竹红菌甲素（HA）光敏致实作用的研究

以中国苍鼠卵巢成纤维细胞（ＣＨＯ）为材料,通过ＨＡ光敏诱变,乌本甙筛选研究了竹红菌甲素光敏反应对细胞Ｎａ＾＋／Ｋ＾＋ＡＴＰ酶基因的诱变致突作用。结果表明ＨＡ光敏反应可造成细胞Ｎａ＾＋／Ｋ＾＋ＡＴＰ酶基因点突变,并具有遗传稳定性。另外通过测定光敏反应细胞脂质过氧化程度衣其产物与细胞ＤＮＡ形成的荧光产物表明脂质过氧化在细胞ＤＮＡ的损伤突变过程中,起着重要的作用。     

竹红菌甲素（HA）光敏致实作用的研究

以中国苍鼠卵巢成纤维细胞（ＣＨＯ）为材料,通过ＨＡ光敏诱变,乌本甙（ｏｕａｂａｉｎ）筛选研究了竹红菌甲素光敏反应对细胞Ｎａ＋／Ｋ＋ＡＴＰ酶基因的诱变致灾作用。结果表明ＨＡ光敏反应可造成细胞Ｎａ＋／Ｋ＋ＡＴＰ酶基因点突变,并具有遗传稳定性。另外通过测定光敏反应细胞脂质过氧化程度及其产物与细胞ＤＮＡ形成的荧光产物表明脂质过氧化在细胞ＤＮＡ的损伤突变过程中,起着重要的作用。     

天然抗氧化剂丹参酮Ⅱ—A对肝细胞脂质过氧化产物与DNA相互作用的影响

［＾３Ｈ］花生四烯酸标记的肝细胞,经ＦｅＣｌ２－ＤＴＰＡ启动脂质过氧化后,细胞ＤＮＡ出现放射性,并随保温时间增加而逐渐增高,表明在细胞内脂质过氧化产物与ＤＮＡ发生相互作用,生成了一种ＤＮＡ加成物,经测定它具有特征荧光光谱,显示较低的增色效应和Ｔｍ值。用高度敏度荧光图象显微镜直接观察发现丹参酮Ⅱ－Ａ经细胞摄取后主要滞留在细胞膜与胞浆中。它能有效地抑制细胞脂质过氧化,减少脂质－ＤＮＡ加成物的产生,并阻     

天然抗氧化剂丹参酮Ⅱ—A对肝细胞脂质过氧化产物与DNA相互作用的影响

［３Ｈ］花生四烯酸标记的肝细胞,经ＦｅＣｌ２－ＤＴＰＡ启动脂质过氧化后,细胞ＤＮＡ出现放射性,并随保温时间增加而逐渐增高,表明在细胞内脂质过氧化产物与ＤＮＡ发生相互作用,生成了一种ＤＮＡ加成物,经测定它具有特征荧光光谱,显示较低的增色效应和Ｔｍ值。用高度敏度荧光图象显微镜直接观察发现丹参酮Ⅱ－Ａ经细胞摄取后主要滞留在细胞膜与胞浆中。它能有效地抑制细胞脂质过氧化,减少脂质－ＤＮＡ加成物的产生,并阻止了细胞存活率和Ｏ６甲基鸟嘌呤转移酶活性的降低,其抑制率与ＶｉｔＥ,ＢＨＴ相近,但显著高于ＮａＮ３,甘露醇和ＳＯＤ。上述结果提示丹参酮Ⅱ－Ａ是一种新的有效的细胞内脂质过氧化产物与ＤＮＡ相互作用的抑制剂。它对ＤＮＡ的保护作用可能是通过清除脂类自由基而阻断脂质过氧化的链式反应,抑制ＤＮＡ加成物的生成,从而减少了细胞毒性。     

抗氧化剂对HeLa细胞在竹红菌甲素（HA）光敏损伤下的保护作用的研究

通过细胞台盼兰拒染检测、电镜观察以及＾３Ｈ－Ｔｄｒ同位素掺入等方法,分别从细胞水平及分子水平上,定性定量测定了维生素Ｅ、丁羟基甲苯（ＢＨＴ）、阿魏酸钠（ＳＦ）三种抗氧化剂对于竹红菌甲素（ＨＡ）光敏所致ＨｅＬａ细胞损伤的抑制。结果表明：三种抗氧化剂对于细胞的损伤均有不同程度的抑制作用,其中ＶｉｔＥ的抑制作用最明显,ＢＨＴ及ＳＦ的抑制作用则较弱,提示脂质过氧化在细胞膜和细胞核损伤中起着重要的作用。     

Cd^2＋,Al^3＋作用下蚕豆UDS与微核相关性分析及高等植物UDS技？…

为探讨金属离子对高等植物非按期ＤＮＡ合成（Ｕｎｓｃｈｅｄｕｌｅｄ ＤＮＡ Ｓｙｔｈｅｓｉｓ,简称ＵＤＳ）和微核（ＭＣＮ）的诱导作用,二者之间的关联性以及利用高等培植物ＵＤＳ技术检测环境诱变物的可行性,利用＾３Ｈ－ＴｄＲ前体掺入法研究了Ｃｄ＾２＋,Ａｌ＾３＋作用下蚕豆的ＵＤＳ效应。     

竹红菌乙素修饰物（－乙醇胺）对于腹水肝癌细胞光敏损伤的研究

通过竹红菌乙素修饰物（－乙醇胺,简称ＨＢ－Ｅ）对小鼠腹水肝癌细胞光敏损伤以及ＨＢＥ被ＡＨ细胞摄取过程的研究,结果表明,ＨＢ－Ｅ在６４０ｎｍ光照的条件下对ＡＨ细胞产生很强的光动力作用,细胞对ＨＢ－Ｅ的摄取速度快（室温下２ｍｉｎ,达到平衡）在饱和时每个细胞可摄取ＨＢ－Ｅ分子５×１０＾９个;经多种竹红菌素修饰物光敏能力的比较中ＨＢ－Ｅ光敏作用明显要大于其它的光敏剂,光敏机理的研究中确定了活性氧的作用是存     

.OH在博莱霉素A5介导的DNA断链反应中的作用

利用ＥＳＲ技术研究了博莱霉素Ａ５（ＢＬＭＡ５）、Ｆｅ＾２＋,Ｏ２体系中．ＯＨ的产生。并用ＴＢＡ反应检测了该系对ＤＮＡ的断链作用。以Ｆｅ＾３＋,Ｃｕ＾＋或Ｃｕ＾２＋替换Ｆｅ＾２＋,则体系中无．ＯＨ产生,同时也失去对ＤＮＡ的断链作用;一定浓匠．ＯＨ清除剂可完全清除．ＯＨ,地对ＤＮＡ断链影响甚微;还原剂可淬灭体系中的．ＯＨ,却能促进ＤＮＡ的断链反应;ＳＯＤ可清除体系中的．ＯＨ,失活的ＳＯＤ夫此功能,但两     

辐射及活性氧对DNA的损伤以及芥子碱的保护作用

在Ｘ射线照射下,小牛胸腺ＤＮＡ的碱基损伤及链断裂随着剂量升高而增加,其损伤主要集中于链断裂;活性氧可以引起ＤＮＡ损伤,Ｈ２Ｏ２仅造成少量伤害,当在含有Ｈ２Ｏ２的体系中加入微量的Ｃｕ＾２＋、Ｆｅ＾２＋时损伤急剧增加,这是由反应产生的．ＯＨ所致,Ｃｕ＾２＋的致损伤效果明显高于Ｆｅ＾２＋。ＯＨ清除剂芥子碱具有很强的抗辐射及抗氧化作用,且对ＤＮＡ无伤害。这说明ＯＨ在ＤＮＡ的氧化损伤中起重要作用。     

缺氧预处理对乳鼠心肌细胞蛋白激酶C活性的影响

在培养的乳鼠心肌细胞缺氧／复氧模型上,观察了缺氧预处理的细胞保护作用及其对细胞蛋白激酶Ｃ活性和蛋白磷酸化的影响。结果表明,ＡＰＣ可减轻心肌细胞的Ｈ／Ｒ损伤;提高细胞存活率,减少细胞脂质过氧化产物生成及细胞内乳酸脱氢酶和蛋白质漏出。模拟ＡＰＣ的短暂缺氧显著激活ＰＫＣ,使心肌细胞内分子量为６６ｋＤ和３１ｋＤ的蛋白条带＾３２Ｐ掺入增加;ＰＫＣ抑制剂Ｈ７完全消除ＡＰＣ对心肌细胞的保护作用,并抑制了短暂缺氧     

The effect of hypericin and hypocrellin-A on lipid membranes and membrane potential of 3T3 fibroblasts

Hypericin (HY) and Hypocrellin-A (HA) photosensitization induce rapid depolarization of plasma membrane in 3T3 cells as revealed by confocal microspectrofluorimetry using diO-C5(3) fluorescent probe. HY and HA are also able to rigidify the lipid membrane of DMPC liposomes as indicated by the decrease of pyrene excimer fluorescence used as a marker of the lipid membrane fluidity. We have also observed a nonspecific inhibition of Na+,K+-ATPase activity due to the HY and HA photosensitization. The described effects are concentration- and light dose-dependent and generally more pronounced for HA than for HY. All these observations suggest that the lipid membranes can play an important role in the photosensitization process induced by HY and HA at the cellular level. It can be hypothesized that for HA and HY the secondary mechanism following type I or type II photosensitization process can be the peroxidation of membrane lipids as well, and thus intracellular membranes seem to be one of the most important targets of these photosensitizers.     

Error prone translesion synthesis past gamma-hydroxypropano deoxyguanosine, the primary acrolein-derived adduct in mammalian cells

8-Hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin- 10(3H)-one,3-(2'-deoxyriboside) (1,N(2)-gamma-hydroxypropano deoxyguanosine, gamma-HOPdG) is a major DNA adduct that forms as a result of exposure to acrolein, an environmental pollutant and a product of endogenous lipid peroxidation. gamma-HOPdG has been shown previously not to be a miscoding lesion when replicated in Escherichia coli. In contrast to those prokaryotic studies, in vivo replication and mutagenesis assays in COS-7 cells using single stranded DNA containing a specific gamma-HOPdG adduct, revealed that the gamma-HOPdG adduct was significantly mutagenic. Analyses revealed both transversion and transition types of mutations at an overall mutagenic frequency of 7.4 x 10(-2)/translesion synthesis. In vitro gamma-HOPdG strongly blocks DNA synthesis by two major polymerases, pol delta and pol epsilon. Replicative blockage of pol delta by gamma-HOPdG could be diminished by the addition of proliferating cell nuclear antigen, leading to highly mutagenic translesion bypass across this adduct. The differential functioning and processing capacities of the mammalian polymerases may be responsible for the higher mutation frequencies observed in this study when compared with the accurate and efficient nonmutagenic bypass observed in the bacterial system.     

Endogenous formation and significance of 1,N2-propanodeoxyguanosine adducts

The detection of 1,N2-propanodeoxyguanosine adducts in the DNA of rodent and human tissues as endogenous lesions has raised important questions regarding the source of their formation and their roles in carcinogenesis. Both in vitro and in vivo studies have generated substantial evidence which supports the involvement of short- and long-chain enals derived from oxidized polyunsaturated fatty acids (PUFAs) in their formation. These studies show that: (1) the cyclic propano adducts are common products from reactions of enals with DNA bases; (2) they are formed specifically from linoleic acid (LA; omega-6) and docosahexaenoic acid (omega-3) under in vitro stimulated lipid peroxidation conditions; (3) the levels of propano adducts are dramatically increased in rat liver DNA upon depletion of glutathione; (4) the adduct levels are increased in the liver DNA of the CCl4-treated rats and the mutant strain of Long Evans rats which are genetically predisposed to increased lipid peroxidation; and (5) adduct levels are significantly higher in older rats than in newborn rats. These studies collectively demonstrate that tissue lipid peroxidation is a main endogenous pathway leading to propano adduction in DNA. The possible contribution from environmental sources, however, cannot be completely excluded. The mutagenicity of enals and the mutations observed in site-specific mutagenesis studies using a model 1,N2-propanodeoxyguanosine adduct suggest that these adducts are potential promutagenic lesions. The increased levels of the propano adducts in the tissue of carcinogen-treated animals also provide suggestive evidence for their roles in carcinogenesis. The involvement of these adducts in tumor promotion is speculated on the basis that oxidative condition in tissues is believed to be associated with this process.     

Formation of acrolein-derived 2'-deoxyadenosine adduct in an iron-induced carcinogenesis model

Acrolein is a representative carcinogenic aldehyde found ubiquitously in the environment and formed endogenously through oxidation reactions, such as lipid peroxidation and myeloperoxidase-catalyzed amino acid oxidation. It shows facile reactivity toward DNA to form an exocyclic DNA adduct. To verify the formation of acrolein-derived DNA adduct under oxidative stress in vivo, we raised a novel monoclonal antibody (mAb21) against the acrolein-modified DNA and found that the antibody most significantly recognized an acrolein-modified 2' -deoxyadenosine. On the basis of chemical and spectroscopic evidence, the major antigenic product of mAb21 was the 1,N6-propano-2' -deoxyadenosine adduct. The exposure of rat liver epithelial RL34 cells to acrolein resulted in a significant accumulation of the acrolein-2' -deoxyadenosine adduct in the nuclei. Formation of this adduct under oxidative stress in vivo was immunohistochemically examined in rats exposed to ferric nitrilotriacetate, a carcinogenic iron chelate that specifically induces oxidative stress in the kidneys of rodents. It was observed that the acrolein-2' -deoxyadenosine adduct was formed in the nuclei of the proximal tubular cells, the target cells of this carcinogenesis model. The same cells were stained with a monoclonal antibody 5F6 that recognizes an acrolein-lysine adduct, by which cytosolic accumulation of acrolein-modified proteins appeared. Similar results were also obtained from myeloperoxidase knockout mice exposed to the iron complex, suggesting that the myeloperoxidase-catalyzed oxidation system might not be essential for the generation of acrolein in this experimental animal carcinogenesis model. The data obtained in this study suggest that the formation of a carcinogenic aldehyde through lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress.     

Glucose induces lipid peroxidation and inactivation of membrane-associated ion-transport enzymes in human erythrocytes in vivo and in vitro.

Erythrocytes of diabetic subjects (non-insulin dependent) were found to have eight- to ten-fold higher levels of endogenously formed thiobarbituric acid reactive malonyldialdehyde (MDA), thirteen-fold higher levels of phospholipid-MDA adduct, 15-20% reduced Na(+)-K(+)-ATPase activity with unchanged Ca+2-ATPase activity, as compared with the erythrocytes from normal healthy individuals. Incubation of normal erythrocytes with elevated concentrations (15-35 mM) of glucose, similar to that present in diabetic plasma, led to the increased lipid peroxidation, phospholipid-MDA adduct formation, reduction of Na(+)-K(+)-ATPase (25-50%) and Ca+2-ATPase (50%) activities. 2-doxy-glucose was 80% as effective as glucose in the lipid peroxidation and lipid adduct formation. However, other sugars, such as fructose, galactose, mannose, fucose, glucosamine and 3-O-methylmannoside, and sucrose, tested at a concentration of 35 mM, resulted in reduced (20-30%) lipid peroxidation without the formation of lipid-MDA adduct. Kinetic studies show that reductions in Na(+)-K(+)-ATPase and Ca+2-ATPase activities precede the lipid peroxidation as the enzyme inactivation occur within 30 min of incubation of erythrocytes with high concentration (15-35 mM) of glucose, while lipid peroxidation product, MDA appears at 4 hr and lipid-MDA adducts at 8 hr. The lipoxygenase pathway inhibitors, 5,8,11-eicosatriynoic acid and Baicalein (5,6,7-trihydroxyflavone), reduced the glucose-induced lipid peroxidation by 30% and MDA-lipid adduct formation by 26%. Indomethacin, a cyclooxygenase pathway inhibitor, had no discernible effect on the lipid peroxidation in erythrocytes. However, the inhibitors of lipid peroxidation, 3-phenylpyrazolidone, metyrapone, and the inhibitors of lipoxygenase pathways did not ablate the glucose-induced reduction of Na(+)-K(+)-ATPase and Ca+2-ATPase activities in erythrocytes. Erythrocytes produce 15-HETE (15-hydroxy-eicosatetraenoic acid), which is augmented by glucose. These results suggest that the formation of lipoxygenase metabolites potentiate the glucose-induced lipid peroxidation and that the inactivation of Na(+)-K(+)-ATPase and Ca+2-ATPase occurs as a result of non-covalent interaction of glucose with these enzymes.     

DNA adduct profiles: chemical approaches to addressing the biological impact of DNA damage from small molecules

Diverse small molecules alkylate DNA and form covalently linked adducts that can influence crucial biological processes, contributing to toxicity and mutation. Understanding the chemical reactivity dictating DNA alkylation and interactions of adducts with biological pathways can impact disease prevention and treatment. The ambident reactivity of DNA-alkylating small molecules, and of DNA itself, often results in formation of multiple adducts. Determining which structures impart biological responses is important for understanding the underlying relationships between small-molecule structure and biology. With application of sensitive and structure-specific experimental and analytical methodology, such as heteronuclear NMR spectroscopy and mass spectrometry, there are increasing numbers of studies that evaluate DNA alkylation from the perspective of resulting adduct profiles. DNA adduct profiles have been examined for both exogenous and endogenous reactive small molecules. Examples of recent findings are in the areas of tobacco-specific carcinogens, lipid peroxidation products, environmental and dietary chlorophenols, and natural-product-derived antitumor therapies. As more profile data are obtained, correlations with biological impact are being observed that would not be identified by a simplified single agent/single adduct approach.     

Comparison of DNA adduct levels associated with oxidative stress in human pancreas

DNA adducts associated with oxidative stress are believed to involve the formation of endogenous reactive species generated by oxidative damage and lipid peroxidation. Although these adducts have been reported in several human tissues by different laboratories, a comparison of the levels of these adducts in the same tissue samples has not been carried out. In this study, we isolated DNA from the pancreas of 15 smokers and 15 non-smokers, and measured the levels of 1,N⁶-etheno(2′-deoxy)guanosine (edA), 3,N⁴-etheno(2′-deoxy)cytidine (edC), 8-oxo-2′-deoxyguanosine (8-oxo-dG), and pyrimido[1,2-]purin-10(3H)-one (m₁G). Using the same DNA, the glutathione S-transferase (GST) M1, GSTT1, and NAD(P)H quinone reductase-1 (NQO₁) genotypes were determined in order to assess the role of their gene products in modulating adduct levels through their involvement in detoxification of lipid peroxidation products and redox cycling, respectively. The highest adduct levels observed were for m₁G, followed by 8-oxo-dG, edA, and edC, but there were no differences in adduct levels between smokers and non-smokers and no correlation with the age, sex or body mass index of the subject. Moreover, there was no correlation in adduct levels between edA and eC, or between edA or edC and m₁G or 8-oxo-dG. However, there was a significant correlation (r=0.76; p<0.01) between the levels of 8-oxo-dG and m₁G in human pancreas DNA. Neither GSTM1 nor NQO₁ genotypes were associated with differences in any of the adduct levels. Although the sample set was limited, the data suggest that endogenous DNA adduct formation in human pancreas is not clearly derived from cigarette smoking or from (NQO₁)-mediated redox cycling. Further, it appears that neither GSTM1 nor GSTT1 appreciably protects against endogenous adduct formation. Together with the lack of correlation between m₁G and edA or edC, these data indicate that the malondialdehyde derived from lipid peroxidation may not contribute significantly to m₁G adduct formation. On the other hand, the apparent correlation between m₁G and 8-oxo-dG and their comparable high levels are consistent with the hypothesis that m₁G is formed primarily by reaction of DNA with a base propenal, which, like 8-oxo-dG, is thought to be derived from hydroxyl radical attack on the DNA.