Oxidative Damage to DNA and Its Relationship With Diabetic Complications
紫外导致的DNA损伤与修复 论文
紫外导致的DNA损伤与修复论文由于臭氧层的损耗到达地球表面的紫外辐射日渐增加。
而关于增加的紫外辐射对生物体的各种作用机制已引起了研究者的极大兴趣。
而DNA在从细菌到人的各种生物体中无疑是紫外线导致的损伤的重要目标。
紫外辐射能够导致两种常见的，具有遗传与细胞双重毒性的DNA损伤：环丁烷-嘧啶二聚体（CPD）和6-4光产物（6-4PP）及其杜瓦异构体。
但是，在长期的进化中，细胞已经进化出了很多修复或耐受机制来抵抗由紫外线及其他因素导致的DNA损伤。
在光复活酶的帮助下进行的光复活作用是很多生物体中最重要和常见的修复机制。
切除修复，它可以划分成碱基切除修复（BER）与核苷酸切除(NER)修复，在一些生物体中也是一种重要的修复途径。
这种途径分别是由一些糖基化酶和聚合酶催化的。
此外，像二聚体旁路，重组修复等其它一些修复途径也在不同的生物体中发挥着作用。
本综述讨论了紫外线导致的DNA损伤及相关的修复途径，并对未来的研究工作做了展望。
紫外线DNA损伤；光复原作用；切除修复；损伤旁路1DNA损伤（DNA damage）是指在外界因素作用下，DNA的结构发生变化，DNA损伤可以通过转录，翻译的产物——RNA与蛋白质来作用于细胞,并影响其代谢。
DNA 损伤可分为两大类型： 1.内源性损伤，如被正常代谢的副产物活性氧物种攻击导致的损伤（自发突变）； 2.外源性损伤，由外部因素引起，例如：（1）来自太阳的紫外射线； (2）其他频率的辐射，包括X射线和γ射线；(3)水解和热解； (4)某些植物毒素； (5)人造的突变物质，如吸烟产生的某些烃等。
如果这些损伤得不到修复，细胞的正常生命活动就会受到破坏，由细胞构成的生命体就会产生突变，并会由此而诱发一系列的遗传疾病。
为了防止DNA损伤而造成突变，生物体的细胞通过自身的DNA修复系统对其进行自我修复——DNA修复（DNA repair）以维持自身遗传的稳定性。
每个正常生物体内都存在着能―医治‖DNA损伤的复杂的修复系统，它们就像一个受过良好训练的维修小组，不停地对受损DNA进行着修复。
精子DNA损伤的研究进展
•488Chin. J Obstet Gynecol Pediatr (Electron. Ed) ^August 2018»V〇1. 14»No. 4•综述•精子DNA损伤的研究进展于鲁华1许琳1张晓梅2【摘要】随着男性不育症发病率的升高，精液常规检测已不足以精确评估男性生育力。
近年来，精子D N A损伤检测作为一项评估精子质量及男性生育力更精准的指标，逐步成为生殖医学领域的研究热点。
精子D N A损伤的机制包括精子发生异常、氧化应激损伤、精子凋亡异常等。
目前，精子染色质结构分析法（S C SA)已经成为检测精子D N A完整性的金标准。
精子D N A损伤可能与男性不育、辅助生殖结局及后代生长、发育相关。
笔者拟就精子D N A损伤的机制、检测方法、对男性生育力的影响及其与辅助生殖技术的关系进行综述，旨在为男性不育症的临床诊断提供依据-【关键词】D N A损伤；不育；精子发生；精液分析（生殖技术，辅助（男（雄）性Research progress of sperm DNA damage Y u L u h u a1，X u L in1, Zhang X iaom ei2. D epartm ent Obstetrics a n d G yn ec o lo g y，D alianM edica l U n iversity，D alian 111044，Liaoning P ro vince，China (2 R eproductive M edicine C enter，N orthern Jiangsu P eople’s H o sp ita l，Yangzhou 223QQ1，JiangsuProvince , ChinaCorresponding author：Z h a n g X ia o m ii，E m ail •.zh ang xiao m eizy@11'. com【Abstract】W ith the increasing incidenceofm ale infertility，sem en analysis is not sufficient toevaluate male infertility accurately. In recent y ears，sperm DNA damage as a more accurateassessm ent of sperm quality and male fertility index, has gradually become a research hotpot inreproductive medicine. T he m echanism of sperm D NA damage includes abnorm al sperm atogenesis，oxidative stress dam age, abnorm al sperm apoptosis, etc. . C urrently, sperm chrom atin structure assayhas been considered as the gold standard for detecting sperm DNA integrity. Sperm DNA be associated w ith m ale infertility，assisted reproductive outcom es ?and grow th and developm ent ofoffspring. This article mainly reviews the m echanism of sperm D NA dam age，detection m ethod，influence on the male fertility and assisted reproductive technology , so as to provide the basis forclinical diagnosis to male infertility.【K eyw ords】DNA dam age; Infertility；Sperm atogenesis; Semen analysis ；Reproductivetechniques，assisted ；MaleFund program s：N ational N atural Science Foundation of China (81100421); Six T alents PeakProject of Jiangsu Province (2014-W SW-080 ); N atural Science Foundation of Yangzhou City(YZ2014050)在不孕不育的病因中，既往研究更多关注女性因素，但是导致不孕不育的病因中约50%为男性因素，并且约15% 男性不育患者的精液常规检查结果显示参数正常[1],这为男性生育力的评估造成了困扰。
《中国老年学杂志》被国际数家数据库、检索性期刊检索机构收录情况
的新作用，该信号传导途径可以保护沉默基因组区域，并能维持基因组的稳定性〔17〕。
研究发现，wnt 信号传导参与睾丸减数分裂前未分化精原细胞的扩增〔18〕。
wnt2b 的过表达即可导致组织畸形生长，这主要是通过抑制细胞的周期和分化实现的〔19〕。
本研究发现了松果菊苷可以通过提高wnt2b 甲基化水平，改变Leydig 细胞的衰老状态。
4参考文献1王玉娟.表观遗传基因对衰老大鼠睾丸组织退行性变的作用及何首乌饮干预机制〔D 〕．保定：河北大学，2016.2韩飞，周孟良.过氧化氢诱导HepG2细胞产生氧化应激细胞模型的建立〔J 〕．食品科学，2011；32（5）：55-7.3李朝晖，王芬，刘水平，等.过氧化氢诱导PCI2细胞损伤的模型的建立〔J 〕．法医学杂志，2007；23（3）：191-2.4da Silva ＲA ，Mariano FS ，Planello AC.HaCaT anchorage blockade leads to oxidative stress ，DNA damage and DNA methylation changes 〔J 〕．Biochem Biophys Ｒeports ，2015；（2）：94-102.5Wang Y ，Wu W ，Yao C.Elevated tissue cr levels ，increased plasma oxidative markers ，and global hypomethylation of blood DNA in male sprague-dawley rats exposed to potassium dichromate in drinking water 〔M 〕．Wiley Periodicals ，Inc．2015：1-11.6Sasaki M ，Kajiya H ，Ozeki S.Ｒeactive oxygen species promotes cellu-lar senescence in normal human epidermal keratinocytes through epi-genetic regulation of p16INK4a 〔J 〕．Biochem Biophys Ｒes Commun ，2014；452：622-8.7Ferioli M ，Zauli G ，Maiorano P ，et al ．Ｒole of physical exercise in the regulation of epigenetic mechanisms in inflammation ，cancer ，neurode-generative diseases ，and aging process 〔J 〕．J Cell Physiol ，2019：epub．8孙静.何首乌饮及其单味药对大鼠Leydig 细胞衰老的表观遗传基因调控作用〔D 〕．保定：河北大学，2018.9Bormann F ，Ｒodríguez-Paredes M ，Hagemann S ，et al ．Ｒeduced DNAmethylation patterning and transcriptional connectivity define human skin aging 〔J 〕．Aging Cell ，2016；15：563-71.10Zampieri M ，Ciccarone F ，Calabrese Ｒ，et al ．Ｒeconfiguration of DNA methylation in aging 〔J 〕．Mech Ageing Dev ，2015；151：60-70.11Heidelbaugh JJ.Management of erectile dysfunction 〔J 〕．Am Fam Physician ，2010；81（3）：305-12.12Chen FF ，Song FQ ，Chen YQ ，et al ．Exogenous testosterone alleviates cardiac fibrosis and apoptosis via Gas6/Axl pathway in the senescent mice 〔J 〕．Exp Gerontol ，2019；119：128-37.13Shen LX ，Zhou S ，Glowacki J.Effects of age and gender on WNT gene expression in human bone marrow stromal cells 〔J 〕．J Cell Bio-chem ，2009；106（2）：337-43.14Lombardi APG ，Ｒoyer C ，Pisolato Ｒ.Physiopathological aspects of the Wnt /β-catenin signaling pathway in the male reproductive sys-tem 〔J 〕．Spermatogenesis ，2013；3（1）：1-8.15Takase HM ，Nusse Ｒ.Paracrine Wnt /β-catenin signaling mediates proliferation of undifferentiated spermatogonia in the adult mouse tes-tis 〔J 〕．Proc Natl Acad Sci U S A ，2016；113（11）：E1489-E1497.16Koch S ，Acebron SP ，Herbst J ，Hatiboglu G ，Niehrs C.Post-tran-scriptional Wnt signaling governs epididymal sperm maturation 〔J 〕．Cell ，2015；163：1225-36.17Theka I ，Sottile F ，Cammisa M ，et al ．Wnt /β-catenin signaling path-way safeguards epigenetic stability and homeostasis of mouse embry-onic stem cells 〔J 〕．Sci Ｒep ，2019；9：948.18Chassot AA ，Le Ｒolle M ，Jourden M ，et al ．Constitutive WNT /CT-NNB1activation triggers spermatogonial stem cell proliferation and germ cell depletion 〔J 〕．Dev Biol ，2017；426（1）：17-27.19Takase HM ，Nusse Ｒ.Paracrine Wnt /β-catenin signaling mediates proliferation of undifferentiated spermatogonia in the adult mouse tes-tis 〔J 〕．Proc Natl Acad Sci U S A ，2016；113（11）：E1489-97.〔2018-11-17修回〕（编辑杜娟）《中国老年学杂志》被国际数家数据库、检索性期刊检索机构收录情况根据国际检索机构给中国科学技术期刊编辑学会国际交流工作委员会、中国高等学校自然科学学报研究会对外联络委员会发来的电子邮件及其附件统计整理，《中国老年学杂志》2009年又被5种国际重要检索系统列为来源期刊：1）美国化学文摘（CA ），CODEN ZLZHAO ，http ：// /layersec.asp ；2）波兰《哥白尼索引》（IC ，Index of Copernicus ），http ：// /karta.Php ；3）日本《科学技术社（中国文献数据库）》（JST ，Japan Science ＆Technology Agency ）（Chinese Bibliographic Database ）；4）美国《乌利希期刊指南》（UPD ，Ulrich's Periodicals Directory ），http ：// /ulrichsweb /；5）美国《剑桥科学文摘：生物科学》（CSA ：BS ）。
三丁基氯化锡对小鼠胸腺细胞凋亡及其Fas蛋白表达的影响
(上接第32页)根据大鼠血清中T BH Q及其代谢物一级质谱峰面积的高低,推算T BH Q在大鼠体内代谢程度高,主要经Ⅱ相反应(与硫酸或葡萄糖醛酸结合)代谢,少量T BH Q还会发生Ⅰ相反应,推测为氧化反应,生成的二羟基化物与谷氨酸结合。
参考文献:[1] Nakagawa Y.E ffects of dicoumarol on cytotoxicity caused bytert2butylhydroquinone in is olated rat hepatocytes.T ox Lett,1996,84:63268.[2] Y unbo L,Andrew S,Periannan K,et al.C opper redox2dependent activation of22tert2butyl(1,4)hydroquinone:formation of reactive oxygen species and induction of oxidativeDNA damage in is olated DNA and cultured rat hepatocytes.Mutat Res,2002,518:1232133.[3] Okubo T,Y okoyama Y,K ano K,et al.Cell death induced bythe phenolic antioxidant tert2butylhydroquinone and itsmetabolite tert2butylquinone in human m onocytic leukemiaU937cells.F ood Chem T oxicol,2003,41:6792688.[4] Ikeda G J,Sapienza PP,R oss I A.Distribution and excretion ofradiolabelled tert2butylhydroquione in fischer344rats,F oodChem T oxicol,1998,36:9072914.[5] 包金风,刘国卿.中药血清药理学的方法学研究概述.药学进展,2000,24:89292.[6] 郝鹏鹏,倪晋仁,孙卫玲,等.液相色谱Π离子阱质谱法检测不同食用植物油中的叔丁基对苯二酚(T BH Q).质谱学报,2005,26:2222227.[7] 刘烨.关于中药血清药理学实验方法的讨论.贵阳中医学院学报,2004,26:53256.[8] 姜浩,姜泓,江骥,等.高效液相色谱2电喷雾离子阱质谱法鉴定人尿中奥美拉唑代谢物.分析化学,2002,30:143121434.[9] 杜宗敏,黄海华,陈笑艳,等.人尿中苯丙哌林羟基化代谢物的研究.药学学报,2000,35:9162920.[10] G u J,Zhong D,Chen X.Analysis of O2glucuronide conjugatesin urine by electrospray ion trap mass spectrometry.Fresenius’J Anal Chem,1999,365:5532558.(收稿日期:2006-05-04)中图分类号:R99416 文献标识码:A 文章编号:1002-3127(2007)01-0033-04・论著・三丁基氯化锡对小鼠胸腺细胞凋亡及其F as蛋白表达的影响陈庆1,张振军2,康维钧1,连靠奇1(1.河北医科大学公共卫生学院卫生毒理与卫生化学教研室,河北石家庄050017;2.西安交通大学医学院劳动卫生与环境卫生学教研室)【摘要】 目的 研究三丁基氯化锡(T BT C L)对小鼠胸腺细胞凋亡及Fas蛋白表达的影响,探讨T BT C L的免疫毒性机制。
组蛋白H3K36me3在砷致大鼠肝氧化损伤中的作用
/artide/cn/10.13213/kj.jeom.2021.20542
Funding This study was funded.
Correspondence to ZHANG Aihua, E-mail: aihuagzykd@
Ethics approval Obtained Competing interests None declared Received 2020-11-18 Accepted 2021-04-15
►Link to this article
/artide/en/10.13213/ki.jeom.2021.2 of Environmental and Occupational Medicine | 2021, 38(6)
median lethal dose (LD50) of sodium arsenite in rats was 41 mg-kg1. Accordi ng to the principle of dose design for subchronic toxicity test, the three arsenic dose groups were given 2.5 (1/16 LD5o), 5.0 (1/8 LDSo), and 10.0 (1/4 LD5o) mg-kg1 (calculated by body weight, thereafter) sodium arsenite solution respectively. The rats in the control group were given lOmL-kg1 deionized water by intragastric administratlon, 6d a week. After 4 months, urine and liver tissue samples were collected. The content of arsenic in liver was measured by inductively coupled plasma mass spectrometry (ICP-MS). Histone was extracted from the liver of rats by acid extract!on, and the modification level of H3K36me3 was detected by en zyme-linked immunosorbe nt assay (ELISA). The level of urinary 8-hydroxy-2-deoxygua no sine (8-OHdG) was tested by the ultra-performa nee liquid chromatography tan dem mass spectrometry (UPLC-MS/MS). The content of liver malon dialdehyde (MDA) was determined by thiobarbituric acid (TBA) method. The linear relationships between liver arsenic, H3K36me3z 8-OHdG, and MDA were evaluated. A "liver arsenic-H3K36me3-8-OHdG/MDA" mediation model was established to explore potential mediating role of H3K36me3 in arsenic induced liver oxidative injury.
姜黄素对长波紫外线所致DNA损伤的拮抗作用
・基础研究・姜黄素对长波紫外线所致DNA损伤的拮抗作用太原市中心医院(030009) 周 敏山西省疾病预防控制中心 周 慧【摘 要】 目的 观察长波紫外线引起人表皮样癌细胞DNA链断裂损伤的情况,以及姜黄素对这种损伤的拮抗作用。
方法 用长波紫外线照射细胞,用单细胞凝胶电泳法检测DNA损伤。
结果 长波紫外线剂量依赖性地诱发表皮细胞的DNA链断裂损伤,损伤的高峰出现在照射后1h。
而姜黄素可以拮抗长波紫外线引起的DNA链断裂,表现为彗星细胞百分比的降低和彗星尾巴长度的缩短,并有量效关系。
结论 姜黄素可以预防长波紫外线照射引起的皮肤细胞DNA链断裂损伤。
【关键词】 姜黄素; 紫外线; DNA损伤; 彗星试验An t agon istic effect of curcu m i n on ultrav iolet A-i nduced D NA damage i n hu man ep ider m o id carc i no ma cells ZH OU M in3,ZH OU H u i.3T aiy uan M unicip al Central H osp ital,T aiy uan030009,Ch ina【Abstract】 Objective To investigate the antagonistic effect of curcum in on DNA dam age in hum anep ider mo id carcinom a cells induced by long2w avelength ultravi o let(UVA).M ethods Cells w ere treated w ithUVA radiati on,and DNA strand break s w ere studied th rough using the com et assay.Results UVA radiati on ledto a do se2dependently increased DNA strand breakage,and the peak dam age occurred at1h after treatm ent.A tthe sam e ti m e,curcum in do se2dependently inh ibited the DNA strand break s induced by16kJ m2UVA,as show nby the decrease of com et cells and the w eakness of the com et tails.Conclusion Curcum in can p revent the DNA strand break s induced by UVA.【Key words】 Curcum in; U ltravi o let; DNA dam age; Com et assay 按照波长不同,紫外线可以分为长波、中波和短波三类。
OxiSelect 氧化DNA损害定量试验(AP位)产品说明书
Product ManualOxiSelect™ Oxidative DNA Damage Quantitation Kit (AP Sites)Catalog NumberSTA-324 50 assaysFOR RESEARCH USE ONLYNot for use in diagnostic proceduresIntroductionFree radicals and other reactive species are constantly generated in vivo and cause oxidative damage to biomolecules, a process held in check only by the existence of multiple antioxidant and repair systems as well as the replacement of damaged lipids and proteins. DNA is probably the most biologically significant target of oxidative attack, and it is widely thought that continuous oxidative damage to DNA is a significant contributor to the age-related development of the major cancers, such as those of the colon, breast, rectum, and prostate. Among numerous types of oxidative DNA damage,apurinic/apyrimidinic (AP or abasic) site is one of the prevalent lesions of oxidative DNA damage. Abasic sites arise in DNA at a significant rate by spontaneous base loss as in depurination, by DNA oxidation, or by the action of DNA glycosylases. Estimates of the number of abasic sites generated per mammalian cell run as high as 50,000 to 200,000 per day. Unrepaired abasic sites inhibit topoisomerases, replication, and transcription and can be mutagenic because of bypass synthesis on nontemplated DNA.Cell Biolabs’ Oxidative DNA Damage Quantitation Kit (AP sites) uses an Aldehyde Reactive Probe (ARP) to react specifically with an aldehyde group on the open ring form of AP sites. This allows for the AP sites to be tagged with biotin which is later detected with Streptavidin-Enzyme conjugate. The quantity of AP sites in unknown DNA sample is determined by comparing its absorbance with a standard curve generated from the provided DNA standard containing predetermined AP sites. The kit has a detection sensitivity range of 4 to 40 AP sites per 1 x 105 bp. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and 50 tests for unknown samples. Related Products1.STA-303: OxiSelect™ Nitrotyrosine Immunoblot Kit2.STA-305: OxiSelect™ Nitrotyrosine ELISA Kit3.STA-308: OxiSelect™ Protein Carbonyl Immunoblot Kit4.STA-310: OxiSelect™ Protein Carbonyl ELISA Kit5.STA-315: OxiSelect™ Protein Carbonyl Spectrophotometric Assay6.STA-331: OxiSelect™ MDA Immunoblot Kit7.STA-332: OxiSelect™ MDA ELISA Kit8.STA-334: OxiSelect™ HNE Adduct ELISA Kit9.STA-320: OxiSelect™ Oxidative DNA Damage ELISA Kit (8-OHdG)10.STA-325: OxiSelect™ Oxidative RNA Damage ELISA Kit (8-OHG)Kit Components1.Glycogen Solution (Part No. 232401): One 100 µL vial of 10 mg/mL glycogen.2.Sodium Acetate Solution (Part No. 232402): One 1.0 mL vial of 3M Sodium Acetate, pH 5.5.3.ARP Solution (Part No. 232403): One 250 µL vial of 10 mM ARP.24.DNA High-Binding Plate (Part No. 232404): One 96-well strip plate.5.DNA Binding Solution (Part No. 232405): One 6 mL bottle.6.10X Wash Buffer (Part No. 232406): One 30 mL bottle.7.Streptavidin-Enzyme Conjugate (Part No. 310803): One 20 µL vial.8.Substrate Solution (Part No. 310807): One 12 mL amber bottle.9.Stop Solution (Part. No. 310808): One 12 mL bottle.10.Reduced DNA Standard (Part No. 232407): One 1.0 mL vial of 6 µg/mL fully reduced in TEBuffer (0 ARP/100,000 bp).11.ARP-DNA Standard (Part No. 232408): One 400 µL vial of 6 µg/mL ARP-DNA in TE Buffer (40ARP/100,000 bp).Materials Not Supplied1.DNA samples from cell or tissue for measuring DNA damage2.TE Buffer: 10 mM Tris, pH 7.5, 1 mM EDTA3.100% and 70% Ethanol4.10 µL to 1000 µL adjustable single channel micropipettes with disposable tips5.50 µL to 300 µL adjustable multichannel micropipette with disposable tips6.37 0C Incubator7.Multichannel micropipette reservoir8.Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length) StorageUpon receipt, aliquot and store both the Reduced DNA and ARP-DNA Standards at -20ºC to avoid multiple freeze/thaw cycles. Store all other components at 4ºC until their expiration dates.Preparation of Reagents•1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir to homogeneity.•Streptavidin-Enzyme Conjugate: Immediately before use, dilute the Streptavidin-Enzyme Conjugate 1:1000 with 1X Wash Buffer. Do not store diluted solutions.Preparation of Standard CurvePrepare a dilution series of ARP-DNA standards in the concentration range of 0 – 40 ARP/100,000 bp according to table 1.TubesARP-DNAStandard (µL)Reduced DNAStandard (µL)TEBuffer(µL)TotalVolume(µL)DNAConcentration(µg/mL)AP Sitesper 100,000bp1 20 0 100120 1 402 16 4 100120 1 323 12 8 100120 1 244 8 12 100120 1 165 4 16 100120 1 86 2 18 100120 1 47 1 19 100120 1 28 0 20 100120 1 0 Table 1. Preparation of ARP-DNA StandardsAssay ProtocolI. ARP Reaction1.Isolate genomic DNA with desired method and dissolve the genomic DNA in TE buffer.Dilute the genomic DNA with TE buffer to 100 µg/mL.Note: During DNA extraction, avoid heating the DNA solution, or any procedure will introduceAP sites. We recommend using DNAZOL reagent to extract DNA and dissolve DNA in TEbuffer.2.Mix 5 µL of purified genomic DNA (100 µg/mL) with 5 µL of ARP solution in amicrocentrifuge tube and incubate 1 hr at 37 0C.3.Add 90 µL of TE buffer and 1 µL of Glycogen Solution to each tube and mix well.4.Add 10 µL of Sodium Acetate Solution to each tube, mix well.5.Add 300 µL of absolute ethanol to each tube and mix well and incubate at -20 0C for 30minutes.6.Centrifuge for 10-20 minutes at 14,000 g and carefully wash the pellet three times with 70%ethanol.7.Dissolve the DNA pellet in 10-50 µL of TE buffer and determine the DNA concentration withdesired method. ARP-derived DNA can be stored at -20 0C for up to one year.Note: It is important that the ARP-derived DNA concentration is determined precisely for the accurate measurement of AP sites. We recommend using Invitrogen’s Quanti-iT™ DNA assay kit to measure D NA concentration.II. Determination of AP sites in DNA:1.Dilute the ARP-derived DNA sample to 1 µg/mL with TE buffer.2.Add 50 µL of ARP-derived DNA sample or each dilution of the prepared ARP-DNA standardsto the DNA High-binding plate. Add 50 µL of DNA Binding Solution to each well. Mix well by pipetting and incubate at room temperature for 2 hrs or overnight on an orbital shaker. Each sample including unknown and standard should be assayed in duplicate.3.Wash microwell strips 3 times with 250 µL 1X Wash Buffer per well with thorough aspirationbetween each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.4.Add 100 µL of diluted Streptavidin-Enzyme Conjugate to each well and incubate at 37 0C for 1hr.5.Wash microwell strips 3 times with 250 µL 1X Wash Buffer per well with thorough aspirationbetween each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.6.Warm Substrate Solution to room temperature. Add 100 µL of Substrate Solution to each well,including the blank wells. Incubate at room temperature for 5 to 20 minutes on an orbital shaker.7.Stop the enzymatic reaction by adding 100 µL of Stop Solution into each well, including theblank wells. Results should be read immediately (color will fade over time).8.Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wavelength.Example of ResultsThe following figures demonstrate typical Oxidative DNA Damage Quantitation results. One should use the data below for reference only. This data should not be used to interpret actual results.Figure 1: ARP-DNA Standard Curve.References1.Croteau D L, Bohr V A. (1997) J Biol Chem. 272:25409–25412.2.Lindahl T. (1993) Nature 362:709–715.3.Kubo K, Ide H, Wallace S S, Kow Y W. (1992) Biochemistry 31:3703–3708.WarrantyThese products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions. THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CELL BIOLABS’ sole obligation and purchaser’s exclusive remedy for breach of this warranty shall be, at the option of CELL BIOLABS, to repair or replace the products. In no event shall CELL BIOLABS be liable for any proximate, incidental or consequential damages in connection with the products.Contact InformationCell Biolabs, Inc.7758 Arjons DriveSan Diego, CA 92126Worldwide: +1 858-271-6500USA Toll-Free: 1-888-CBL-0505E-mail: ********************2007-2008: Cell Biolabs, Inc. - All rights reserved. No part of these works may be reproduced in any form without permissions in writing.。
DNA损伤的原因
错配修复（mismatch repair）
CH3
CH3
mutL, mutS, mutH, ATP
CH3 CH3
Exonuclease, mutL, mutS, Helicase, ATP
CH3 CH3
DNA polymerase III, SSB, DNA ligase
CH3 CH3
Methyl transferase
3' 5'
碱基切除修复（BER） ---糖基化酶修复
释放出损伤碱基， 产生AP位点 （2）AP内切酶在AP位点切 开磷酸二酯键 （3）外切酶进一步切割 （4）DNA聚合酶、DNA连接 酶填充缺口
3' （1）糖基化酶切断糖苷键，
5'
3' 5' 3'
5 Pol β 5' 3'
3' 5'
三、直接修复 （一）光复活（photoreactivation）
四、重组修复（Recombination repairing） 通过从另一双链中获得同源单链来修补双链 DNA一条链上缺口的模式。
复制时遇到一个未修复的DNA 损伤（DNA分子的损伤
面较大，还来不及修复完善就进行复制 ）
跳过该损伤并重新起始复制 留下一个子链缺口
Recombination repairing
使损伤限制在原来的链上，不至影响新合成的DNA
五、SOS修复（SOS response）五
一种能够引起误差的紧急修复，是在无模板
DNA情况下诱导产生的修复作用。
特点：
1、DNA受到严重损伤、细胞处于危急状态时所诱导的一种 DNA修复方式 2、修复结果只是能维持基因组的完整性，提高细胞的生 成率，但留下的错误较多，故又称为错误倾向修复
天然产物诱导肿瘤细胞DNA损伤应答的研究进展
天然产物诱导肿瘤细胞DNA损伤应答的研究进展[摘要] 细胞在生长增殖过程中受到?仍椿蛲庠葱缘囊蛩囟?使DNA结构发生变化或受损，如果不能在短时间内修复，将引起周期阻滞，甚至凋亡。
利用肿瘤细胞快速增殖的特性，通过诱导其DNA损伤、细胞周期阻滞和凋亡已成为抗肿瘤药物研究的重要策略。
研究显示，一些天然化合物可通过诱导肿瘤细胞DNA损伤抑制肿瘤细胞增殖，具有抗肿瘤药物研发价值。
[关键词] 天然产物；DNA损伤；肿瘤细胞；周期阻滞；凋亡[Abstract] The DNA structures could be altered or even damaged by exogeous or endogenous factors during cell proliferation. Failure of effective and timely repair will lead to cell cycle arrest or apoptosis. By taking the advantage of the quick proliferation of cancer cells，DNA damage induction，cell cycle arrest and apoptosis promotion have become important strategies for ant-cancer chemotherapy. Previous reports showed that an array of natural compounds inhibit cancer cell proliferation by inducing DNA damage，which have therapeutic potentials for anti-cancer drug research anddevelopment.[Key words] natural products；DNA damage；cancer cell；cell cycle arrest；apoptosisdoi：10.4268/cjcmm201524141 DNA损伤DNA损伤是指由内源性或外源性因素导致的DNA分子结构的异常改变。
Neuronal Death
Pyruvate Protection Against-Amyloid-Induced Neuronal Death:Role of Mitochondrial Redox StateGema Alvarez,Milagros Ramos,Francisca Ruiz,Jorgina Satru´stegui,andElena Bogo´nez*Centro de Biologı´a Molecular“Severo Ochoa,”CSIC-Universidad Auto´noma de Madrid,Madrid,SpainThe mechanism by which-amyloid protein(A)causesdegeneration in cultured neurons is not completely un-derstood,but several lines of evidence suggest that A-mediated neuronal death is associated with an enhancedproduction of reactive oxygen species(ROS)and oxida-tive damage.In the present study,we address whethersupplementation of glucose-containing culture mediawith energy substrates,pyruvate plus malate(P/M),pro-tects rat primary neurons from A-induced degenerationand death.We found that P/M addition attenuated celldeath evoked by-amyloid peptides(A25–35and A1–40)after24hr treatment and that this effect was blocked by ␣-ciano-3-hydroxycinnamate(CIN),suggesting that it re-quires mitochondrial pyruvate uptake.P/M supply tocontrol and A-treated neuronal cultures increases cellularreducing power,as indicated by the ability to reduce thedye3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT).The early increases in ROS levels,mea-sured by dichlorofluorescein(DCF)fluorescence,andcaspase-3activity that follow exposure to Awere no-tably reduced in the presence of P/M.These resultsplace activation of caspase-3most likely downstream ofoxidative damage to the mitochondria and indicate thatmitochondrial NAD(P)redox status plays a central role inthe neuroprotective effect of pyruvate.Inhibition of respi-ratory chain complexes and mitochondrial uncouplingdid not block the early increase in ROS levels,suggestingthat Acould initiate oxidative stress by activating asource of ROS that is not accesible to the antioxidantdefenses fueled by mitochondrial substrates.©2003Wiley-Liss,Inc.Key words:Alzheimer’s disease;mitochondria;oxidative stress;pyruvateAccumulation of-amyloid protein(A),the pri-mary protein constituent of senile plaques present in the brains of Alzheimer’s disease(AD)patients,has been pro-posed to play a central role in the development of AD neuropathology(amyloid hypothesis).This hypothesis is supported by genetic,biochemical,and animal modeling studies(for review see Yankner,1996;Selkoe,1999),but the mechanism and the nature of Aspecies responsible for neurodegeneration in AD are not well understood. There are several lines of evidence showing that brain oxidative stress contributes to AD pathogenesis and that oxidative damage is one of the earliest events in this neurodegenerative disorder(for review see Pratico`and Delanty,2000;Butterfield et al.,2001).Most of the evi-dence originates from quantitative analysis of oxidative markers in postmorten AD brain and from the develop-ment of mouse models of AD and cellular models of A-associated neurodegeneration.In culture,neuronal death induced by Ais observed only with aggregated,fibrillar forms of Apeptides(Pike et al.,1991,1993; Mattson et al.,1993;Lorenzo and Yankner,1994).Dif-ferent studies have shown that aggregated Ainduces oxidative stress in culture and that antioxidants provide protection against A-associated neurotoxicity(Behl et al.,1994;Butterfield et al.,1994;Hensley et al.,1994; Schubert et al.,1995;but see also Pike et al.,1997).In support for a role of oxidative stress in the neurotoxic action of A,increased expression and activity of catalase, glutathione peroxidase(GPx),and Mn-dependent super-oxide dismutase(Mn-SOD)in neuronal cell lines and rat embryonic cortical neurons render cells resistant to A(Sagara et al.,1996;Keller et al.,1998;Barkats et al., 2000).Oxidative stress-induced lipid peroxidation seems to be a critical event in the mitochondrial death pathway triggered by apoptotic stimuli,such as tumor necrosis-␣(TNF-␣),ceramide,staurosporine,lipopolysaccharide, hypoglycemia,and growth factor withdrawal(Cai and Jones,1999;Nomura et al.,2000).Mitochondrial defenseContract grant sponsor:Ministerio de Ciencia y Tecnologı´a(MCT);Con-tract grant sponsor:Direccio´n General de Investigacio´n de la Comunidad de Madrid(CAM);Contract grant sponsor:Fundacio´n Ramo´n Areces.*Correspondence to:Dr.Elena Bogo´nez,Departamento de Biologı´a Mo-lecular,Centro de Biologı´a Molecular“Severo Ochoa”(CSIC-UAM), Universidad Auto´noma de Madrid,28049Madrid,Spain.E-mail:ebogonez@cbm.uam.esReceived12December2002;Revised26March2003;Accepted27March 2003Journal of Neuroscience Research73:260–269(2003)©2003Wiley-Liss,Inc.against ROS and oxidative damage requires the concerted action of MnSOD,GPx,thioredoxin peroxidase(TPx), and phospholipid hydroperoxide glutathione peroxidase (PHGPx;Sies,1993;Miranda-Vizuete et al.,2000).Ox-idized glutathione(GSSG)produced during the GPx, TPx,and PHGPx reactions is reduced by glutathione reductase(GR),which uses NADPH as electron donor. Thus,peroxide detoxification by mitochondria is highly dependent on the availability of NAD(P)H and,therefore, on energy substrates.The beneficial effect of pyruvate on neuronal sur-vival in culturefirst reported by Selak et al.(1985)and Facci et al.(1985)has been extended to cellular models of neurodegeneration induced by several different insults, such as hydrogen peroxide,glutamate,zinc,and copper/ cysteine(Eimerl and Schramm,1995;Desagher et al., 1997;Ruiz et al.,1998;Sheline et al.,2000;Wang and Cynader,2001).We have previously shown that the sup-ply of culture media with pyruvate plus malate,in addition to unlimited glucose availability,protects hippocampal and cortical embryonic neurons against glutamate excitotox-icity mediated by N-methyl-D-aspartate(NMDA)recep-tor overactivation(Ruiz et al.,1998).These survival-promoting effects and the preferential metabolism of pyruvate and malate(P/M)over glucose(Ruiz et al., 1998)suggested that provision of pyruvate from glucose may be limited in cultured neurons,especially under con-ditions of high energy demands.Schurr et al.(1988)have shown,in line with these observations,that glutamate stimulation of hippocampal slices induces an increase in glial glycolyticflux and lactate output that could serve to meet the increased energy requirements of glutamate-activated neurons.The aim of the present study was threefold:(1)to test the hypothesis that neuronal damage induced by Ain culture,if partially associated with oxidative stress and overwhelmed antioxidant defenses,could be counteracted by the supply of energy substrates(pyruvate plus malate);(2)to evaluate the capacity of exogenous substrates to block A-induced apoptotic events;and(3)to study the mechanism of protection provided by these substrates.MATERIALS AND METHODSCell CulturePrimary neuronal cultures from rat cerebral cortex were established from embryonic day18fetuses as previously de-scribed(Ruiz et al.,1998;Alvarez et al.,1999).Culture medium was based in a serum-free defined medium[minimal essential medium(MEM;Gibco,Grand Island,NY),1%glutamax-I (Gibco),and B18supplements(Brewer and Cotman,1989), ommitting glutamate,glutamine,retinol,retinylacetate,super-oxide dismutase,catalase,and triodo-1-thyronine].Cells were plated at a density of1ϫ105cells/cm2on poly-L-lysine-and laminin-coated24-well plates(Ruiz et al.,1998)and maintained at37°C in a humidified incubator with7%CO2/93%room air. To obtain adequate numbers of neurons for caspase-3assays, cultures were prepared by using the same protocol,with one minor alteration;i.e.,cells were plated on poly-L-lysine-and laminin-coated six-well plates.Cells were seeded in definedmedium containing20%heat-inactivated horse serum for2–3hrto let the cells attach to the surface,and then the medium wasreplaced,reducing serum concentration to5%.One-half of theculture medium was replaced every second day with serum-freedefined medium.This gradual reduction in serum concentrationyields highly enriched neuronal cultures(neurons represent Ͼ80%of the cell population)as assesed by immunocytochem-istry for a neuronal marker(microtubule-associated protein-2)and glial markers for astrocytes(glialfibrillary acidic protein)andmicroglia(OX-42;Ruiz et al.,1998).Cultures were used forexperimentation between7and10days in vitro(DIV),exceptwhere otherwise indicated.Treatments With APeptides and Viability Analysis A25–35(CBMSO Protein Synthesis Service)and A1–40 (QCB)stock solutions were prepared as2.5mM and0.25mM stocks in sterile,deionized water,respectively.Preaging of Apeptide stocks was performed at37°C for15min(A25–35)or 7days(A1–40)as described elsewhere(Pike et al.,1993).Rat cortical neurons were exposed to Aby removing the culture medium and replacing it with minimal essential medium(MEM) with Earle’s salts(Gibco)supplemented with N2components (Bottenstein and Sato,1979)containing Afor the indicated times.Cell viability was assesed by counting the number of live/dead neurons in three differentfields per well(aproximately 300neurons/field)using the propidium iodide/calcein-AM procedure as previously described(Mattson et al.,1995).To test the implication of caspase-3in neuronal death,we have used N-acetyl-Asp-Glu-Val-Asp-aldehyde(DEVD-CHO;Bachem, Torrance,CA).The specificity of this and other caspase inhib-itors was tested together with their control peptides,in the context of delayed neuronal death elicited by transient exposure to low concentrations of glutamate(Ruiz et al.,2000). Cytosolic pH DeterminationCytosolic pH was measured with thefluorescent probe2Ј,7Ј-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein(BCECF;Molecular Probes,Eugene,OR).Cells grown onto coverslipswere incubated in Ca2ϩ-free HCSS(in mM:NaCl,120;KCl,5.4;MgCl,0.8;glucose,15;HEPES,25;pH7.4)containing4M BCECF-AM and0.025%pluronic F.127for20min at37°C and rinsed with HCSS containing1mM CaCl2for20minbefore use.Fluorescence signals were recorded using anAminco-Bowman Series2spectrofluorometer equipped with amagnetic stirring and temperature-statted cell holder.Excitationand emission wavelengths were set at490/439and530nm,respectively.Cytosolic pH was calibrated as described by James-Kracke(1992).MTT Reduction AssayMeasurement of initial rates of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)reduction was amodification of the method of Hansen et al.(1989).Briefly,0.125ml of MTT stock solution[5mg/ml in phosphate-buffered saline(PBS)]was added to0.5ml of culture media(24-well plates).Neuronal cultures were incubated for30min at37°C in a humidified incubator.Cells were solubilized byadding0.5ml of lysis buffer[20%sodium dodecyl sulfate(SDS)/ Mitochondria Redox State and AToxicity26150%N,N-dimethylformamide,pH 4.7],and the formazan product was extracted by overnight incubation at37°C.Absor-bance at570nm was used to quantify the amount of soluble formazan formed.Caspase-3Activity AssayColorimetric caspase-3activity assays were performed ac-cording to the manufacturer’s manual(ApoAlert CPP32/ Caspase-3Assay Kits;Clontech,Palo Alto,CA).In brief,cul-tures were rinsed with PBS,and neuronal cells(2ϫ106)were harvested in50l lysis buffer and left for10min on ice.The lysates were centrifuged for5min at11,000g,and the superna-tants were collected and incubated with50M N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide(DEVD-pNA)at37°C for1hr. Formation of pNA was measured at400nm in a spectropho-tometer.Absorbance data were converted to nanomoles pNA per106cells using a standard curve generated with free pNA. Quantification of Intracellular ROSTo measure net intracellular accumulation of ROS,the peroxide sensitivefluorescent probe5-(and-6)-carboxy-2Ј,7Ј-dichlorodihydrofluorescein diacetate(carboxy-H2DCFDA; Molecular Probes)was used,following procedures previously utilized to estimate-amyloid-induced ROS production in neurons(Ueda et al.,1997;Wang et al.,2001).In brief, carboxy-H2DCFDA[10mM in dimethyl sulfoxide(DMSO)] was added to control or A-treated wells(24-well plates)to a final concentration of10M,and the cultures were returned to the37°C incubator for50min.After two washes with pre-warmed PBS,cells were solubilized with1%SDS,5mM Tris HCl,pH7.4.DCFfluorescence in the lysate was measured in an Aminco-Bowman Series2spectrofluorometer(excitation at 504nm and emission at529nm).RESULTSRespiratory Substrates Protect Neurons Against Delayed Death Induced by ATo examine whether supplementation of culture media with the respiratory substrates P/M had any effect on neuronal survival after exposure to A,we tested initially the neurotoxic fragment comprising amino acids 25–35of A(A25–35).To this end,cultured neurons from cerebral cortex(7–9DIV)were treated with increas-ing concentrations of A25–35in the presence or absence of P/M,and cell death was assessed by the calcein/ propidium iodide(PI)assay after24hr of treatment.In accordance with other studies,A25–35-treated cultures displayed PI-positive condensed and fragmented nuclei (not shown),a morphological characteristic of apoptosis. A25–35caused a concentration-dependent increase in cell death,which approached60%at25M A25–35(Fig. 1A).No cell death was seen in neuronal cultures treated with a control peptide with the amino acid sequence inverted(data not shown).However,in the presence of P/M(2.5mM each),A25–35-induced cell death was significantly lower at all concentrations of peptide studied (Fig.1A).In our previous work on the trophic and protective effects of exogenous respiratory substrates on neurons in culture,we used malate,as a source of oxaloacetate,along with pyruvate to optimize pyruvate oxidation by mito-chondria(Villalba et al.,1994;Ruiz et al.,1998).To determine whether P/M promotion of cell survival re-quired the supply to culture media of both respiratory substrates,we analyzed the effects of pyruvate and malate individually.To this end,neuronal cultures were exposed to10M A25–35,a concentration that induced29.8%Ϯ2.4%neuronal loss(Fig.1A),a level of injury at which P/M protection was highly significant(38.9%reduction in cell death,PϽ.0005).As shown in Figure1B,although the presence of exogenous pyruvate provided the same attenuation of neuronal loss as did the addition of P/M, the supply of malate was ineffective.We also examined the neuroprotective capacity of other energy substrates,such as lactate or3-hydroxybutyrate (3HB).Paradoxically,lactate,which through the action of lactate dehydrogenase renders pyruvate in the cytosolic compartment,did not improve neuronal viability(data not shown).However,3HB(2.5mM),an energy substrate that is oxidized within mitochondria to acetoacetate by 3HB dehydrogenase,was as effective as P/M at reducing A-induced cell death(Fig.1C).There is evidence indicating that,although the neu-rodegenerative processes activated by A25–35and the pathophysiological peptides A1–40and A1–42in vitro may not be identical,they share,among other biochemical events,an increased generation of ROS(Yankner,1996; Pike et al.,1997).To determine whether the reduction in cell death provided by exogenous substrates could be extended to physiologically relevant peptides,cortical neuronal cultures were exposed to aggregated A1–40in the absence or presence of P/M or3HB,and cell death was examined after24hr of treatment.Both P/M and 3HB supply to culture media improved cell survival,the reduction in specific cell death being74.8%and94% respectively(Fig.1D,E).The Neuroprotective Effect of P/M Supplement Requires Pyruvate Transport Into Mitochondria In addition to serving as an energy substrate for neurons,pyruvate is a potent antioxidant;as with other ␣-ketoacids,it directly neutralizes hydrogen peroxide and lipid peroxides in a nonenzymatic reaction(Constanto-poulos and Barranger,1984).Because oxidative stresss could be mediating Aneurotoxicity(Behl et al.,1994; Butterfield et al.,1994;Hensley et al.,1994),protection against A25–35-induced cell death by P/M supply could arise from the ability of pyruvate to act as scavenger of peroxides and/or from its use as energy substrate.To evaluate the latter possibility,we analyzed the neuropro-tective effect of P/M supply in the presence of␣-ciano-3-hydroxycinnamate(CIN),a specific inhibitor of the monocarboxylate transporters,at concentrations that se-lectively block mitochondrial pyruvate uptake(10–4M) but are lower than concentrations inhibiting the plasma membrane carrier(Halestrap and Denton,1975;Juntti-Berggren et al.,1994).Neuronal cultures were exposed to 10M A25–35in the presence or absence of P/M plus262Alvarez et al.0.2mM CIN,and cell death was determined at 24hr posttreatment.Figure 2A shows that,although exposure to 0.2mM CIN to control cultures did not affect neuronal viability (cell death in the presence of CIN was not sig-nificantly different from that shown by vehicle-treated controls),P/M protection against A 25–35neurotoxicitywas completely abolished in cultures cotreated with CIN.The same results were obtained in neuronal cultures treated with 25M A 1–40(not shown).To exclude that inhibition of pyruvate transport at the plasma membrane was responsible for the loss of P/M protective effects in the presence of 0.2mM CIN,we examined the cytosolic acidification produced by pyruvate/H ϩcotransport,by using the pH-sensitive flu-orescence probe BCECF.Figure 2B shows that addition of pyruvate (2.5mM)to neuronal cultures induced a slight decrease in cytosolic pH (⌬pH was 0.05Ϯ0.005units,n ϭ4),which was preserved in cells preincubated with 0.2mM CIN (⌬pH was 0.13Ϯ0.02units,n ϭ4).These results suggest that the protective effect of exogenous substrates requires the entry of pyruvate into mitochondria and presumably its metabolic oxidation.This is consistent with earlier findings that P/M supply increased the respi-ration rate of dissociated cells from adult rat brain,indi-cating a specific mitochondrial response to P/M in neu-rons (Villalba et al.,1994).Cellular Redox State and Oxidative Damage in Neuronal Cultures Supplemented With Respiratory SubstratesThe survival-promoting activity of P/M supply could rely on an indirect antioxidant effect of pyruvate,if mitochondrial utilization of pyruvate,by increasing NAD(P)H concentrations,strengthens the antioxidant de-fense systems within the organelle.To search for changes in cellular reducing power of cortical neurons supple-mented with P/M,we used a modified tetrazolium salt assay (MTT assay;see Materials and Methods).The MTT assay is thought to measure the reduction of MTT by electrons flowing down the electron transport chain of mitochondria (Slater et al.,1963),but it also measures the enzymatic and nonenzymatic reduction of the MTT mol-ecule to a formazan product involving hydride ion transfer from mitochondrial or cytosolic NADH or NADPH(Alt-ŠFig.1.Effect of energy substrates supply on cell death induced by A .A:Cerebral cortex neurons were exposed to increasing concentrations of A 25–35in the absence or presence of pyruvate plus malate (P/M;2.5mM each).After 24hr,neuronal death was evaluated by calcein/propidium iodide staining.Data were expressed as percentage of dead cells among the total number of cells.The results reflect specific A -induced cell death,i.e.,the percentage of dead cells in A -exposed cultures minus the nonspecific death in vehicle-treated cultures,and represent mean ϮSEM from three separate cultures.B,C:Effect of individual addition of pyruvate (P;2.5mM),malate (M;2.5mM),and 3-hydroxybutyrate (3HB;2.5mM)on cell death induced by 24hr of exposure to A 25–35(10M).Specific cell death was estimated as described for A.Data represent means ϮSEM of three or four separate cultures.The statistical significance of the difference between cell death obtained in the presence or absence of energy substrates was *P Ͻ.05,**P Ͻ.005,***P Ͻ.0005,paired t -test.D,E:Cortical cultures were treated with A 1–40(25M)for 24hr in the presence or absence of P/M and 3HB.Data represent the mean ϮSEM from seven (D)and four (E)separate experiments.*P Ͻ.05,***P Ͻ.0005vs.A -exposed cultures,paired t -test.Mitochondria Redox State and A Toxicity 263man,1976;Shearman et al.,1994).Therefore,MTT reduction could be considered as an index of cellular reducing power or redox state.Because some formazan is eliminated by exocytosis in neural cells and this can be enhanced in the presence of A (Liu and Schubert,1997),we have used very short incubation times (30min),with which this effect in control cells was minimal.Figure 3A shows that MTT reduction was significantly higher when neuronal cultures were supplemented with P/M (33%Ϯ6.8%increase relative to controls,P Ͻ.05).Coincubation with 0.2mM CIN induced a decrease in MTT reduction both in the absence and in the presence of P/M to 76.8%of vehicle-treated controls,results consistent with an in-hibition by CIN of the mitochondrial-and pyruvate-dependent component of cellular MTT reduction.To evaluate whether the enhanced cellular redox state provided by exogenous substrates improves the ca-pacity to limit oxidative damage associated with A ex-posure,we determined MTT reduction and ROS accu-mulation in neuronal cultures exposed to A in the presence or absence of P/M.Because we wanted to com-pare cellular status under different experimental condi-tions,and both parameters (MTT and ROS)are affected by cell loss,experiments were performed at early time points in treatment with A 1–40(25M),when changes in neuronal viability were still undetectable (not shown).Figure 3B shows that MTT reduction capacity of neuronal cultures at 3and 6hr of A treatment was not significantly different from vehicle-treated controls,and cells were competent to respond to the addition of respiratory sub-strates with an enhanced reduction of MTT (133%and 136%in A -treated vs.145%and 151%in control cultures at 3and 6hr,respectively).To determine ROS accumulation,cells were loaded with the fluorescent probe carboxy-H 2DCFDA (see Ma-terials and Methods)during the last 50min of 1,3,and 6hr of exposure to A 1–40,in the absence or presence of P/M,and DCF fluorescence was quantitated by spec-trofluorimetry.Differences in time course and concentra-tion dependence of A neurotoxicity have previously been found and have been related to lot-to-lot variability in aggregation kinetics and type and degree of fibril gen-eration (May et al.,1992).Indeed,A -induced increases in DCF fluorescence at a given time of exposure were highly variable,being dependent on peptide lot and neu-ronal culture.For example,the increase in A -induced increase in DCF fluorescence at 3hr of treatment was 203%Ϯ18%(mean ϮSEM,n ϭ8)with respect to control cultures.The presence of P/M consistenty re-duced this increase to 169%Ϯ11%.To illustrate better the effects of P/M,we have performed linear regression anal-ysis of the increase in DCF induced by A and the inhibition of this increase provided by P/M.Figure 3C shows that there was a significant correlation between both magnitudes (R ϭ0.5561,P Ͻ.05).Taken together,these results suggest that,early in the course of A expo-sure,neuronal mitochondria are functionally competent to metabolize pyruvate,and attenuation of oxidative stress in the presence of pyruvate and other respiratory substrates could arise from the provision of reducing equivalents,within the mitochondrial compartment,essential for de-toxification of ROS and repair of oxidative damage.However,inhibition by P/M was never complete (Fig.3C),even though the cellular redox state obtained with P/M was always higher (Fig.3B).This may indicate that A treatment activates an extramitochondrial source of ROS inaccessible to the antioxidant systems fueled by respiratory substrates.The Mitochondrial Electron Transport Chain Is Not Involved in the Early Generation of ROS Induced by A A major source of intracellular ROS,specifically superoxide anion radical,is generated by oneelectronFig.2.Protective effect of P/M supply is abolished in the presence of ␣-cianno-3-hydroxycinnamate (CIN).A:Cortical cultures (9–10DIV)were treated for 24hr with 10M A 25–35in the presence or absence of P/M (2.5mM each).Sets of sister cultures were preincubated with CIN (0.2mM)for 1hr before A 25–35or vehicle addition.Specific cell death was determined by calcein/PI staining as described in the legend to Figure 1.Values represent the mean ϮSEM of determinations made in three separate cultures.The difference between A -exposed cultures in the absence or presence of P/M was significant (*P Ͻ.0005,paired t -test).Data shown in gray bars (A -,A ϩCIN-,and A ϩP/M ϩCIN-treated cultures)were not statistically different (P Ͼ.05).B:pHi traces representative of four separate experiments showing that the decrease in pHi produced by pyruvate (pyr)addition is maintained in cortical cultures preincubated with 0.2mM CIN.264Alvarez et al.transfer to molecular oxygen at sites on the mitochondria electron transport chain.To assess whether mitochondria-derived superoxide was at the origin of the early A -dependent ROS accumulation,we preincubated cortical neurons in the presence of respiratory chain inhibitors or the protonophore FCCP to abolish mitochondrial func-tion and then studied A 1–40-induced DCF fluorescence.Cultures were preincubated for 1hr with rotenone plus thenoyltrifluoroacetone (TTFA)to inhibit both complex I and complex II;antimycin A,which inhibits complex III;or FCCP before exposure to A 1–40for 3hr.Oligomycin was also present during treatments to avoid cellular aden-osine triphosphate (ATP)depletion by reversal of ATP synthase activity.Under control conditions,a 4-hr incu-bation with respiratory chain inhibitors or FCCP resulted in an increase in the DCF signal (Fig.4).DCF fluores-cence in these experiments (last 50min of the 4-hr time period)is most probably an index of ROS accumulation/oxidative stress rather than a measure of mitochondrial radical production.Interestingly,the A -inducedROSFig.3.MTT reduction and ROS accumulation in neuronal cultures supplemented with mitochondrial substrates.A:Effect of P/M supply on cellular MTT reduction.Cortical cultures (9–10DIV)were assayed for cellular MTT reduction in the absence and presence of P/M (2.5mM each)ϮCIN (0.2mM).When present,CIN was added 1hr before MTT reduction assay.Data represent the mean ϮSEM of triplicate samples from three separate cultures.*P Ͻ.05relative to control cells,paired t -test.B:MTT reduction in cortical cultures following 3and 6hr of exposure to 25M A 1–40in the presence and absence of P/M.Data are the mean ϮSEM of triplicate samples from two separate cultures.*P Ͻ.005,**P Ͻ.0005relative to control cells;#P Ͻ.005relative to P/M-treated cells,paired t -test.C:Correlation between the increase in DCF fluorescence induced by exposure to 25M A 1–40and the inhibition of A -dependent DCF increase produced by coincubation with P/M.Data represent the mean of triplicate values corresponding to 1(n ϭ5),3(n ϭ8),or 6(n ϭ4)hr of treatment from eight separatecultures.Fig.4.A -induced ROS accumulation in the presence of mitochon-drial inhibitors.Cultures of cortical neurons (6DIV)were preincubated for 1hr with rotenone/TTFA/oligomycin (R/T/O),FCCP/oligomycin (F/O),and antimycin A/oligomycin (AA/O),and then 25M A 1–40was added for 3hr.Cells were loaded with carboxy-H 2DCFDA during the last 50min of the 3-hr period as described in Materials and Methods.Concentrations used were rotenone,2M;TTFA,10M;oligomycin,2M;antimycin A,1M;FCCP,1M.The results were normalized to vehicle-exposed values as 100.Data are means ϮSEM for triplicate determinations from two separate cultures.The difference between the DCF increase in A -exposed cultures relative to the corresponding control was *P Ͻ.05,**P Ͻ.005,paired t -test.A -specific DCF increase:Values represent ROS accumulation induced by A 1–40expressed as percentage of the increase produced by the peptide in the absence of mitochondrial inhibitors.Mitochondria Redox State and A Toxicity 265。