褪黑素对苯妥英诱发胚胎脑组织氧化性损伤的拮抗作用

目的　研究褪黑素 (MT)对苯妥英 (Phe)诱发的胚胎脑组织氧化性损伤有无保护作用。方法　妊娠Wistar母鼠于妊娠 (GD11～ 14 )ig 0和 10 0mg·kg- 1Phe ,4 0mg·kg- 1MT及 4 0mg·kg- 1MT +10 0mg·kg- 1Phe ;GD15脱颈椎处死动物 ,测定胎鼠脑组织各种与氧化性损伤有关的指标。结果　妊娠期染毒Phe导致胚胎脑组织H2 O2 水平升高 ,脂质过氧化和蛋白质氧化产物增加 ,超氧化物岐化酶、过氧化氢酶和谷胱甘肽过氧化物酶活性下降 ,总还原型谷胱甘肽(GSH)含量及总抗氧化力降低。妊娠期MT处理减少正常胎鼠脑中H2 O2 及产率 ;同时MT和Phe共同处理可阻断Phe诱发的氧化性损伤 ,GSH耗竭和抗氧化酶活性的下降。结论　MT对Phe诱发的胎鼠脑组织氧化性损伤有一定的拮抗作用。     

大鼠出生前接触苯妥英和(或)褪黑素对学习记忆的影响

目的　探讨褪黑素对苯妥英诱发的仔代学习记忆功能发育异常的影响。方法　Wistar孕大鼠于妊娠d 11～ 14ig苯妥英 10 0 ,2 0 0mg·kg- 1·d- 1或合并ig褪黑素 4 0mg·kg- 1(每日 3次 ) ,观察F1代仔鼠的味觉回避反射、穿梭反射和Morris水迷宫空间识别能力。结果　出生前染毒仔鼠成年后味觉回避反射、主动回避反射及空间识别等学习和记忆能力下降。褪黑素和苯妥英合并用药组仔鼠上述 3种学习和记忆能力均有不同程度的改善。结论　褪黑素对苯妥英诱发的大鼠仔代学习记忆功能发育异常具有拮抗作用 ,该效应可能与其拮抗胚胎脑组织中氧化应激反应有关。     

大鼠出生前接触苯妥英和褪黑素对仔代反射功能发育及自发运动的影响

目的 :探讨苯妥英 (DPH)神经发育毒性与胚胎脑组织中自由基产生和氧化应激反应的关系。方法 :Wistar孕鼠于妊娠d 11～ 14经 0 ,10 0 ,2 0 0mg·kg-1DPH或合并 4 0mg·kg-1褪黑素 (MT)染毒处理 ,研究MT对DPH的仔代反射功能发育及自发运动损害作用的拮抗效应。结果 :孕鼠在染毒期及染毒后增重下降 ,仔代体重减轻 ,哺乳期死亡率增高 ;DPH染毒仔鼠的转身运动增多 ,空中翻正反射及游泳能力发育延迟 ,成年后行走次数、站立次数、刻板动作等自发活动增多 ,旋转手比率增多 ,对阿朴吗啡“激发”反应性增强。MT和DPH合并处理可明显拮抗上述DPH仔鼠的行为异常。结论 :氧化性损伤在DPH神经发育毒性发生中发挥重要作用 ,而MT可拮抗其毒性作用。     

金属硫蛋白对阿霉素致小鼠心脏氧化损伤的影响

目的研究金属硫蛋白(metallothionein,MT)对阿霉素(Doxorubicin,DOX)心脏氧化损伤的影响。方法雄性野生型小鼠(MT / )及敲除MT基因的转基因小鼠(MT-/-)随机分成4组,即对照组、给药组(DOX)、锌预处理组(Zn)、预处理给药组(Zn DOX),每组6只动物。动物单次腹腔注射DOX(15 mg/kg)或生理盐水(NS),此前24及48 h分别给予ZnSO4(20 mg/kg,sc)或用生理盐水预处理。DOX给药4 d后处死动物,测定血浆中肌酸激酶(CK)及乳酸脱氢酶(LDH)活力,取心脏制备组织匀浆,测定脂质过氧化产物丙二醛(MDA)以及蛋白羰基产物含量。结果DOX能引起MT / 小鼠及MT-/-小鼠血浆CK、LDH活力升高(P<0.01),心脏组织MDA以及蛋白羰基产物含量增加(P<0.01),而且MT-/-小鼠变化更为明显(P<0.01)。Zn预处理能显著降低DOX引起的MT / 小鼠CK、LDH活力升高,同时抑制心脏组织的脂质过氧化以及蛋白的羰基化。然而,这种抑制效应在MT-/-小鼠中没有出现。结论Zn诱导MT表达增强可抑制DOX引起的心脏氧化损伤,MT缺失可导致DOX心脏氧化损伤加重,提示体内MT对DOX诱发的心脏氧化损伤具有保护作用。     

氧自由基与肝细胞损伤

在各种原因所致的肝细胞初始和后续损伤过程中,氧自由基(OFR)起了非常重要作用.肝细胞在缺血再灌注时,OFR及其诱发的脂质过氧化产物(LPO)可引起激烈的链式反应,产生几十种毒性分子和活性基因,再次诱发生成更多的OFR,继而加速加重肝细胞膜、细胞器、蛋白质以及DNA的损伤[1-4].OFR致肝细胞损伤的病理生理过程及其致病机理目前尚未完全清楚,清除及拮抗OFR是在各种肝病治疗中不可或缺的手段之一[1、4、5].本文就近年有关OFR致肝细胞损伤的分子生物学方面研究进展作一简述.     

草乌的体外胚胎发育毒性研究

目的探讨生草乌对胚胎发育的毒性作用及机制。方法应用大鼠着床后体外全胚胎培养模型,将9.5dSD大鼠胚胎与含不同浓度生草乌的大鼠即刻离心血清共培养48h,观察其对大鼠胚胎生长发育和组织器官形态分化的影响。生草乌终浓度分别为:0、0.63、1.25、2.5和5mg生药/ml。结果随着生草乌剂量增加,胚胎生长发育和器官分化的各项指标均呈现下降趋势,有一定的剂量-效应关系。生草乌最大无作用剂量为1.25,2.5mg/ml以上剂量可诱发卵黄囊生长和血管分化不良、生长迟缓及形态分化异常,严重者出现体节紊乱、小头、心脏发育迟滞(心小,停留在心管期)及心脏空泡等。结论较高剂量生草乌对体外培养大鼠胚胎具有一定的毒性作用,建议孕妇妊娠期间(特别是妊娠前3个月)慎用或禁用草乌。     

乙醇和乙醛对培养大鼠胚胎卵黄囊细胞膜脂质流动性的影响

为探讨酒精对发育中胚胎卵黄囊 ( VYS)膜功能的影响 .孕 9.5d大鼠胚胎于体外分别给予不同剂量的酒精及其代谢产物乙醛 ,培养 48h后 ,分离胚胎 VYS细胞 .以 1 ,6-二苯己三烯 ( DPH)为荧光探针研究二者对 VYS细胞膜脂质流动性的影响 .结果表明 0 .4g· L-1乙醇 ,2 .0 g· L-1乙醛并不影响 VYS细胞膜脂质的荧光偏振度 ( P)和细胞膜流动度 ( LFU) ,随剂量增高可导致 VYS荧光偏振度降低 ,膜脂质流动度增高 ,相同剂量 ( 2 .0 g· L-1)的乙醇 ,乙醛对 VYS细胞作用无显著性差异 .乙醇 ,乙醛均能改变 VYS细胞膜脂质流动性引起 VYS细胞功能紊乱 ,它很可能在诱发胎儿酒精综合征起重要作用 .     

葛根素对红细胞膜脂质过氧化损伤的防护作用

目的:研究葛根素对红细胞膜脂质过氧化损伤的防护作用.方法:采用3种活性氧产生体系诱发脂质过氧化为实验模型,观察了葛根素对膜脂质过氧化作用的防护作用.结果:葛根素对黄嘌呤-黄嘌呤氧化酶(Xan-Xo)系统、H2O2及UV照射3种方法引起的细胞膜脂质过氧化产物丙二醛(MDA)的生成增加均有抑制作用,其作用呈剂量依赖性,IC50分别为16.95,14.44和14.90 mg·L-1.结论:葛根素对自由基引起的细胞膜脂质过氧化损伤有防护作用.     

硫酸铝在体外对大鼠胚胎组织谷胱甘肽含量和卵黄囊细胞膜流动性的影响

目的　为探讨铝的发育毒性及机理。方法孕 9.5d大鼠胚胎于体外培养系统中给予不同剂量的硫酸铝 ,培养 4 8h后 ,观察胚胎生长发育和器官形态分化状况 ;应用二硫代双硝基苯甲酸 (DTNB)直接法测定胚胎组织谷胱甘肽 (GSH)含量 ;以 1,6 二苯己三烯为荧光探剂 ,用荧光偏振技术测定卵黄囊细胞膜脂质流动性。结果　当培养液中铝浓度为1.2mg·L- 1时 ,胚胎生长发育和分化明显被抑制 ;3.0mg·L- 1时 ,畸形胚胎发生率明显升高 ,主要有神经管闭合不全 ,脑发育不良和体翻转不全 ;6 .0mg·L- 1时 ,胚胎组织GSH含量和卵黄囊细胞膜脂质流动性显著降低。上述效应均呈现出一定的剂量 效应 (反应 )关系。结论　铝有潜在的致畸性和胚胎毒性 ,胚胎组织GSH含量和卵黄囊细胞膜流动性降低可能在铝致胚胎发育毒性中起重要作用     

金粉蕨素对脂质过氧化损伤内皮细胞的保护作用

目的 :探讨金粉蕨素对脂质过氧化损伤人内皮细胞株 (ECV30 4 )的保护作用及其可能的分子作用机制。方法 :在铜离子诱发的低密度脂蛋白氧化修饰的基础上 ,建立内皮细胞 (endothelialcell ,EC)的脂质过氧化损伤模型 ;采用MTT比色法、硝酸还原酶法等方法 ,测定不同浓度金粉蕨素 (1.0、5 .0、10 .0 μmol·L-1)对EC生长抑制率、硫代巴比妥反应物 (thiobarbituricacid reactivesubstances ,TBARS)、LDH及NO含量的影响。结果 :氧化修饰低密度脂蛋白 (oxidativelymodifiedlowdensitylipoprotein ,ox LDL)作用 2 4h后 ,对EC的生长增殖有明显的抑制作用 ,细胞培养液中TBARS、LDH水平显著升高 ,NO含量降价 ;而不同浓度金粉蕨素与oxLDL共同孵育2 4h ,呈剂量依赖性对抗oxLDL对EC的损伤 ,并促进EC增殖 ,同时使培养液中TBARS、LDH含量降低 ,NO含量回升。结论 :脂质过氧化能直接损伤内皮细胞 ,金粉蕨素对脂质过氧化损伤的内皮细胞有保护作用     

Evidence for Ras-dependent signal transduction in phenytoin teratogenicity

Reactive oxygen species (ROS) can transduce intracellular signals or damage macromolecules, including mutational activation of the K-ras oncogene, particularly in A/J mice, which are reported to be highly sensitive to the ROS-initiating teratogen phenytoin. Here, we determined in embryo culture whether the Ras pathway mediated phenytoin embryopathy at the protein or gene level. Embryos from pregnant inbred A/J dams and outbred CD-1 dams were cultured with a therapeutic concentration of phenytoin, with or without alpha-hydroxyfarnesylphosphonic acid, an inhibitor of the enzyme farnesyl-protein transferase, which is required for posttranslational Ras activation. A/J and CD-1 embryos were similarly susceptible to phenytoin embryopathies, with reductions in anterior neuropore closure, turning, yolk sac diameter, and somite development (p < 0.05). The farnesyl-protein transferase inhibitor blocked phenytoin embryotoxicity in A/J embryos for all parameters except yolk sac diameter (p < 0.05) and completely blocked embryotoxicity in CD-1 embryos (p < 0.05). Embryonic DNA did not show phenytoin-initiated mutations in codon 12 of the K-ras gene in either A/J or CD-1 embryos, but phenytoin substantially increased the levels of GTP-bound Ras in both CD-1 and A/J embryos. These results provide the first direct evidence that Ras proteins may be involved in the teratogenicity of phenytoin, likely via a mechanism other than mutational activation.     

A coupled microsomal-activating/embryo culture system: Toxicity of reduced β-nicotinamide adenine dinucleotide phosphate (NADPH)

An NADPH-dependent microsomal-activating system has been coupled to a rat embryo culture in vitro. No embryonic morphological abnormalities or decreases in final yolk sac or embryo DNA and protein contents occurred when 0.2 mM NADPH was used in this coupled system. In contrast, 1.0 mM NADPH alone, or 0.2 mM NADPH in the presence of microsomes and a glucose-6-phosphate dehydrogenase-based NADPH-generating system, greatly reduced embryo and yolk sac growth in vitro. The toxicity of NADPH was not due to lipid peroxidation. Only minor decreases in final yolk sac protein levels occurred when embryos were grown in media containing male rat microsomes and 1.0 mM NADPH. The protective effect of rat hepatic microsomes on NADPH toxicity does not seem to have been due to the oxidation of NADPH to the less toxic NADP. Although cyclophosphamide alone was not toxic to rat embryos cultured in vitro, in the coupled microsomal-activating/embryo culture system, cyclophosphamide reduced yolk sac and embryo growth and caused abnormal embryonic differentiation. The uses of the coupled microsomal-activating/embryo culture system to study mechanisms in anomalous development, as well as its possible use in embryo toxicity and teratogenicity testing, are discussed.     

Modulation of acrylonitrile-induced embryotoxicity in vitro by glutathione depletion

The effects of glutathione (GSH) depletion on the embryotoxicity of acrylonitrile were assessed in vitro using the rat whole-embryo culture system. Day 10 rat embryos were cultured in rat serum medium for 6 h in the presence of 250 Ml-buthionine-S,R-sulfoximine (BSO), a specific inhibitor of GSH synthesis, to deplete GSH in both embryo and visceral yolk sac. Following pretreatment, conceptuses were cultured for an additional 21 h in the presence of 152, 228, or 304 M acrylonitrile. At the end of the culture period, conceptuses were assessed for survival, growth and development, malformations, and the protein and glutathione content of embryos and yolk sacs were assayed. Acrylonitrile alone produced concentrationrelated and statistically significant decreases in yolk sac diameter, crown-rump length, head length and number of somite pairs, as well as in embryonic and yolk sac proteins. The chemical also caused dysmorphogenesis of the brain and of the caudal extremity, and a concentration-related and statistically significant increase in GSH content in the yolk sac. Pretreatment with BSO significantly enhanced the embryotoxic effects of acrylonitrile. The conceptuses displayed further decreases in functional yolk sac circulation, yolk sac diameter, crown-rump and head length, when compared to either acrylonitrile or BSO alone. The incidence of caudal malformations and the severity of brain malformations produced by acrylonitrile were also increased. Marked decreases in embryonic and yolk sac GSH contents were observed after exposure to BSO alone or in combination with acrylonitrile. Thus, depletion in embryonic and yolk sac GSH by BSO enhanced teratogenic and growth retarding effects of acrylonitrile in vitro, suggesting that GSH plays a critical role in modulating acrylonitrileelicited embryotoxicity.     

Microinjection of cultured rat embryos: a new technique for studies in chemical dysmorphogenesis

The utility of a new technique for exposure of cultured whole rat embryos to potential dysmorphogens was demonstrated with nitrosofluorene (NF), a cytotoxic and mutagenic metabolite of 2-acetylaminofluorene (AAF). At an initial concentration in the culture medium of 41 microM, NF produced a 100% incidence of defects in axial rotation with no significant effect on prosencephalic development, consistent with previous reports. This route of exposure was also associated with a significant decrease in yolk sac vasculature and protein content. However, when 2 to 20 ng of NF was microinjected directly into the amniotic space, the predominant malformation observed was prosencephalic hypoplasia. Injection of 10 ng NF resulted in approximately equivalent decreases in viability as 41 microM NF dissolved in the culture medium, but produced only a 41% incidence of rotation defects and a 27% incidence of open neural tubes in the rhombencephalic region. The protein content of injected conceptuses was significantly reduced in the embryo, but not in the visceral yolk sac. When 10 ng of NF was injected inside the yolk sac but outside the amnion, the incidence of abnormal rotation was increased to 75%, and the severity of prosencephalic hypoplasia as well as the incidence of neural tube abnormalities was attenuated. The protein content of both the embryo and yolk sac was significantly decreased relative to that of the controls. The data are consistent with the suggestion that NF elicits defects in axial rotation primarily via its effects on the visceral yolk sac and demonstrate the capacity of this technique to provide insights into mechanistic aspects of chemical dysmorphogenesis.     

Apoptosis in Embryos of Diabetic Rats

Abstract: The aim of the present study was to determine whether maternal diabetes affects rat embryo and yolk sac apoptosis during the postimplantation period. Severely malformed and growth-retarded embryos of gestational day 12 from diabetic rats exhibited pronounced DNA laddering on agarose gels. On the other hand, no DNA laddering could be observed in any of the non-malformed embryos from control and diabetic rats, or in their corresponding yolk sacs. Analysis of embryos of gestational day 10 revealed only a few scattered TUNEL positive cells mainly located in the allantois, the foregut epithelium, the cranial neuroepithelium and in the cranial mesenchyme. Embryonic tissue of gestational day 12 showed numerous aggregates of TUNEL-positive cells, indicating developmental remodelling of multiple organs. Analysis of non-malformed embryos of day 10 and 12 revealed a distribution and frequency of TUNEL positive cells unaffected by the diabetic state of the mother on both days. In vitro incubation (2–8 hr) of normal day-12 yolk sacs resulted in strong DNA laddering, but not in the corresponding embryos. Dispersed yolk sac cells generated higher levels of reactive oxygen species than dispersed embryonic cells. Reactive oxygen species levels in both embryonic and yolk sac cells were unaffected by the diabetic state of the mother. Moreover, immunoblot analysis showed high Bcl-2 and undetectable caspase-1 levels in embryos from both normal and diabetic rats and low Bcl-2 and high caspase-1 levels in the corresponding yolk sacs. Immunohistochemical analysis of embryos demonstrated caspase-1-reactivity in a small subpopulation of cells located in proximity to TUNEL-positive cells. We conclude that the inherent capacity of embryonic cells to enter apoptosis in vitro is low as compared to yolk sac cells, and that wide-spread apoptosis is not likely to play a major role in diabetes-induced dysmorphogenesis but rather in early phases of resorption of severely malformed and developmentally retarded embryos.     

Influence of electrophilic character and glutathione depletion on chemical dysmorphogenesis in cultured rat embryos

To examine the importance of reduced intracellular glutathione (GSH) in the modulation of dysmorphogenesis and to gain insight into the electrophilic character of the embryotoxic intermediates generated in the rat embryo from N-acetoxy-2-acetylaminofluorene (AAAF) and acetaminophen (APAP) in cultured embryos, the effects of GSH depletion on the embryotoxicity, dysmorphogenesis and covalent binding of these agents were examined. Both AAAF (90 microM) and APAP (500 microM) produced concentration-dependent, statistically significant (P less than or equal to 0.05) decreases in embryonic length as well as embryonic and visceral yolk sac protein content when rat embryos were exposed in vitro between days 10 and 11 of gestation. The predominant malformations observed upon exposure to AAAF and APAP were prosencephalic hypoplasia and abnormal neurulation respectively. Exposure of conceptuses to [3H]APAP followed by separation and fractionation of the cellular RNA, DNA and protein via density gradient centrifugation resulted in detectable binding in fractions that contained protein, but not DNA or RNA. This suggested that the rat conceptus is capable of bioactivating APAP to a soft electrophile that selectively arylates protein. In contrast, conceptuses exposed to [3H]AAAF exhibited detectable binding to RNA, DNA and protein, indicative of conversion to both hard and soft electrophiles. Depletion of GSH was accomplished by pretreating conceptuses with 500 microM L-buthionine-S,R-sulfoximine (BSO) from the start of the culture period (day 9.5) until the morning of day 10. When conceptuses were depleted previously of GSH by BSO, exposure to APAP resulted in significant potentiation (relative to APAP alone) of the observed embryotoxicity. These conceptuses displayed further decreases in both embryonic size and protein content of the embryo and yolk sac, as well as increased incidence of abnormally open anterior neuropores and increased binding (3-fold) of [3H]APAP to protein. In contrast, pretreatment with BSO did not potentiate the AAAF-elicited decreases in embryonic size or protein content, nor the severity of prosencephalic hypoplasia, although a slight increase in binding of [3H]AAAF to DNA was observed. Taken together, these data are consistent with the concept that abnormal neurulation elicited by APAP results from the generation of one or more soft electrophilic species, whereas elicitation of prosencephalic hypoplasia by AAAF appears to be a consequence of conversion to a relatively hard electrophile(s).