Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone

[Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD50) of ZEN with tetrazolium-based colorimetric assay (MTT assay). Comet assay was carried out to detect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 μmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%, 52.00% and 64.67% under 0, 1, 5, 10 and 20 μmol/L of ZEN, respectively; the differences between the percentages of cell tail in various experimental groups had extremely significant statistical significance compared with the negative group (P<0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60±4.78, 57.75±6.25, 78.97±5.83, 100.50±6.94 and 146.83±12.31 μm, respectively; Tail DNA % in various groups was 21.29±2.25%, 22.24±2.43%, 31.21±6.27%, 37.45±4.33% and 60.68±9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 μmol/L showed significant differences (P<0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.

The Superiority of Like-rocket Immunoelectrophoresis Using in Single Cell Gel Electrophoresis Assay

[Objective] The like-rocket immunoelectrophoresis was used to explore a new feasible electrophoresis method for single cell gel electrophoresis assay （comet assay）.[Method] The like-rocket immunoelectrophoresis was used for single cell gel electrophoresis assay to detect DNA damage at single cell level,then it was compared with traditional electrophoresis method to analyze its advantage and disadvantages.[Result] Under cell DNA undamaged state,the results of two electrophoresis methods were consistent.When cell DNA was damaged,the comet tail divergence of some cells under traditional electrophoresis method were drifted,however,the comet tail image of like-rocket immunoelectrophoresis was concentrated and not shifted.[Conclusion] The like-rocket immunoelectrophoresis had some advantages.

Evaluation of Genotoxicity of Abrus mollis Hance with Single-cell Gel Electrophoresis Assay

[ Objective] This study aimed to evaluate the genotoxicity ofAbrus mollis Hance by using single cell gel electrophoresis. [Method] Forty mice were di- vided into five groups randomly, including positive control group （ cyclophosphamide group ）, negative control group （ physiological saline group）, high-dose A. moles Hance group （30 g/kg）, moderate-dose A. mollis Hance group （20 g/kg） and low-dose A. mollis Hance group （10 g/kg）. Tail DNA% and Tail Moment of mouse liver, kidney, lung and testicular cells were analyzed by using single cell gel electrophoresis assay, to investigate the effect of A. mollis Hance on DNA in mouse cells. [Result] Compared with positive control group, Tail DNA% and Tail Moment of moose liver, kidney, lung and testicular cells in A. moles Hance groups were significantly lower （ P 〈 0.01 ）. Compared with negative control group, Tail DNA% and Tail Moment of mouse liver, kidney, lung and testicular ceils in high-dose A. mollis Hance group were significantly lower （ P 〈 0.01 ）, while the other A. mollis Hance groups showed no statistically significant difference （ P 〉0.05 ）. [ Conclusion] A. mollis Hance has no damage effect on DNA in mouse cells within this experimental dose range.

Detection of Sperm DNA Damage in Workers Exposed to Benzene by Modified Single Cell Gel Electrophoresis

Objective To assess the effect of benzene on sperm DNA damage ;Methods Twenty-seven benzene-exposed workers were selected as exposed group and 35 normal sperm donors as control group. Air concentration of benzene series in workshop was determined by gas chromatography. As an internal exposure dose of benzene, the concentration of trans, trans-muconic acid （ttMA） was determined by high performance liquid chromatography. DNA was detected by modified single cell gel electrophoresis （SCGE）. Results The air concentrations of benzene, toluene and xylene at the workplace were 86.49±2.83 mg/m^3, 97.20±3.52 mg/m^3 and 97.45± 2.10 mg/m^3, respectively. Urinary ttMA in exposed group （1.040 ± 0.617 mg/L） was significantly higher than that of control group （0.819 ± 0.157 mg/L）. The percentage of head DNA, determined by modified SCGE method, significantly decreased in the exposed group （n=13, 70.18% ± 7.36%） compared with the control （n=16, 90.62% ± 2.94%）（P〈0.001）. Conclusion The modified SCGE method can be used to investigate the damage of sperm DNA. As genotoxin and reprotoxins, benzene had direct effect on the germ cells during the spermatogenesiss.

DNA degradation within mouse brain and dental pulp cells 72 hours postmortem

In this study, we sought to elucidate the process of DNA degradation in brain and dental pulp cells of mice, within postmortem 0-72 hours, by using the single cell gel electrophoresis assay and professional comet image analysis and processing techniques. The frequency of comet-like cells, the percentage of tail DNA, tail length, tail moment, Olive moment and tail area increased in tandem with increasing postmortem interval. In contrast, the head radius, the percentage of head DNA and head area showed a decreasing trend. Linear regression analysis revealed a high correlation between these parameters and the postmortem interval. The findings suggest that the single cell gel electrophoresis assay is a quick and sensitive method to detect DNA degradation in brain and dental pulp cells, providing an objective and accurate new way to estimate postmortem interval.

Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

General anesthesia-associated DNA damage in peripheral blood mononuclear cells of surgical patients

Objective: To evaluate retrospectively the effect of general anesthesia on DNA damage in the blood mononuclear cells (PBMCs) of surgical patients in order to provide evidence for a better nursing care during the procedure. Methods: Clinical charts of 76 patients who underwent operation under general anesthesia and 76 healthy control subjects with documented results of DNA damage extent in PBMCs from the single-cell gel electrophoresis (SCGE) or comet assay and serum contents of superoxide dismutase (SOD) and malondialdehyde (MDA) from biochemical analyses were reviewed. The percentage of comet PBMCs and tail DNA and serum contents of SOD and MAD were analyzed by student t-test. Results: Compared with healthy control subjects, generally anesthetized surgical patients had significantly higher % comet PBMCs and % tail DNA (P < 0.05) and significantly lower serum concentrations of SOD (P < 0.05) and significantly higher serum concentrations of MAD (P < 0.05). Compared with levels before general anesthesia in surgical patients, % comet PBMCs, % tail DNA, and serum levels of MAD were significantly higher (P < 0.05 or 0.01), and serum levels of SOD were significantly lower (P < 0.05), after general anesthesia. Conclusions: General anesthesia during surgery causes a certain degree of hypoxia and PBMC damage. Particular attention should be paid to monitoring and maintenance of blood oxygen saturation in patients undergoing surgery under general anesthesia.

十二烷基苯磺酸钠暴露对黄颡鱼稚鱼的遗传毒性

【目的】评估十二烷基苯磺酸钠(SDBS)暴露对黄颡鱼(Pelteobagrus fulvidraco)稚鱼的遗传毒性。【方法】将黄颡鱼稚鱼(孵化120 h)暴露于质量浓度分别为0、0.2、0.4、0.6、0.8 mg/L的SDBS中96 h后,采用微核实验和单细胞凝胶电泳技术测定不同暴露浓度的黄颡鱼稚鱼红细胞微核率和细胞尾部DNA损伤率,检测稚鱼组织中DNA氧化损伤的特异性产物8-羟基脱氧鸟苷的含量。【结果】黄颡鱼稚鱼红细胞微核率变化范围为(0.22±0.02)%~(0.59±0.03)%,且随着SDBS暴露浓度的升高,对应黄颡鱼稚鱼红细胞微核率也显著增加(P<0.05)。SDBS暴露对黄颡鱼稚鱼细胞均具有不同程度的遗传损伤,DNA损伤率在9.4%~24.7%。SDBS暴露浓度(x)与稚鱼细胞尾部DNA含量(y)呈现一定的剂量效应关系,其对应的回归方程为y^(^)=4.6x+1.48(R2=0.9396,n=24,P<0.05)。黄颡鱼稚鱼组织中8-羟基脱氧鸟苷质量浓度变化范围为(1.7±0.36)~(8.0±0.58)μg/L,随着SDBS暴露浓度的升高,对应黄颡鱼稚鱼组织中8-羟基脱氧鸟苷质量浓度也显著增加(P<0.05)。【结论】0.2~0.8 mg/L的SDBS暴露对黄颡鱼稚鱼具有遗传毒性。

基于SCGE的五氯酚对稀有鮈鲫DNA损伤的研究

采用单细胞凝胶电泳(SCGE)技术,研究了在不同的染毒时间内不同浓度的五氯酚(PCP)对稀有鮈鲫(Gobiocypris rarus)血细胞和肝脏细胞DNA的损伤.结果显示,各PCP染毒组细胞彗星尾部DNA含量(%DNAT)和彗星尾长(TL)显著增加,与空白对照组比较,差异极显著(P<0.05).随着浓度的增加,细胞彗星尾部%DNAT和TL逐渐增加,其相关系数>0.926,说明在实验浓度范围内,存在显著的浓度-效应关系.在同一浓度下,随着暴露时间的增加,处理组%DNAT和TL逐渐增加,存在显著的时间-效应关系.由于PCP可引起稀有鮈鲫血细胞和肝脏细胞DNA的严重损伤,因此稀有鮈鲫血细胞和肝脏细胞DNA的损伤可作为PCP遗传毒性的指示.

番茄红素对大鼠细胞DNA氧化损伤修复的影响

目的 研究番茄红素对大鼠淋巴细胞和肝细胞DNA损伤的保护作用。方法 大鼠灌胃番茄红素 4周后 ,采用单细胞凝胶电泳技术对外周血淋巴细胞和肝细胞DNA损伤进行分析。结果 番茄红素组大鼠淋巴细胞和肝细胞DNA的迁移率和尾长显著低于对照组。结论 番茄红素对淋巴细胞和肝细胞DNA损伤具有一定的保护作用。

镉胁迫对大弹涂鱼(Boleophthalmus pectinirostris)血细胞遗传损伤的研究

以大弹涂鱼为研究对象,应用单细胞凝胶电泳技术并结合外周血基因组DNA琼脂糖凝胶电泳,研究了镉胁迫对大弹涂鱼外周血细胞的遗传损伤。结果表明:(1)镉对大弹涂鱼外周血细胞是遗传毒性而非细胞毒性,并且产生的遗传损伤存在显著(P<0.01或P<0.05)的剂量效应,染毒1h的大弹涂鱼的全长和尾长与浓度的相关方程分别是y=29.592x-10.576(r2=0.9786),y=28.417x-14.859(r2=0.9857),染毒2h的大弹涂鱼的全长和尾长与浓度的相关方程分别是y=32.044x-5.0235(r2=0.941),y=29.911x-9.539(r2=0.9635)。(2)DNA的损伤程度和污染胁迫之间存在着时间效应。以上结果表明,DNA损伤可作为大弹涂鱼受镉胁迫时的生物指标。

白藜芦醇和抗坏血酸对预防非典型肺炎方剂Ⅰ和Ⅵ所致小鼠外周血液淋巴细胞DNA损伤的保护作用

目的 研究白藜芦醇和抗坏血酸对预防非典型肺炎方剂 和 致小鼠外周血液淋巴细胞 DNA损伤的保护作用。方法 采用单细胞凝胶电泳方法研究白藜芦醇和抗坏血酸对预防非典型肺炎方剂 和 致小鼠外周血液淋巴细胞 DNA损伤的保护作用。结果 白藜芦醇 ( 5 0 ,10 0 ,2 0 0 m g/ kg× 3d)和抗坏血酸 ( 5 0 ,10 0 ,2 0 0 mg/ kg×3d)对方剂 和 (临床等效量× 3d)所致的小鼠外周血液淋巴细胞 DNA损伤呈剂量依赖性的保护作用 ( P<0 .0 5 )。结论 白藜芦醇和抗坏血酸能够保护预防非典型肺炎方剂 和 对小鼠外周血液淋巴细胞 DNA的损伤。

枸杞多糖对小鼠睾丸细胞DNA损伤的保护作用

目的 :研究枸杞多糖 (LBP)对过氧化氢 (H2 O2 )引起的睾丸细胞DNA氧化损伤的保护作用。方法 :睾丸细胞先用不同浓度LBP作用 1h ,再加入 10 0 μmol LH2 O2 共同培养 2 5min ,然后用单细胞凝胶电泳技术(SCGE)测定DNA链断裂情况 ,分析LBP的抗氧化损伤作用。结果 :H2 O2 作用一定时间后 ,可引起DNA链断裂。 50、10 0、2 0 0、40 0 μg mL的LBP预处理可使H2 O2 染毒细胞的拖尾百分率和尾长显著降低。结论 :LBP本身没有致氧化损伤作用 ,它能够清除自由基 。

硫胺素和核黄素对H_2O_2诱导的DNA氧化损伤的影响

目的探讨硫胺素和核黄素对H2O2诱导人脐静脉内皮细胞ECV304的DNA氧化损伤的影响。方法于细胞培养液中分别加入硫胺素(终浓度为10、100、500、1000mg/L)或核黄素(终浓度为20、100、300、500nmol/L)预处理24h,给予H2O2(终浓度为25μmol/L)反应30min,采用单细胞凝胶电泳技术(SCGE)分析DNA氧化损伤情况。以不加H2O2、硫胺素和核黄素者为阴性对照组;以加H2O2而不加硫胺素、核黄素者为阳性对照组。结果H2O2诱导ECV304细胞DNA断裂损伤,SCGE分析显示,阳性对照组拖尾细胞率、彗星尾长、尾部DNA含量和Olive尾矩等指标均较阴性对照组升高;经10、100、500mg/L硫胺素或20、100、300nmol/L核黄素预处理后,各DNA损伤指标均较阳性对照组显著下降(P<0.05或P<0.01);经1000mg/L硫胺素或500nmol/L核黄素预处理后,各DNA损伤指标与阳性对照组比较差异无统计学意义(P>0.05)。结论适宜浓度的硫胺素和核黄素能降低H2O2诱导的DNA氧化损伤,高浓度的硫胺素和核黄素不能保护H2O2诱导的DNA氧化损伤。

水中有机提取物对V79细胞DNA损伤的研究

为研究饮水中有机提取物对人群健康的影响，采用单细胞凝胶电泳试验，对Ａ市三个自来水厂枯水期的水源水和出厂水中的有机提取物引起的Ｖ７９细胞ＤＮＡ损伤效应进行了研究。结果表明：各水厂的水源水和出厂水中的有机提取物在一定浓度范围内均可诱导体外培养的Ｖ７９细胞ＤＮＡ链断裂，且在相同浓度下，出厂水引起的ＤＮＡ迁移长度大于水源水。

抗菌肽CM4组分对K562癌细胞染色质DNA断裂作用的SCGE研究

单细胞凝胶电泳法（ｓｉｎｇｅｃｅｌｇｅｌｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ，ＳＣＧＥ）是一种快速，敏感的检测单个哺乳动物细胞ＤＮＡ断裂的技术，也叫彗星实验（ｃｏｍｅｔａｓｓａｙ）．此实验首次通过ＳＣＧＥ法观察抗菌肽ＣＭ４组分对人髓样白血病Ｋ５６２细胞和正常人白细胞核染色质ＤＮＡ的影响，从而进一步研究抗菌肽抗癌作用的机制．荧光显微镜观察显示经抗菌肽ＣＭ４组分处理过的Ｋ５６２癌细胞核染色质ＤＮＡ出现断裂，形成一个亮的荧光头部和彗星似的尾部，而经同样处理的正常人白细胞和未经抗菌肽处理的Ｋ５６２癌细胞核染色质ＤＮＡ未出现断裂，核完整，呈圆形．经彗星尾长分析，前者ＤＮＡ损伤率平均为７３６２％，统计学处理Ｐ＜０００１，具高度显著性差异．这表明，抗菌肽ＣＭ４对Ｋ５６２癌细胞核染色质ＤＮＡ有明显的断裂作用，而对正常人白细胞则没有断裂作用．

应用蝌蚪快速检测环境变异的两种方法——微核试验和单细胞凝胶电泳

本文介绍了应用无尾两栖类动物的蝌蚪进行环境监测的两种方法———微核试验和单细胞凝胶电泳。此两种环境检测的方法与其他环境检测方法相比具有快速 ,简便 ,易操作 ,适于检测现场应用 ,可大面积推广等优点。

邻苯二甲酸二乙基己酯(DEHP)对金鲫鱼脑细胞DNA的损伤

为了研究邻苯二甲酸二乙基己酯(DEHP)对金鲫鱼(Carassius auratu)脑细胞DNA的损伤作用,采用不同浓度的DEHP溶液(0、25、50、100、200mg·L-1)对体外培养脑细胞进行染毒,应用单细胞凝胶电泳(彗星实验)检测脑细胞DNA的损伤效应.结果表明,染毒1.5h后,与对照组相比,DEHP各染毒组细胞尾部DNA百分率(Tail DNA%)和尾矩(Tail Moment)均显著升高(p<0.01),即DEHP染毒引起了脑细胞DNA的严重断裂;随着DEHP浓度的增加,DNA损伤程度加剧,细胞尾部DNA百分率及尾距与DEHP染毒浓度呈明显的剂量-效应关系.以上结果表明,DEHP可导致金鲫鱼脑细胞DNA损伤。

铜暴露下赤子爱胜蚓(Eisenia foetida)活体基因的损伤研究

通过碱性单细胞凝胶电泳法研究了Cu2+暴露剂量对赤子爱胜蚓(Eiseniafoetida)活体基因损伤的动态影响.结果显示:不添加Cu2+的对照组和添加Cu2+的处理组蚯蚓体腔细胞尾部DNA含量和尾长均呈非正态分布(p<0.05);在暴露72h时,125mg·L-1Cu2+处理组尾部DNA含量值最大,为41.44%,100mg·L-1Cu2+处理组尾长值最大,为33.79μm;随着Cu2+暴露剂量的增加,尾部DNA含量和尾长损伤频率增加;对照组和处理组的尾部DNA含量和尾长之间均存在显著性差异(p<0.05),Spearman非参数相关分析表明,尾部DNA百分含量和尾长之间呈显著相关(p<0.01,n=21),Cu2+暴露浓度与尾部DNA百分含量、尾长具有良好的剂量-构效关系(p<0.01).在125mg·L-1Cu2+浓度下暴露72h时蚯蚓的基因损伤程度达到最大,损伤程度为3级.可见,蚯蚓DNA生物标志物是重金属污染基因毒理诊断的重要指标,碱性SCGE试验是检测Cu2+暴露对赤子爱胜蚓活体基因损伤的有效手段.