natl Acad Sci USA, 1998, 95 (8): 4882-4887.
[25] Uchida K. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog Lipid Res, 2003, 42 (4): 318-343.
[26] Stark G. Functional consequences of oxidative membrane damage. J Membrane Biol, 2005, 205: 1-16.
[27] Del Rio D, Stewart AJ, Pellegrini N. A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr Metab Cardiovasc Dis, 2005, 15 (4): 316-328.
[28] 文镜, 李晶洁, 郭豫等. 用蛋白质羰基含量评价抗氧化保健食品的研究[J] . 中国食品卫生杂志, 2002 ,14 :13 - 17.
[29] T. Akasaka, S. Kurosaka, Y. Uchida, M. Tanaka, K. Sato, I. Hayakawa, Antibacterial activities and inhibitory effects of sitafloxacin (DU-6859a) and its optical isomers against type II topoisomerases, Antimicrob. Agents Chemother. 1998, 42: 1284–1287.
[30] Q. Liu, J.C. Wang, Similarity in the catalysis of DNA breakage and rejoining by type IA and IIA DNA topoisomerases, Proc. Natl. Acad. Sci. U.S.A. 1999, 96: 881–886.
[31] M. Fukuda, M. Inomata, K. Nishio, K. Fukuoka, F. Kanzawa, H. Arioka, T. Ishida, H. Fukumoto, H. Kurokawa,M. Oka, N. Saijo, A topoisomerase II inhibitor, NK109, induces DNA single- and double-strand breaks and apoptosis, Jpn. J. Cancer Res. 1996, 87: 1086–1091.
[32] Fridovich I. Superoxide radical and superoxide dismutases. Ann Rev Biochem, 1995, 64: 97-112.
[33] Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev, 1979, 59: 527-605.
[34] Masella R, Benedetto RD, Varì R, Filesi C, Giovannini C. Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione-related enzymes. J Nutr Biochem, 2005, 16: 577-586.
[35] Sies H. Glutathione and its role in cellular functions. Free Radic Biol Med, 1999, 27: 916-921.
[36] Armstrong, RN. Structure, catalytic mechanism, and evolution of the glutathione transferases. Chem Res Toxicol, 1997, 10: 2-18.
[37] van Bladeren PJ. Glutathione conjugation as a bioactivation reaction. Chem Biol Interact, 2000, 129: 61-76.
[38] V.L. Gabai, A.B. Meriin, J.A. Yaglom, V.Z. Volloch, M.Y. Sherman. Role of Hsp70 in regulation of stress-kinase JNK: implications in apoptosis and aging, FEBS Lett, 1998, 438: 1-4.
[39] Geske, F.J., Gerschenson, L.E. The biology of apoptosis. Hum. Pathol, 2001, 32: 1029–1038.
[40] 刘戎,宣昭林。活性氧、线粒体与细胞凋亡[J]。中国地方病防治杂志, 2005,15:285-288
[41] Ma L.X., Huang Y.H., Cheng L.M., Lei J. and Wang Q.R. Inducing effects of macrophagestimulating protein on the expansion of early hematopoietic progenitor cells in liquid culture, Chin Med J, 2007, 120(13):1192-1197.
[42] Lefeuvre M, Amjaad W, Goldberg M, Stanislawaski L. TEGDMA induces mitochondrial damage and oxidative stress in human gingival fibroblasts. Biomaterials, 2005, 26: 5130-5137.
[43] Ribeiro, C.; Vignes, M.; Brehelin, M. Xenorhabdus nematophila (enterobacteriacea) secretes a cation-selective calcium-independent porin which causes vacuolation of the rough endoplasmic reticulum and celllysis. J. Biol. Chem, 2003, 278:3030–3039.
[44] J Kslle DJ,Sarfian TA,Hahn H,et a1.Bcl一2 inhibition of neural death:decreased generation of reactive oxygen species.Science, 1993, 262(5137):1274—1277
[45] Batandier C, Fontaine E, Keriel C, Leverve XM. Determination of mitochondrial reactive oxygen species: methodological aspects. J Cell Mol Med, 2002, 6 (2): 175-187.
[46] Compton M, Virji S, Ward JM. Cyclophlin-D binds strongly to complexes of the voltage-dependent anion channel and the adeninine nucleotide translocase to form the permeability transition pore. Eur J Biochem, 1998, 258: 729-735.
[47] Zoratti M, Szabo I. The mitochondria permeability pore. Biochim Biophys Acta, 1995, 1241 (1): 139-176.
[48] Kowaltowski AJ, Castilho RF, Vercesi AE. Mitochondrial permeability transition and oxidative stress. FEBS Lett, 2001, 495 (1-2): 12-15.
[49] Jae SK, Lihua H, John JL. Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochem Biophys Res Commun, 2003, 304: 463-470.
[50] Halestrap AP. The mitochondrial permeability transition: a pore way to die. J Clin Basic Cardiol, 2002, 5: 29-41.
[51] Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, Crowe RA, Cascio WE, Bradham CA, Brenner DA, Herman B. The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta, 2002, 1366: 177-196.
[52] Kowaltowski AJ, Vercesi AE. Mitochondrial damage induced by conditions of oxidative stress. Free Radic Biol Med, 1999, 26 (3-4): 463-471.
[53] Ding WX, Shen HM, Ong CN. Critical role of reactive oxygen species and mitochondria permeability transition in microcystin-induced rapid apoptosis in rat hepatocytes. Hepatology, 2000, 32 (3): 547-555.
[54] Shirlee Tan,Tutaka Sash ,Yuanbin Liu,et a1. Regulation of reactive oxygen species production during programmed ceel death.J cell Biol, 1998, 141(6):1423 1432.
[55] MaconkeyDJ,Orrenius S. role of calcium in the regulation of apoptosis.Biolchem Biophys Res Commun,1997, 239: 357—366.