亚硒酸钠诱发的晶状体上皮细胞DNA损伤及修复

观察了亚硒酸钠（Ｎａ２ＳｅＯ３）在体外作用于大鼠晶状体上皮细胞（ＲＬＥ ｃｅｌｌｓ）而造成的ＤＮＡ单链断裂（ＳＳＢ）,并对其ＤＮＡ损伤、修复动力学做了初步研究。发现ＳＳＢ严重程度与亚硒酸钠的浓度呈线性相关,其ＳＳＢ重接修复约在３０￣６０ｍｉｎ内完成,还作了有关非程序ＤＮＡ合居（ＵＤＳ）的检测,发现与ＳＳＢ相比,ＵＤＳ发生迟且持续时间更长,提示Ｎａ２ＳｅＯ３可能在体外对大鼠晶状体上皮细胞除造成ＳＳＢ     

Se和环境中富里酸对小麦种子发芽的影响及其生理特性

利用种子发芽实验研究了亚硒酸钠（ＮＶ２ＳｅＯ３）和从土壤中提取的富里酸（Ｆｕｌｖｉｃ ａｃｉｄ, ＦＡ）对小麦种子发芽和生长的影响,以及 ＦＡ对亚硒酸钠毒性的抑制作用．结果表 明．ＦＡ和低浓度Ｎａ２ＳｅＯ３对种子发芽及发芽后的生长有促进作用,高浓度 Ｎａ２ＳｅＯ３明显 降低种子发芽率、种子活力、α－淀粉酶活性及幼苗的生长,ＦＡ对亚硒酸钠的毒性有一定的 抑制作用．     

亚硒酸钠对大鼠晶体上皮细胞αA晶体蛋白基因转录的影响

在体外观察了亚硒酸钠（Ｎａ２ＳｅＯ３）作用于大鼠晶体上皮细胞（ＲＬＥｃｅｌｌｓ）而引起其晶体蛋白基因转录的改变,对不同浓度的硒在体外对αＡ晶体蛋白基因转录的影响作了初步的研究,结果发现,随着亚硒酸钠浓度的升高,αＡ基因的转录下降;而当亚硒酸钠浓度升至５×１０＾－５ｍｏｌ／Ｌ时,αＡ基因的转录又呈反跳性回升,提示硒在致障过程中对晶体蛋白基因转录的影响作用不可忽视。同时αＡ晶体蛋白在晶体细胞内,至少应     

亚硒酸钠对大鼠晶体上皮细胞αA晶体蛋白基因转录的影响

在体外观察了亚硒酸钠（Ｎａ＿２ＳｅＯ＿３）作用于大鼠晶体上皮细胞（ＲＬＥｃｅｌｌｓ）而引起其晶体蛋白基因转录的改变,对不同浓度的硒在体外对αＡ晶体蛋白基因转录的影响作了初步的研究。结果发现,随着亚硒酸钠浓度的升高,αＡ基因的转录下降;而当亚硒酸钠浓度升至５×１０（－５）ｍｏ１／Ｌ时,αＡ基因的转录又呈反跳性回升。提示硒在致障过程中对晶体蛋白基因转录的影响作用不可忽视;同时αＡ晶体蛋白在晶体细胞内,至少应答于高浓度的硒,可能作为一种应激蛋白表达。     

NNK诱发BEP2D细胞产生活性氧及其对DNA的损伤

通过测定细胞内和细胞上清中活性氧（ｒｅａｃｔｉｖｅ ｏｘｙｇｅｎ ｓｐｅｃｉｅｓ,ＲＯＳ）水平,以及ＤＮＡ 加合物——８－羟基脱氧鸟嘌呤核苷（８－ｈｙｄｒｏｘｙｄｅｏｘｙｇｕａｎｏｓｉｎｅ,ＯＨ８ｄＧ）含量,对烟草特异亚硝胺类化合物４－甲基亚硝胺－１（３－吡啶基）－１－丁酮（４－（ｍ ｅｔｈｙｌｎｉｔｒｏｓａｍ ｉｎｏ）－１－（３－ｐｙｒｉｄｙｌ）－１－ｂｕｔａｎｏｎｅ,ＮＮＫ）诱发人乳头状病毒永生化的人支气管上皮细胞（ｈｕｍ ａｎ ｐａｐｉｌｌｏｍ ａｖｉｒｕｓ－ｉｍ ｍ ｏｒｔａｌｉｚｅｄ ｈｕｍ ａｎｂｒｏｎｃｈｉａｌｅｐｉｔｈｅｌｉａｌｃｅｌｌｌｉｎｅ,ＢＥＰ２Ｄ）产生的ＲＯＳ及其对ＤＮＡ 的氧化损伤进行研究,并观察纳米硒的保护作用．结果表明,ＢＥＰ２Ｄ 细胞经不同浓度的ＮＮＫ 作用后,细胞内和细胞上清中ＲＯＳ以及ＯＨ８ｄＧ含量均显著增加,并有较好的剂量效应关系．１ μｍ ｏｌ·Ｌ－ １纳米硒（ｎａｎｏｓｅｌｅｎｕｉｍ ,ＮＳ）能明显抑制ＮＮＫ 诱发ＢＥＰ２Ｄ细胞产生的ＲＯＳ及ＯＨ８ｄＧ 水平．揭示ＮＮＫ 能造成细胞的氧化损伤,而ＮＳ对ＮＮＫ 所致细胞的氧化损伤有保护作用．     

亚硒酸钠对四氯化碳损伤草鱼肝原代细胞与肝组织的保护作用

不同浓度四氯化碳（ＣＣｌ４）对草鱼肝原代细胞的损伤实验中,ＣＣｌ４浓度为１０μｌ／ｍｌ可引起细胞血清中丙氨酸氨基转移酶（ＡＬＴ）、天门冬氨酸氨基转移酶（ＡＳＴ）、乳酸脱氢酶（ＬＤＨ）逸出量与细胞破损率显著增高,培养液中添加亚硒酸钠（Ｎａ２ＳｅＯ３）０．２μｇ／ｍｌ,则可降低ＡＬＴ、ＡＳＴ、ＬＤＨ的逸出量,减轻细胞破损程度。Ｎａ２ＳｅＯ３保护实验中,Ｎａ２ＳｅＯ２＋ＣＣｌ４组预先腹腔注射（ｉｐ）０．１ｍｇ／ｋｇ．ｂｗ连续三日,末次ｉｐＣＣｌ４混合液１ｍｌ／ｋｇ．ｂｗ,２４ｈ内肝组织超氧物歧化酶（ＳＯＤ）相对活性比ＣＣｌ４组提高达９１．５％,第七日仍提高达５４．５％,与对照组的水平基本接近;血清中丙氨酸氨基转氨酶（ＡＬＴ）水平逐渐降低。本实验还观察到Ｎａ２ＳｅＯ３可引起肝脂质过氧化物显著降低,肝微粒体蛋白含量与细胞色素Ｐ—４５０活性升高;组织切片观察显示肝组织损伤程度减轻,７２ｈ后细胞核增多。表明Ｎａ２ＳｅＯ３可提高草鱼肝清除自由基能力,增强肝脏解毒功能。     

TNT体外大鼠白内障模型中的DNA单链断裂及其重接修复的研究

在大鼠晶状体器官培养的条件下,运用单细胞电泳法（ＳＣＧ）,对远在晶状体混浊之前的晶状体上皮细胞进行了有关ＴＮＴ致其ＤＮＡ损伤（ＳＳＢ）与修复的初步观察,提示ＤＮＡ损伤也是体外ＴＮＴ性白内障中的早期变化．     

TNT体外大鼠白内障模型中的DNA单链断裂及其重接修复的研究

在大鼠晶状体器官培养的条件下,运用单细胞电泳法（ＳＣＧ）,对远在晶状体混浊之前的晶状体上皮细胞进行了有关ＴＮＴ致其ＤＮＡ损伤（ＳＳＢ）与修复的初步观察。提示ＤＮＡ损伤也是体外ＴＮＴ性白内障中的早期变化。     

铜锌超氧物歧化酶对几种羟自由基产生系统的影响

应用脱氧核糖降解法研究了ＣｕＺｎ－ＳＯＤ对几种·ＯＨ产生系统的作用机理．结果证明：ＳＯＤ对Ｆｅ（３＋）·Ｏ·Ｈ２Ｏ２系统中·ＯＨ的产生有明显的抑制作用,而失活ＳＯＤ或ＢＳＡ对它的抑制作用不大;在Ｆｅ（２＋）·Ｈ２Ｏ２和ＣＵ（２＋）·Ｈ２Ｏ２系统中,ＳＯＤ、失活ＳＯＤ和ＢＡＳ均能抑制·ＯＨ的产生;在Ｆｅ（２＋）·Ｏ系统中,ＳＯＤ对·ＯＨ产生作用不大,而失活ＳＯＤ或ＢＳＡ对它有明显的抑制作用．由此推测ＳＯＤ对·ＯＨ形成可能有三方面的影响：１．对Ｏ的清除作用,阻断Ｈａｂｅｒ－Ｗｅｉｓｓ反应;２．对金属离子的络合作用,降低·ＯＨ的产额;３．促进Ｈ２Ｏ２的积累,加快Ｆｅｎｔｏｎ反应．     

箬叶多糖及其化学修饰物、亚硒酸钠和GSH对Cu~(2+)诱导的LDL氧化修饰的保护作用

研究了箬叶多糖ＦⅢ－ａ及其化学修饰物、亚硒酸钠和ＧＳＨ对Ｃｕ２＋诱导的低密度脂蛋白氧化修饰的保护作用．其结果表明箬叶多糖、硫酸酯多糖、硒酸酯多糖可显著抑制脂质过氧化产物（ＴＢＡＲＳ）及荧光物质的生成,彼此之间无明显差异．但对ＶＥ的消耗有着不同的保护作用,其顺序是ＦⅢ－ａ＞Ｓ－ＦⅢ－ａ＞Ｓｅ－ＦⅢ－ａ,并且具有明显的量效关系．硒或ＧＳＨ对Ｃｕ２＋诱导的ＬＤＬ氧化修饰无明显的抑制,但联合使用在０．１２５ｍｍｏｌ／ＬＮａ２ＳｅＯ３和０．２ｍｍｏｌ／ＬＧＳＨ及１２．５μｍｏｌ／ＬＮａ２ＳｅＯ３和０．０２ｍｍｏｌ／ＬＧＳＨ的浓度下能强烈地抑制ＴＢＡＲＳ的生成,甚至比正常的ＬＤＬ还要低．但是对ＶＥ的消耗只有较弱的保护作用,硒酸酯多糖与此相似．Ｎａ２ＳｅＯ３在０．１２５ｍｍｏｌ／Ｌ时可以明显抑制荧光物质的生成．     

Expression changes in mRNAs and mitochondrial damage in lens epithelial cells with selenite

An overdose of sodium selenite induces cataracts in young rats. The mid-stage events producing the cataract include calpain-induced hydrolysis and precipitation of lens proteins. Apoptosis in lens epithelial cells has been suggested as an initial event in selenite cataracts. Expression levels of two genes associated with apoptosis were altered in lens epithelial cells from selenite-injected rats. The purpose of the present experiment was to perform a more comprehensive search for changes in expression of mRNAs in lens epithelial cells in order to more fully delineate the early events in selenite-induced cataracts. Lens epithelial cells were harvested at 1 and 2 days after a single subcutaneous injection of sodium selenite (30 mumol/kg body weight) into 12-day-old rats. Gene expression was analyzed using a commercial DNA array (Rat Genome U34A GeneChip array, Affymetrix). Of approximately 8000 genes assayed by hybridization, 13 genes were decreased and 27 genes were increased in the rat lens epithelial cells after injection of selenite. Some of the up-regulated genes included apoptosis-related genes, and a majority of the down-regulated genes were mitochondrial genes. Previously observed changes in expression of EGR-1 mRNA were also confirmed. Changes in the expression patterns of mRNAs were also confirmed by RT-PCR. To determine the mechanism for damage of lens epithelial cells (alpha TN4 cell) by culture in selenite, leakage of cytochrome c from mitochondria was measured. Selenite caused significant leakage of cytochrome c into the cytosol of alpha TN4 cells. Our data suggested that the loss of integrity of lens epithelial cells by selenite might be caused by preferential down-regulation of mitochondrial RNAs, release of cytochrome c, and impaired mitochondrial function. Up-regulation of mRNAs involved in maintenance of DNA, regulation of metabolism, and induction of apoptosis may also play roles.     

Unscheduled DNA synthesis in rat tracheal epithelial cells, hepatocytes and spermatocytes following exposure to methyl chloride in vitro and in vivo

Measurement of DNA repair as unscheduled DNA synthesis (UDS) in vitro following exposure in vivo in multiple tissues from the same treated animal can provide valuable information relating to the tissue- and organ-specificity of chemically induced DNA damage. UDS was evaluated in primary cultures of rat tracheal epithelial cells, hepatocytes and pachytene spermatocytes after exposure in vitro to methyl chloride (MeCl), and after isolation from the same treated animal following inhalation exposure in vivo. Concentrations of 1-10% MeCl in vitro induced UDS in hepatocytes and spermatocytes, but not in tracheal epithelial cells. Inhalation exposure to MeCl in vivo (3000-3500 ppm 6 h/day for 5 successive days) failed to induce DNA repair in any cell type. In vivo exposure to 15 000 ppm MeCl for 3 h also failed to induce UDS in tracheal epithelial cells and spermatocytes, but did cause a marginal increase in UDS in hepatocytes. Thus, MeCl appears to be a weak, direct-acting genotoxicant. While activity could be measured in hepatocytes and spermatocytes directly in vitro, only extremely high concentrations of MeCl elicited a response in the whole animal, and then only in hepatocytes.     

Neutrophils cause oxidative DNA damage in alveolar epithelial cells.

Inflammation has been recognized as a contributing factor in the pathogenesis of some cancers. In the lung, inflammation is characterized by an influx of polymorphonuclear leukocytes (PMN) that release a variety of reactive oxygen species (ROS). The aim of the present study was to investigate the direct effect of PMN on oxidative DNA damage in lung target cells. Therefore, rat alveolar epithelial cells (RLE) were coincubated with PMN or hydrogen peroxide. Known to be correlated with the incidence of cancer, 7-hydro-8-oxo-2'deoxyguanosine (8-oxodG) was used as an effect marker for oxidative damage. Viability of the RLE, when coincubated with PMN, decreased to 43%, dependent on the ratio between PMN and RLE. After washing off PMN, 8-oxodG levels were significantly increased in RLE, but the highest levels were observed in the washed off PMN fraction. In addition, to avoid washing off procedures, immunohistochemical analysis was used to measure the 8-oxodG levels specifically in the RLE and similar results were obtained. In addition, inhibitor experiments showed that antioxidants ameliorated oxidative DNA damage. Our data provide evidence that ROS released by PMN as well as H2O2, cause oxidative DNA damage in epithelial cells.     

Measurement of genotoxic activity in multiple tissues following inhalation exposure to dimethylnitrosamine

Chemically-induced DNA repair was measured as unscheduled DNA synthesis (UDS) in selected tissues isolated from rats following in vivo exposure to inhaled dimethylnitrosamine (DMN). UDS was evaluated in epithelial cells from rat nasal turbinates and trachea, in hepatocytes and in pachytene spermatocytes from the same treated animal. At nominal concentrations of 500 and 1000 ppm of DMN in air, chemically-induced DNA repair was observed in the epithelial cells of the upper respiratory system. DMN also entered the circulation, as evidenced by a strong DNA-repair response in hepatocytes. No DNA repair was observed in pachytene spermatocytes indicating either that DMN or its active metabolites did not reach the testes in sufficient concentration to induce DNA repair or that the testes lacked the capability to metabolically activate the compound. These results illustrate the potential of this approach to assess the organ-specific genotoxicity of environmental chemicals.     

Repair of X-ray induced DNA strand damage by isolated rat splenic lymphocytes.

Although a number of chemicals can alter DNA repair function, little is known about the effect of chronic, low dose exposure to environmental agents on DNA repair capacity. Lymphocytes provide a potential target population to study the effects of chronic exposures to low doses of toxic chemicals since they are an easily obtainable cell population. Prior to investigating the repair capacity of chemically exposed lymphocytes, the repair by chemically naive lymphocytes has been characterized. In the present study, the DNA repair capacity of isolated rat lymphocytes was characterized. The capacity of these cells to repair single-strand DNA breaks (SSB) was determined after in vitro treatments with X-rays. The effect of in vitro exposure to 3-aminobenzamide (3-AB) on DNA repair capacity was also assessed. The levels of induced SSB and their repair were determined using the alkaline elution technique. Splenic lymphocytes were isolated and placed in culture medium 18 h prior to assessment of repair capacity, but were not stimulated with mitogens. A dose-dependent increase in SSB was observed following exposure of lymphocytes to 300 or 600 rad. The rate of SSB repair was analyzed after a dose of 400 rad. Approximately 80% of the DNA strand break repair was completed within 60 min. The half-time for repair of these lesions by lymphocytes was determined to be 21.3 min. Exposure to 3-AB resulted in a decrease in the rate of repair of the X-ray-induced strand breakage. Although no SSB were detected at the end of a 1-h 3-AB treatment of non-irradiated cells, significant accumulation of SSB was observed after a 2-h treatment. The characterization of DNA repair in rat lymphocytes following in vitro exposure to X-rays will allow us to investigate the effects of chronic, in vivo toxicant exposure on the capacity of isolated lymphocytes to repair DNA damage produced by X-rays.     

An autoradiographic study of unscheduled DNA synthesis in the germ cells of male mice treated with X-rays and methyl methanesulfonate

Unscheduled DNA synthesis (UDS) in the germ cells of male mice after in vivo treatment with X-rays or methyl methanesulfonate (MMS) was assayed by use of a quantitative autoradiographic procedure. MMS induced UDS in meiotic through type III elongating spermatid stages, whereas X-rays induced UDS in meiotic through round spermatid stages. No UDS was detected in the most mature spermatid stages present in the testis with either MMS or X-rays. Taking into account differences in DNA content of the various germ-cell stages studied, we concluded that X-rays induced a maximum UDS response in spermatocytes at diakinesis--metaphase I. The level of UDS induced by MMS was about the same in all the stages capable of repair. Chromosome damage and UDS were measured simultaneously in the same spermatocytes at diakinesis 90 min after X-irradiation or MMS treatment. The level of UDS in most of the X-irradiated cells paralleled the extent of chromosome damage induced. A statistical analysis of these results revealed a positive correlation. As expected, MMS induced no chromosome aberrations above control levels. Therefore no correlation was determined between UDS and chromosome damage in this case. The distribution of UDS over the chromosomes treated at diakinesis with MMS or X-rays was studied. It was found that UDS occurred in clusters in the irradiated cells, whereas it was uniformly distributed in the MMS-treated cells.     

PHA刺激对淋巴细胞修复DNA损伤的影响

本文报道PHA刺激对淋巴细胞DNA修复的影响的实验结果。以254nm波长的UV照射细胞(30J/m~2)引起DNA损伤,以[~3H]-TdR掺入实验测定非程序DNA合成,用超微量法测定细胞的NAD~+含量,并以[~(35)S]-蛋氨酸掺入,聚丙烯酰胺凝胶电泳及放射自显影术测定蛋白质生物合成,其结果如下: (1)在被PHA转化的淋巴细胞内非程序DNA合成,随PHA刺激的时间加长而增高;PHA处理淋巴细胞42小时,合成的速率约增加4倍;(2)在转化的淋巴细胞内,非程序DNA合成及程序DNA合成都被N-乙基马来酰亚胺(一种DNA聚合酶α的抑制剂)抑制,表明在DNA修复过程中DNA聚合酶α可代替DNA聚合酶β发挥作用; (3)UV照射后,被PHA刺激的淋巴细胞内NAD~+含量大约减少43.2％,而对照淋巴细胞内NAD~+的含量只减少25％,似乎说明PHA刺激能促进淋巴细胞内的P-ADP-核糖化作用;(4)在受PHA刺激72小时的淋巴细胞内有多种蛋白质合成,这些细胞在UV照射后以含10μg/ml嘌呤霉素的培养基培养,则非程序DNA合成被明显抑制(P<0.01),这提示DNA修复是一需要蛋白质合成的过程。此外,在受UV照射后10-45小时的淋巴细胞内,诱导产生一种分子量大约34000道尔顿的蛋白质。 上述结果表明,当PHA使淋巴细胞从静止状态转化为增殖状态时,有多种酶被诱导。由于这些酶,如DNA聚合酶α及P-ADP-核糖聚合     

Mechanisms of neutrophil-induced DNA damage in respiratory tract epithelial cells

Reactive oxygen species (ROS) released by neutrophils have been suggested to play an important role in cancer development. Since the mechanisms underlying this effect in the respiratory tract are still unclear, we evaluated DNA damage induced by neutrophils in respiratory tract epithelial cells in vitro and in vivo. For in vitro studies, rat lung epithelial cells (RLE) were co-incubated with activated neutrophils, neutrophil-conditioned medium, or hydrogen peroxide. For in vivo studies, we considered the human nose as a target organ, comparing neutrophilic inflammation in the nasal lavage fluid with the oxidative DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in epithelial cells obtained by nasal brush. Our in vitro data show that human neutrophils are able to induce both 8-OHdG and strand breaks in DNA from RLE cells. Our data also suggest that DNA damage induced by neutrophils is inhibited when neutrophil-derived H2O2 is consumed by myeloperoxidase. In contrast, in the nose no association between neutrophil numbers and 8-OHdG was found. Therefore, it remains unclear whether neutrophils pose a direct genotoxic risk for the respiratory tract epithelium during inflammation, andmore in vivo studies are needed to elucidate the possible association between neutrophils and genotoxicity in the lung.     

Unscheduled DNA synthesis after treatment with 20-methylcholanthrene and N-nitrosodimethylamine in primary culture of human gallbladder epithelial cells

Unscheduled DNA synthesis (UDS) induced by treatment with 20-methylcholanthrene (MCA) and N-nitrosodimethylamine (DMN) was measured in cultured human gallbladder epithelial cells. MCA induced UDS very efficiently, while DMN was far less effective than MCA. Addition of rat S9 mixture did not affect the amount of UDS by the chemicals. Differences between the present results in human cells and our previous findings in bovine cells could be due to species and tissue specificity.