氟通过胞外信号调控激酶通路对成骨细胞增殖作用的影响

目的 观察氟通过胞外信号调控激酶(ERK)通路对成骨细胞(MC3T3-E1)增殖作用的影响.方法 取小鼠成骨细胞(MC3T3-E1)进行体外培养,加入不同浓度的氟(Fˉ,0、200、400、600、1000、2000、4000、8000、10 000 μmol/L)培养24、48 h,甲基噻唑蓝(MTT)法筛选出促进细胞增殖的最适浓度.根据最适浓度,将成骨细胞单纯随机分为3组:对照组(Fˉ,0 μmol/L)、染氟组(Fˉ,400 μmol/L)、染氟抑制组(Fˉ,400 μmol/L+PD9805,10 μmmol/L).培养48 h后应用流式细胞术检测各组细胞周期;Western bolt法和免疫荧光法检测各组磷酸化ERK(P-ERK)蛋白表达.结果 400 μmol/L的氟是促进成骨细胞增殖的最适合浓度.与对照组比较[(76.12±10.08)％、(2.06±0.31)％],染氟组G0/G1期细胞数[(63.04±8.12)％]明显减少,S期细胞数[(9.13±2.08)％]明显增多(P均＜ 0.05);而染氟抑制组G0/G1期细胞数[(92.11±9.01)％]明显增多(P＜0.05).Western blot结果表明,与对照组[(100.00±0.00)％]比较,染氟组成骨细胞P-ERK蛋白表达水平[(131.24±13.88)％]明显增高(P＜0.05),染氟抑制组P-ERK蛋白表达[(91.33±9.68)％]未见明显改变(P＞0.05);免疫荧光法检测结果与Western blot法相似.结论 400 μmol/L氟可以促进成骨细胞增殖,ERK通路在氟促进成骨细胞的增殖作用中起到了关键的作用.     

氟对体外培养乳鼠软骨细胞增殖的影响

目的 探讨氟对体外培养乳鼠软骨细胞增殖的影响.方法 采用细胞培养的方法,原代培养昆明乳鼠软骨细胞,传第3代后按染氟剂量不同分为O(对照)、5、10、20、40 ms/L组,10 d后光、电镜观察软骨细胞形态学变化;采用生长曲线、噻唑蓝(MTT)方法,在染氟24、48、72 h测定细胞数量变化及细胞增殖率.结果 染氟10 d后,镜下0、5、10mg/L组软骨细胞表现为增殖,细胞生长旺盛,均可见部分细胞核中核仁数增加:40mg/L组部分软骨细胞中有染色质固缩或凝结成块状,可见到凋亡细胞.在染氟24 h,细胞增殖活力各染氟组组间比较差异无统计学意义(F=2.313,P＞0.05).在染氟48、72 h,0 mg/L组[(23.5±4.6)%、(29.9±1.7)%]、5mg/L组[(34.6±4.7)%、(45.3±5.9)%]、10mg/L组[(39.9±4.8)%、(56.8±5.5)%]、20mg/L组[(31.8±4.1)%、(38.3±6.5)%]、40 mg/L组[(28.3±4.3)%、(33.4±4.8)%]组问比较差异有统计学意义(F值分别为11.401、25.671,P＜0.05);各染氟组细胞增殖活力与对照组比较差异均有统计学意义(P＜0.05),其中5、10ms/L组明显高于40mg/L组(P＜0.05).结论 低剂量氟在较短作用时间内可以促进体外培养小鼠软骨细胞的增殖,剂量升高时表现为抑制.     

氟中毒大鼠骨组织中内质网应激实验研究

目的 观察内质网应激在氟中毒大鼠骨组织中的变化,探索内质网应激在氟骨症发病机制中的可能作用.方法 48只Wistar大鼠,按体质量分成4组,每组12只.对照组和低钙组分别饲以常食饲料(含钙量为0.790%)和自制低钙饲料(含钙量为0.063%),饮用自来水(含NaF＜1 mg/L);高氟组和低钙高氟组分别饲以常食饲料和自制低钙饲料,饮用加氟(NaF,221 mg/L)自来水.实验期间动物自由进食、进水,每周测体质量1次.实验期3个月.生化方法检测大鼠血清氧化应激酶、尿酸(URIC)和碱性磷酸酶(ALP)活性.抽提大鼠一侧股骨骨干的总RNA,利用RT-PCR技术分析内质网应激相关基因BIP、Xbp1、CHOP和PDI的表达水平.结果 低钙高氟组血清丙二醛(MDA)水平高于对照组[(14.74±3.11)μmol/L比(10.15±1.96)μmol/L,P＜0.05];高氟组血清谷胱甘肽过氧化物酶(GPx)的活性高于对照组[(3.87±0.41)×103 U/L比(2.85±0.55)×103U/L,P＜0.05];高氟组和低钙高氟组的尿酸(URIC)分别低于对照组和低钙组[(73.95±9.52)μmol/L比(110.43±25.48)μmol/L,(54.32±22.09)μmol/L比(101.71±17.01)μmol/L/L,P＜0.05].低钙高氟组大鼠的ALP活性高于对照组[(24.77±4.57)×103 U/L比(12.91±3.97)×103 U/L,P＜0.01)].低钙组和低钙高氟组BIP/GAPDH的表达高于对照组(1.38±0.24、1.35±0.12比1.14±0.06,P＜0.05).低钙高氟组的Xbp1/GAPDH的表达高于对照组和低钙组(1.48±0.20比1.02±0.25、1.07±0.25,P＜0.01);低钙高氟组的CHOP/GAPDH的表达高于对照组(0.84±0.18比0.52±0.07,P＜0.05).结论 氟中毒大鼠机体内氧化应激态和骨形成有明显的增强,并伴有骨组织细胞的内质网应激.说明内质网应激与氧化应激很可能都参与了氟骨症的发病机制.     

亚砷酸钠诱导人膀胱上皮永生化细胞氧化损伤作用观察

目的 观察亚砷酸钠(NaAsO2)诱导人膀胱上皮永生化细胞(SV-HUC-1)氧化损伤作用.方法 以不同浓度NaAsO2[0(对照)、1、2、4、8、10μmol/L]对SV-HUC-1细胞染砷24 h,利用流式细胞仪检测细胞内活性氧(ROS)水平,采用酶联免疫吸附实验(EHSA法)检测细胞内硝基酪氨酸(NT)含量和细胞培养液中8-羟基脱氧鸟嘌呤核苷(8-OHdG)水平.结果 1、2、4、8、10 μmol/L染砷组SV-HUC-1细胞ROS水平(81.76±4.91、95.23±2.17、126.61±17.95、126.74±27.77、114.18±9.65)明显高于对照组(69.84±1.28,P＜ 0.05或＜ 0.01),ROS水平与染砷剂量呈显著正相关(r=0.818,P＜ 0.01).10 μmol/L染砷组SV-HUC-1细胞内NT含量[ (919.66±206.33)μg/L]显著高于对照组[(238.19±38.28) μg/L,P＜ 0.01],NT含量与染砷剂量呈显著正相关(r=0.617,P＜0.01).各组细胞培养液中8-OHdG含量比较,差异无统计学意义(F=2.127,P＞0.05).结论 NaAsO2能够引起SV-HUC-1细胞氧化损伤.     

氟对原代培养猪甲状腺细胞功能的影响

目的 探讨氟对原代培养甲状腺细胞功能的影响.方法 用猪甲状腺细胞,采用原代培养方法,按染氟(NaF)剂量不同分为:0(对照组)、40、80、160 mg/L组.染氟48 h后,采用噻唑蓝(MTT)法测定细胞存活率,改良愈创木酚法测定甲状腺过氧化物酶(TPO)活性,放射免疫法测定甲状腺素(T4)水平.结果 甲状腺细胞培养48 h后,细胞贴壁,多呈六边形,呈聚集性趋势生长,并可见典型的滤泡结构.甲状腺细胞染氟培养48 h后,细胞存活率组间比较差异有统计学意义(F=149.092,P＜0.05);40mg/L组细胞存活率为(96,04±2.08)%,与对照组(100%)比较,差异无统计学意义(t=2.157,P＞0.05);80、160 mg/L组细胞存活率分别为(86.59±1.79)%、(70.12±1.49)%,与对照组比较,差异有统计学意义(t值分别为7.621、17.697,P＜0.05).甲状腺细胞T4水平随着染氟剂量的升高而明显降低[(44.940±8.212)、(31.442±4.384)、(22.742±4.504)、(15.193±1.633)nmol/L],组间比较差异有统计学意义(F=978.255,P＜0.05).甲状腺细胞TPO活性也随着染氟剂量的升高而明显降低[(3.103±0.090)、(1.944±0.025)、(1.361±0.008)、(0.668±0.026)U/L],组间比较差异有统计学意义(F=1563.864,P＜0.05).TPO活性与染氟剂量呈负相关(r=-0.955,P＜0.05).结论 氟可通过降低T4水平而影响甲状腺功能,其发生机制可能是通过诱导细胞凋亡,降低细胞存活率和抑制TPO活性有关.     

慢性氟中毒大鼠脑组织细胞外调节蛋白激酶表达及大鼠学习记忆能力的变化

目的 观察慢性氟中毒大鼠脑组织中分裂原激活的蛋白激酶(MAPK)信号转导系统细胞外调节蛋白激酶(ERK1/2)的表达与活性,及其与学习记忆能力改变之间的关系,从而进一步探讨氟中毒神经系统损害的发病机制.方法 72只SD大鼠按性别和体质量随机分为3组.每组24只.对照组:自由饮用自来水,含氟量低于0.5 mg/L;低剂量染氟组:饮水含氟量为5.0 mg/L;高剂量染氟组:饮水含氟量为50.0 mg/L.实验6个月后.取大鼠脑组织,用蛋白印迹方法检测细胞外ERK1/2的蛋白表达与活性;用逆转录-聚合酶链反应(RTPCR)方法测定ERK1/2的mRNA水平;用Morris水迷宫检测大鼠学习记忆能力改变.结果 对照组、低、高剂量染氟组的ERK1/2蛋白水平分别为0.94±0.10、1.25±0.02、1.95±0.07,任意两组间比较,差异均有统计学意义(P＜0.05);对照组、低、高剂量染氟组的phospho-ERK1/2蛋白水平分别为0.73±0.08、0.77±0.07、1.28±0.11,任意两组间比较,差异有统计学意义(P＜0.05);低、高剂量染氟组的ERK1/2活化率[(68.4±3.8)%、(64.1±3.2)%]低于对照组[(82.3±10.7)%],组间比较差异均有统计学意义(P＜0.05);在第2、3、5、6天,低剂量染氟组的大鼠逃避潜伏期[(46.0±8.0)、(24.0±2.7)、(8.9±5.3)、(7.4±4.1)s]长于对照组[(39.3±6.9)、(19.1±9.1)、(8.3±3.4)、(4.8±2.7)s],组间比较差异均有统计学意义(P＜0.05),高剂量染氟组的大鼠逃避潜伏期[(36.9±16.8)、(37.7±12.9)、(19.7±7.6)、(12.2±5.7)s]也长于对照组(P＜0.05),第3、5、6天,高剂量染氟组的大鼠逃避潜伏期长于低剂量染氟组(P＜0.05);低剂量组、高剂量染氟组的大鼠第1次穿越平台位置时间[(1.17±0.75),(4.18±1.10)s]长干对照组[(5.89±0.56)s],组间比较差异均有统计学意义(P＜0.05),低、高剂量染氟组的大鼠在原平台所在象限逗留时间[(17.05±4.25)、(18.20±4.57)s]短于对照组[(25.37±5.65)s],组间比较差异均有统计学意义(P＜0.05);大鼠脑组织中ERK1/2的活化率与空间探索实验第1次到达平台区域时间呈正相关(r=0.364,P＜0.05).与第6天定向航行实验找到平台时间呈正相关(r=0.497,P＜0.05).结论 慢性氟中毒导致大鼠脑组织中ERK1/2蛋白表达水平及活性改变,与慢性氟中毒大鼠学习记忆能力的降低有一定关系.     

复方中药对慢性氟中毒大鼠氟骨症治疗效果的显微CT观察

目的 采用显微CT技术评价复方中药方剂对慢性氟中毒大鼠氟骨症的治疗效果.方法 断乳2周的纯系Wistar大鼠88只,体质量(91.1±10.0)g,按体质量采用随机数字表法分为对照组、中氟组、高氟组、高氟低钙低蛋白组,分别为16、24、24、24只大鼠.中氟组、高氟组、高氟低钙低蛋白组染氟剂量分别为50、100、100 mg/kg,高氟低钙低蛋白组饲料中蛋白质与钙的含量为中氟组和高氟组的1/2.染氟6个月后,每组采用股动脉放血法处死8只大鼠;3个染氟组剩余16只大鼠又分为两小组,一组为持续染氟对照组,另一组模拟氟中毒病区实际情况在持续染氟的基础上用复方中药进行治疗,每天每只大鼠按100 g体质量给药194 mg,每周灌服6d;分别于治疗前和治疗后30、60d收集大鼠24h尿样.大鼠连续灌服90 d,股动脉放血法处死大鼠,分离四肢骨.氟离子选择电极法检测大鼠尿氟;高温灰化-氟离子选择电极法检测骨氟;显微CT技术检测大鼠四肢骨的骨矿物质密度(BMD)、组织骨密度(TMD)、结构模型指数(SMI)、骨小梁厚度(Tb.Th)、骨小梁分离度(Tb.Sp)、各向异性(a1/a3)、骨小梁连接密度(Conn.D)、骨小梁与全部骨组织体积比(BV/TV)、骨表面积与体积比(BS/BV)、骨小梁数目(Tb.N).结果 复方中药治疗后60d,高氟低钙低蛋白治疗组尿氟[(11.01±3.67)mg/L]低于高氟低钙低蛋白对照组[(34.32±9.50)mg/L,t=3.13,P＜ 0.05].复方中药治疗后90d,高氟治疗组骨氟[(275.38±171.65) mg/kg]低于高氟对照组[(701.67±178.16)mg/kg,t=5.42,P＜ 0.05],高氟低钙低蛋白治疗组骨氟[(313.26±124.51)mg/kg]低于高氟低钙低蛋白对照组[(794.66±261.35) mg/kg,t=3.25,P＜0.05].复方中药治疗后90d,各组大鼠Tb.Th、Tb.Sp、a1/a3、Conn.D、BV/TV、BS/BV、Tb.N组间比较差异有统计学意义(F值分别为2.785、2.681、3.039、27.231、2.595、2.854、5.050,P均＜0.05).其中中氟治疗组大鼠Tb.Th、Tb.Sp[(0.04±0.01)、(0.03±0.01)mm]高于中氟对照组[(0.02±0.00)、(0.02±0.00)mm,P均＜0.05],a1/a3、Conn.D、BV/TV、Tb.N[(0.77±0.61),(510.91±304.99)mm-3,(0.42±0.06),(13.58±2.48) mm-1]低于中氟对照组[(1.11±0.01),(2 403.69±124.02)mm-3,(0.46±0.03),(18.12±0.69)mm-1,P均＜0.05];高氟治疗组大鼠BV/TV(0.44±0.04)低于高氟对照组(0.49±0.00,P＜0.05),Tb.Th[(0.04±0.01)mm]高于高氟对照组[(0.03±0.00)mm,P＜ 0.05].结论 复方中药对大鼠氟骨症存在一定的治疗效果.     

过量氟暴露对成骨细胞微小染色体维系蛋白3及成骨相关基因表达的影响

目的 观察氟对体外培养人成骨肉瘤细胞内微小染色体维系蛋白(minichromosone maintenance,MCM)3和成骨相关基因表达的影响,探讨MCM3基因能否作为诊断及监测成骨细胞染氟过量的生物学标志.方法 采用McCoy5A培养液,体外培养人成骨肉瘤细胞(Saos-2).按染氟(NaF)剂量将骨肉瘤细胞分为O(对照)、0.625、1.250、2.500、5.000、10.000、20.000、40.000 mg/L组,染氟培养24 h后收集细胞,实时荧光定量(real-time.RT-PCR)PCR测定成骨细胞MCM3和骨涎蛋白(bone sialoprotein,BSP)、骨钙素(osteocalcin,OC)、骨桥蛋白(osteopontin,OP)3种成骨相关基因mRNA的表达,化学比色法测定碱性磷酸酶(alkaline phosphatase,ALP)活性,用双标准曲线法计算基因表达的相对比值.结果 成骨肉瘤细胞MCM3 mRNA表达在0.625、1.250、2.500、5.000、20.000、40.000 mg/L组(0.059±0.003、0.027±0.001、0.272±0.004、0.115±0.002、0.137±0.004、0.754±0.002)低于对照组(1.000±0.020,P均>0.05),但10.000 mg/L组(21.300±1.200)显著高于对照组(P<0.01),也高于其他染氟组(P均<0.01),组间比较差异有统计学意义(F=305.842,P<0.01).BSPmRNA表达在0.625、1.250、2.500、5.000、10.000 mg/L组(71.80±3.60、133.00±7.20、85.50±0.60、80.90±1.20、304.00±21.00)显著高于对照组(1.00±0.04,P均<0.01),尤其10.000 mg/L组明显高于其他染氟组(P均<0.01),组问比较差异有统计学意义(F=159.531,P<0.01).OC mRNA表达在0.625、1.250、2.500、5.000 mg/L组(110.00±12.00、143.00±2.10、90.60±4.10、23.70±1.20)高于对照组(1.00±0.01,P均<0.01),组间比较差异有统计学意义(F=158.734,P<0.01).OP mRNA表达,在0.625、1.250、2.500、5.000、10.000、20.000 mg/L组(167.00±11.20、111.00±12.10、72.50±3.50、134.00±14.00、42.30±2.40、45.20±3.30)高于对照组(1.00±0.04,P均<0.05或<0.01),组间比较差异有统计学意义(F=60.226,P<0.01).ALP的活性虽然在0.625～40.000mg/L组(6.0±0.4、5.8±0.1、5.7±0.4、7.7±1.1、19.2±2.4、8.5±3.0、18.1±4.2)与对照组(4.2±1.2)比较是增加的,但仅10.000、40.000 mg/L组明显高于对照组和其他染氟组(P均<0.01),组间比较差异有统计学意义(F=7.806,P<0.01).结论 MCM3表达不规律,不适合作为诊断及监测成骨细胞在过量氟作用下的生物标志;氟可能通过影响各成骨相关基因的表达而促进成骨分化.     

地拉罗司对小鼠前成骨细胞MC3T3-E1生物学活性的影响

目的探讨铁鳌合剂地拉罗司(deferasirox,DFS)体外对成骨细胞增生、分化、矿化和细胞内铁离子的影响。方法小鼠前成骨样细胞MC3T3-E1在37℃条件下体外培养,在10 mmol/Lβ-甘油磷酸和50 mg/L抗坏血酸的诱导作用下,分化为成骨细胞,同时用不同浓度(10、20、40μmol/L)DFS干预,用CCK-8法检测细胞的增生,碱性磷酸酶(alkaline phosphatase,ALP)活性试剂盒检测细胞ALP活性,Von kossa染色法行细胞钙结节染色,实时定量PCR检测细胞膜转铁蛋白受体(transferrin receptor,TfR)mRNA的表达。结果 MC3T3-E1细胞增生结果显示,DMSO溶剂组、对照组(0μmol/L)、10、20、40μmol/L组A值分别为1.41±0.09、1.41±0.09、1.01±0.01、0.79±0.04、0.67±0.04;ALP活性检测结果显示,对照组(0μmol/L)、10、20、40μmol/L组ALP活性值分别为0.73±0.03、0.65±0.02、0.54±0.03、0.35±0.04;钙结节检测结果显示,对照组(0μmol/L)、10、20、40μmol/L组TfR mRNA钙化面积比值分别为4.22±0.12、3.29±0.14、1.40±0.20、0.86±0.21;TfR mRNA表达检测结果显示,对照组(0μmol/L)、10、20、40μmol/L组TfR mRNA表达比分别为1、1.52±0.23、1.91±0.17、2.98±0.14。MC3T3-E1细胞的增生、ALP活性、钙结节和钙化面积随DFS干预浓度的增加呈剂量依赖性降低(P0.05),TfR mRNA的表达随DFS干预浓度的增加呈剂量依赖性升高(P0.05)。结论 DFS可能通过螯合成骨细胞内的铁离子抑制其增生、分化和矿化。     

GSH/GSSG在染氟成骨细胞中的变化

目的通过对谷胱甘肽(GSH)代谢研究来探索氟对成骨细胞氧化应激的影响。方法取材昆明小鼠乳鼠颅骨来源的成骨细胞进行原代培养并传至第3代,用高效液相色谱仪对不同浓度染氟的成骨细胞内 GSH、氧化型谷胱甘肽(GSSG)含量进行检测。结果在含氟1.0、2.0 mgF~-/L 培养液中作用24～72 h,成骨细胞的 GSH 水平多有不同程度的增高,在12.0、20.0 mgF~-/L 氟水平作用下,GSH 水平达到最高;染氟24～72 h 的成骨细胞在0.5、1.0、2.0 mgF~-/L培养液孵育下,细胞的 GSSG 水平有不同程度的下降(除48 h 的0.5、1.0 mgF~-/L 组细胞外);暴露于氟24 h 的成骨细胞在含氟0.5、2.0 mgF~-/L 培养液孵育下,细胞的 GSH/GSSG 比值有不同程度的提高;氟暴露48 h 组,成骨细胞在2.0、8.0mgF~-/L 氟水平作用下,GSH/GSSG 比值亦明显增加。结论低剂量氟作用下 GSH 含量和 GSH/GSSG 比值增加,而GSSG 水平下降,说明成骨细胞处于活跃的抗氧化状态,进一步证明了成骨细胞内氧化应激水平明显增强。     

Hepatocytes of Zones 1 and 3 conjugate sulfobromophthalein with glutathione

The capacity of hepatocytes of Zones 1 and 3 of the liver acinus to conjugate sulfobromophthalein (BSP) with glutathione and to secrete the conjugate into bile was studied. BSP was infused into the in situ perfused rat liver in concentrations of 0.01 and 0.05 mM. Perfusions were performed either in the portal to hepatic vein direction (forward perfusion) or in the hepatic vein to portal vein direction (retrograde perfusion). Hepatocytes contributing to the uptake, metabolism and biliary secretion of BSP were directly assessed qualitatively by light microscopy, and also semiquantitatively by microspectrophotometry. BSP was taken up predominantly by hepatocytes of Zone 1 during forward perfusion and by those of Zone 3 during the retrograde perfusion of BSP. The biliary products of BSP metabolism by each acinar zone were subsequently assessed. Hepatocytes of both Zones 1 and 3 of the acinus secreted BSP into bile in the form of BSP-glutathione, and BSP-glutathione conjugate represented about 78% of the total BSP secreted into bile by each zone. The rate of BSP biliary secretion by both zones was similar at a concentration of 0.01 mM BSP, while a slight decrease (15%) in BSP secretory rate by hepatocytes of zone 3 was observed at concentrations of BSP near biliary Tm. Liver perfusion with exogenous BSP-glutathione provided results similar to those obtained with BSP. In contrast, the perfusion of 3,6- dibromosulphthalein , a compound which is not conjugated by hepatocytes, resulted in similar biliary secretory rates regardless of the direction of perfusion. These results indicate that: (a) the capacity for glutathione conjugation of BSP is distributed among hepatocytes of all acinar zones; (b) near biliary Tm, the biliary secretory rate of BSP by hepatocytes of Zone 3 is slower than that of hepatocytes of zone 1, and (c) in vivo, all hepatocytes likely contribute to the uptake, metabolism and biliary secretion of BSP.     

Induction of cellular glutathione and glutathione S-transferase by 3H-1,2-dithiole-3-thione in rat aortic smooth muscle A10 cells: protection against acrolein-induced toxicity

There is increasing evidence that aldehydes, including acrolein generated endogenously during the degradation process of biological molecules or the metabolism of foreign chemicals may be involved in the pathogenesis of cardiovascular diseases, such as atherosclerosis. Because glutathione (GSH) and GSH S-transferase (GST) are a major cellular defense against the toxic effects of reactive aldehydes, in this study we have characterized the inducibility of GSH and GST by the unique chemoprotective agent, 3H-1,2-dithiole-3-thione (D3T) and their protective effects against acrolein-induced toxicity in rat aortic smooth muscle A10 cells. Incubation of rat aortic A10 cells with micromolar concentrations of D3T resulted in a concentration- and time-dependent induction of both GSH and GST. Treatment of A10 cells with D3T also led to induction of gamma-glutamylcysteine synthetase, the key enzyme involved in GSH biosynthesis. Notably, the levels of GSH and GST remained higher than basal levels 72 h after removal of D3T from the culture media. To examine the protective effects of D3T-induced GSH and GST against reactive aldehyde-mediated toxicity, A10 cells were pretreated with D3T and then exposed to acrolein. Pretreatment of A10 cells with D3T resulted in a marked decrease of acrolein-induced toxicity as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction assay and morphological changes. To further demonstrate the involvement of GSH and GST in protecting against acrolein-induced toxicity, buthionine sulfoximine (BSO) and sulfasalazine were used to inhibit cellular GSH biosynthesis and GST activity, respectively. Either depletion of cellular GSH by BSO or inhibition of cellular GST by sulfasalazine led to a marked potentiation of acrolein-induced toxicity in A10 cells. Furthermore, co-treatment of cells with BSO was found to greatly abolish the protective effects of D3T on acrolein-induced toxicity. Taken together, our results demonstrate for the first time that both GSH and GST in aortic smooth muscle cells can be induced by D3T, and that this increased cellular defense affords great protection against reactive aldehyde-induced cardiovascular cell injury.     

Maintaining good hearing: Calorie restriction,Sirt3, and glutathione

Reducing calorie intake extends the lifespan of a variety of experimental models and delays progression of age-related hearing loss (AHL). AHL is a common feature of aging and is characterized by age-related decline of hearing associated with loss of sensory hair cells, spiral ganglion neurons, and/or stria vascularis degeneration in the cochlea. Sirtuins are a family of NAD⁺-dependent enzymes that regulate lifespan in lower organisms and have emerged as broad regulators of cellular fate. Our recent study indicated that mitochondrial Sirt3, a member of the sirtuin family, mediates the anti-aging effects of calorie restriction (CR) on AHL in mice. Interestingly, we also found that weight loss alone may not be sufficient for maintaining normal hearing. How does CR slow the progression of AHL through regulation of Sirt3? Here we review the evidence that during CR, Sirt3 slows the progression of AHL by promoting the glutathione-mediated mitochondrial antioxidant defense system in mice. A significant reduction in food consumption in one's daily life may not be a desirable and realistic option for most people. Therefore, identification/discovery of compounds that induce the activation of SIRT3 or glutathione reductase, or that increase mitochondrial glutathione levels has potential for maintaining good hearing through mimicking the anti-aging effects of CR in human inner ear cells.     

The effects of exogenous glutathione on reduced glutathione level, glutathione peroxidase and glutathione reductase activities of rats with different ages and gender after whole-body Γ-irradiation

Age-and gender-related changes on reduced glutathione (GSH) level, glutathione peroxidase (GPx) and glutathione reductase (GR) activities in the liver of rat exposed to different dose of whole-body g-ray irradiation were determined. In addition, the effect of administration of exogenous GSH on endogenous GSH levels, GPx and GR activities was investigated. For this aim, male and female rats aged 1 and 5 moths were divided into two groups as g-ray and g-ray+GSH. Both groups were again divided into four groups as irradiated with 2, 4, 6 and 8 Gy doses. GSH level and GPx activity did not change with age while GR activity was decreased with age. Gender-dependent changes in GPx and GR activities were observed, but GSH values were not affect by sex. GSH levels, GPx and GR activities were not observed dose-associated changes of g-irradiation. GSH level and GPx activity in the 8Gy group were increased by GSH. GR activities of old male rats were found to be increased by glutathione in the 6 and 8Gy groups. These results indicate that radiation and administration of exogenous GSH affect gender-and age-dependent GSH level, GPx and GR activities in the rats.     

Hepatic glutathione homeostasis in the rat: efflux accounts for glutathione turnover

Hepatic glutathione turnover and the efflux of glutathione from the liver into bile and blood were measured in male Sprague-Dawley rats in vivo. In fed rats the efflux of glutathione into blood, calculated from the hepatic arteriovenous concentration gradient and hepatic blood flow, amounted to 12.4 +/- 1.4 nmoles min X gm liver. Together with the excretion of glutathione into bile (3.4 +/- 0.4 nmoles per min X gm liver) total efflux accounted for the hepatic turnover of glutathione of 15.2 +/- 0.9 nmoles per min X gm liver. Fasting animals for 48 hr markedly increased hepatic glutathione turnover to 26.4 +/- 1.2 nmoles per min X gm liver. Increased efflux into blood rather than increased intrahepatic catabolism accounted for this increased turnover. The systemic clearance of glutathione was 3.22 +/- 0.51 ml per min X 100 gm body weight. The efflux of glutathione from liver therefore was calculated to contribute over 90% of total glutathione inflow into the circulation, as determined from the clearance and the arterial concentration of glutathione. Thus, the liver is the major source of plasma glutathione, and turnover of hepatic glutathione in the basal state is accounted for almost entirely by efflux of glutathione from the liver. During fasting, the plasma clearance of exogenous glutathione increased to 5.32 +/- 0.35 ml per min X 100 gm body weight, and the utilization of methionine for glutathione synthesis increased markedly. The increased extrahepatic catabolism during fasting results in a decrease in plasma glutathione, which in turn may account for the observed increase in sinusoidal glutathione efflux with concomitant stimulation of the rate of hepatic glutathione turnover and of synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion of glutathione to glutathione disulfide by cell membrane-bound oxidase activity.

An apparently specific glutathione oxidase activity is present in renal cortex, epididymal caput, jejunal villus tip cells, choroid plexus, and retina (but not in liver). The activity is membrane-bound and is localized on the luminal surface of the brush border membranes of the kidney and jejunum. The distribution and localization of the oxidase are similar to those of gamma-glutamyl transpeptidase, suggesting that there is a significant relationship among the translocation of intracellular glutathione, the extracellular oxidation of glutathione to glutathione disulfide, and the reactions of the gamma-glutamyl cycle. Thus, both glutathione present in the blood plasma and intracellular glutathione translocated to the cell surface are accessible to oxidation and transpeptidation. Acceptor substrates of the transpeptidase (e.g., L amino acids) promote transpeptidation and decrease oxidation of glutathione. Conversion of glutathione to glutathione disulfide is followed by utilization of the latter compound by gamma-glutamyl transpeptidase and dipeptidase. Although intracellular oxidation of glutathione to glutathione disulfide is readily reversed by the action of glutathione reductase, glutathione disulfide formed extracellularly cannot be reduced; instead, it undergoes hydrolytic and transpeptidation reactions leading to gamma-glutamyl amino acid and amino acid products which may be recovered by being transported into the cell.     

Efflux of glutathione and glutathione complexes from human erythrocytes in response to vanadate

The main objective of the present study was to investigate if vanadate is extruded from the cells in a glutathione dependent manner resulting in the appearance of extracellular glutathione and complexes of glutathione with vanadium. Vanadate significantly depleted intracellular non-protein sulfhydryl (NPSH) levels in a time- and concentration-dependent manner. The intracellular NPSH level was decreased to 0.0 ± 0.0 μmol/ml erythrocyte when exposed to 10 mM of vanadate for 4 h. Extracellular NPSH level was increased concomitantly with the intracellular decrease and reached to 0.1410 ± 0.005 μmol/ml erythrocyte in 4 h. Intracellular decrease and extracellular increase in NPSH levels were significantly inhibited in the presence of DIDS, a chloride-bicarbonate exchanger which also mediates phosphate and arsenate transport in erythrocytes. In parallel with the increase in extracellular NPSH levels, significant increases in extracellular glutathione levels were detected following exposure to vanadate. Extracellular glutathione levels reached to 0.0150 ± 0.0.001, 0.0330 ± 0.001, and 0.0576 ± 0.002 μmol/ml erythrocyte with 1, 5, and 10 mM of vanadate respectively. Dimercaptosuccinic acid treatment of supernatants significantly increased the glutathione levels measured in the extracellular media. Utilization of MK571 an MRP inhibitor decreased the rate of glutathione efflux from erythrocytes suggesting a role for this membrane transporter in the process. A known methylation inhibitor periodate oxidized adenosine decreased the rate of glutathione efflux from erythrocytes. This observed decrease in extracellular GSH levels suggests that GSH release partly requires a proper cellular methylation process and that part of GSH detected in the extracellular media may arise from GSH–vandium complexes. The results of the present study indicate that human erythrocyte efflux glutathione in reduced free form and in conjugated form/s that can be recovered with dimercaptosuccinic acid when exposed to vanadate.     

Protecting against peroxynitrite-mediated cytotoxicity in vascular smooth muscle cells via upregulating endogenous glutathione biosynthesis by 3H-1,2-dithiole-3-thione

Peroxynitrite (ONOO⁻) is critically involved in the pathogenesis of cardiovascular diseases. Reaction with glutathione (GSH) was proposed to be a major detoxification pathway of ONOO⁻ in the biological system. This study was undertaken to determine if chemically elevated intracellular GSH affords protection against ONOO⁻-mediated toxicity in vascular cells. Incubation of aortic smooth muscle A10 cells with 3H-1,2-dithiole-3-thione (D3T) led to a concentration- and time-dependent elevation of cellular GSH. Treatment of the cells with D3T also augmented protein and gene expression of γ-glutamylcysteine ligase. To examine the effects of D3T-induced GSH on ONOO⁻-mediated toxicity, we pretreated A10 cells with D3T and then exposed them to either authentic ONOO⁻ or the ONOO⁻ generator, 3-morpholinosydnonimine. We observed that D3T pretreatment of A10 cells resulted in a significant protection against ONOO⁻ cytotoxicity. Conversely, depletion of cellular GSH by buthionine sulfoximine (BSO) caused a marked potentiation of ONOO⁻ cytotoxicity. To further demonstrate the causal involvement of GSH induction in D3T cytoprotection, we cotreated A10 cells with BSO to abolish D3T-induced GSH elevation. BSO cotreatment was found to greatly reverse the protective effects of D3T on ONOO⁻-elicited cytotoxicity. Taken together, our results demonstrate that upregulating GSH biosynthesis by D3T results in a marked protection against ONOO⁻-induced toxicity in vascular cells.     

S-Acetylglutathione normalizes intracellular glutathione content in cultured fibroblasts from patients with glutathione synthetase deficiency

Glutathione synthetase deficiency is an autosomal recessive inherited metabolic defect in the gamma-glutamyl cycle. Decreased intracellular glutathione levels are one of the characteristic biochemical features. In this study we show that addition of S-acetylglutathione to the medium raised intracellular glutathione content in cultured fibroblasts from patients with glutathione synthetase deficiency. This has implications for the treatment of patients with this inborn error of metabolism.