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.

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.

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.

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.

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.

Insight into DNA protection ability of medicinal herbs and potential mechanisms in hydrogen peroxide damages model

DNA damage is one of the most important consequences of oxidative stress in the cells. If DNA repair is unable to modify these inducible DNA damages, genomic instability may lead to mutation, cancer, aging and many other diseases. Single cell gel electrophoresis or comet assay is a common and versatile method to quantify these types of DNA damages. DNA damages induced by hydrogen peroxide(H_2O_2) are one of the proper models for measurement of protective ability of different compounds. So the main aim of this review is to provide an overview about protection ability of medicinal plants and their potential mechanism against H_2O_2 induced DNA damages. In this review, relevant researches on the effect of medicinal plants on DNA damages induced by H_2O_2 and possible molecular mechanisms are discussed.It seems that, medicinal plants are considered as therapeutic key factors to protect DNA from consequences caused by oxidative stress. Sufficient in vitro evidences introduce them as DNA protective agents through different mechanisms including antioxidant activity and some other cellular mechanisms. Moreover, in order to correlate the antigenotoxicity effects with their potential antioxidant property, most of medicinal plants were evaluated in term of antioxidant activity using standard methods. This review highlights the preventive effects of herbal medicine against oxidative DNA damages as well as provides rational possibility to engage them in animal studies and future clinical investigations.

The Protective Effects of N-Acetylcysteine on Exogenous Hydrogen Peroxide and Endogenous Superoxide Anion-induced DNA Strand Breakage in Human Spermatozoa

Objective To explore the protective effects of N Acetylcysteine (NAC) on exogenous hydrogen peroxide and endogenous superoxide anion induced DNA strand breakage in human spermatozoa by using the single cell gel electropherosis (SCGE) Methods Sperm cells were exposed to 0.5 mmol/L of H 2O 2 or 5.0 mmol/L of β NADPH with or without 0.1, 0.5, 1.0 mmol/L of NAC. The percentage of sperm comet cells and the comet tail lengths were measured in the treated sperm cells by using SCGE. Results Both percentage of comet sperm nuclei and mean tail length in sperm cells exposed to 0.5 mmol/L hydrogen peroxide with different concentrations of NAC decrease significantly in a dose dependent manner as compared with sperm cells exposed to H 2O 2 without NAC or catalase. Although mean tail length in sperm cells exposed to 5.0 mmol/L of β NADPH with different concentrations of NAC decreases significantly compared with sperm cells exposed to β NADPH without NAC or SOD, there were no significant differences on the percentage of sperm comet cells between sperm cells exposed to 5.0 mmol/L of β NADPH with different concentrations of NAC and sperm cells exposed to 5.0 mmol/L of β NADPH without NAC. Conclusion NAC has a protective effect on exogenous hydrogen peroxide induced DNA damage, while protective effect of NAC against O - 2 induced DNA strand breakage is significant but very weak.

Comet assay and cytokinesis-blocked micronucleus test for monitoring the genotoxic effects of X-ray radiation in humans

Obejctive To assess the genotoxic effects of X ray radiation on human populations Methods The single cell gel electrophoresis (SCGE) and cytokinesis blocked micronucleus (CBMN) test were applied as biological dosimeters to detect DNA damage and abnormalities in human peripheral lymphocytes of subpopulation exposed to X ray radiation The subjects were divided into four groups: 12 radiation patients; 13 intervention radiation therapy doctors; 32 radiation diagnostians; 28 controls Results The average comet lengths of the four groups were 128 17±4 49?μm, 88 09±5 39?μm, 72 68±2 57?μm and 32 87±0 57?μm, respectively The difference in average comet length between any two groups was highly significant ( P <0 01) The average micronucleated cell (MNC) rates (‰) of the four groups were 12 33±0 85, 9 75±1 02, 8 48±0 66 and 3 18±0 36, respectively The difference of MNC rates of Group 1 vs 3, 1 vs 4, 2 vs 4 and 3 vs 4 was highly significant ( P <0 01), and the difference of Group 1 vs 2 was significant ( P <0 05), but there was no difference of MNC rate in Group 2 vs 3 ( P >0 05) Conclusions This study showed that both the comet assay and the CBMN test could be used to monitor populations exposed to X ray radiation, but the comet assay seems to be more sensitive than the CBMN test

Effects of heavy metals Cd^(2+),Pb^(2+)and Zn^(2+)on DNA damage of loach Misgurnus anguillicaudatus

The effects of heavy metals Cd^(2+),Pb^(2+)and Zn^(2+)at 0.05,0.5 and 5.0 mg/L level and their interactions at 0.5 mg/L level on DNA damage in hepatopancreas of loach Misgurnus anguillicaudatus for 1-35 days exposure were examined by single cell gel electrophoresis(SCGE).For each test group,20 loaches with similar body size(5.17-7.99 g;11.79-13.21 cm)were selected and kept in aquaria with dechlori-nated water at(22±1)℃and fed a commercial diet every 48 h.According to the percentage of damaged DNA with tail and its TL/D(tail length to diameter of nucleus)value,the relationship between DNA damage degree and heavy metal dose and exposure time was determined.Results showed that the percentage of damaged DNA and the TL/D value were increased with the prolonged exposure time.The highest percentage(84.85%)of damaged DNA was shown in 5.0 mg/L Zn^(2+)group after 28 days exposure and the biggest TL/D value(2.50)in all treated groups after 35 days exposure.During the first treated week,the damnification of DNA was mainly recognized as the first level,after that time,the third damaged level was mostly observed and the percentage of damaged DNA was beyond 80%.The joint toxic effects among Cd^(2+),Pb^(2+)or Zn^(2+)revealed much complexity,but it generally displayed that the presence of Cd^(2+)could enhance the genotoxicity of Pb^(2+)or Zn^(2+).In conclusion,the results suggestedthattherewasasignificanttime-anddose-depended relationship between the heavy metal and DNA damage in hepatopancreas of loach,and SCGE could represent a useful means to evaluate the genotoxicity of environmental contamination on aquatic organisms.

Mineral nutrient imbalance,DNA lesion and DNA-protein crosslink involved in growth retardation of Vicia faba L.seedlings exposed to lanthanum ions

Effects of mineral nutrient imbalance,DNA lesion and DNA-protein crosslink on growth of Vicia faba L.seedlings hydroponically cultivated in concentrations of extraneous lanthanum（La） for 20 days were investigated in the present experiment.The results showed that contents of La,Cu or K elements in roots generally changed synchronously with those in leaves,while Ca,Fe,Zn,Mg,Mn or P in the roots altered inversely to those in the leaves.Thus,the extraneous La led to redistribution and imbalance of mineral nutrient elements in the roots and leaves.DNA lesion and DNA-protein crosslink were investigated by single cell gel electrophoresis（SCGE） and sodium dodecyl sulfate/potassium（SDS/K＋） precipitation methods,respectively.The results demonstrated that the increasing La induced DNA break and DNA-protein crosslinks（DPCs） in the seedlings.These results suggested that mineral nutrient imbalance,DNA lesion and DNA-protein crosslink were involved in the growth retardation and growth alteration of the seedlings,which may help to understand the mechanisms of rare earth elements（REEs） on plant growth.