Study of Low-intensity 2450-MHz Microwave Exposure Enhancing the Genotoxic Effects of Mitomycin C Using Micronucleus Test and Comet Assay in vitro

Objective To determine the interaction between 2450-MHz microwaves (MW) radiation and mitomycin C (MMC). Methods The synergistic genotoxic effects of low-intensity 2450-MHz microwave and MMC on human lymphocytes were studied using single cell gel electrophoresis (SCGE) assay (comet assay) and cytokinesis-blocked micronucleus (CBMN) test in vitro. The whole blood cells from a male donor and a female donor were either only exposed to 2450-MHz microwaves (5.0 mW/cm2) for 2 h or only exposed to MMC (0.0125 μ/mL, 0.025 μg/mL, 0.05μg/mL and 0.1 μg/mL) for 24 h; and the samples were exposed to MMC for 24 h after exposure to MW for 2 h. Results In the comet assay, the comet lengths ( 29.1 μm and 25.9 μm) of MW were not significantly longer than those (26.3 μrn and 24.1 μm) of controls (P>0.05). The comet lengths (57.4 μm, 68.9 μm, 91.4 μm, 150.6μm and 50.6 μm, 71.7μm, 100.1 μm, 145.1 μm) of 4 MMC groups were significantly longer than those of controls (P<0.01). The comet lengths (59.1 μm, 92.3 μm, 124.5 μm, 182.7 μm and 57.4 μm, 85.5 μm, 137.5 μm, 178.3 μm) of 4 MW plus MMC groups were significantly longer than those of controls too (P<0.01). The comet lengths of MW plus MMC groups were significantly longer than those of the corresponding MMC doses (P<0.05 or P<0.01) when the doses of MMC were ≥50.025 μg/mL. In the CBMN, the micronucleated cell (MNC) rates of MW were 5% and 6%, which showed no difference compared with those (4‰ and 4‰) of controls (P>0.05). The MNC rates of 4 MMC groups were 8‰, 9‰, 14‰, 23‰ and 8‰, 8‰, 16‰, 30‰ respectively. When the doses of MMC were 3≥0.05 μg/mL, MNC rates of MMC were higher than those of controls (P<0.05). MNC rates of 4 MW plus MMC groups were 12‰, 13‰, 20‰, 32‰ and 8‰, 9‰, 23‰, 40‰. When the doses of MMC were 5≥0.05 μg/mL, MNC rates of MW plus MMC groups were much higher than those of controls (P<0.01). MNC rates of 4 MW plus MMC groups were not significantly higher than those of the corresponding MMC doses. Conclusion The low-intensity 2450-MHz microwave radiation can not induce DNA and chromosome damage, but can increase DNA damage effect induced by MMC in comet assay.

Assessment of Human DNA Repair (NER) Capacity With DNA Repair Rate (DRR) by Comet Assay

Objective Alkaline comet assay was used to evaluate DNA repair (nucleotide excision repair, NER) capacity of human fresh lymphocytes from 12 young healthy non-smokers (6 males and 6 females). Methods Lymphocytes were exposed to UV-C (254 nm) at the dose rate of 1.5 J/m2/sec. Novobiocin (NOV) and aphidicolin (APC), DNA repair inhibitors, were utilized to imitate the deficiency of DNA repair capacity at the incision and ligation steps of NER. Lymphocytes from each donor were divided into three grougs: UVC group, UVC plus NOV group, and UVC plus APC group. DNA single strand breaks were detected in UVC irradiated cells incubated for 0, 30, 60, 90, 120, 180, and 240 min after UVC irradiation. DNA repair rate (DRR) served as an indicator of DNA repair capacity. Results The results indicated that the maximum DNA damage (i.e. maximum tail length) in the UVC group mainly appeared at 90 min. The ranges of DRRs in the UVC group were 62.84%-98.71%. Average DRR value was 81.84%. The DRR difference between males and females was not significant (P<0.05). However, the average DRR value in the UVC plus NOV group and the UVC plus APC group was 52.98% and 39.57% respectively, which were significantly lower than that in the UVC group (P<0.01). Conclusion The comet assay is a rapid, simple and sensitive screening test to assess individual DNA repair (NER) capacity. It is suggested that the time to detect DNA single strand breaks in comet assay should include 0 (before UV irradiation), 90 and 240 min after exposure to 1.5 J·m-2 UVC at least. The DRR, as an indicator, can represent the individual DNA repair capacity in comet assay.

Genotoxicity evaluation of drinking water sources in human peripheral blood lymphocytes using the comet assay

The potential harm of organic pollutants in drinking water to human health is widely focused on in the wodd; more and more pollutants with genotoxic substances are released into the aquatic environment. Water source samples were collected from 7 different localities of Nanjing City. The potential genotoxicity of organic extracts from drinking water sources were investigated by means of the comet assay in human peripheral lymphocytes. The results showed that all the organic extracts from all the water source samples could induce DNA damages of human peripheral blood lymphocytes at different levels. A significant difference （P 〈 0.01） was observed when compared with the solvent control, The DNA damage increased with the increase of the dosage of the original water source. Significant differences of DNA damage were observed in different drinking water sources, as shown by the multiple comparisons analysis at the dosage of 100x; the degree of DNA damage treated by Hushu waterworks （at town level） was the most serious, the arbitrary units （AU） was 141.62±6.96, however, that of Shangyuanmen waterworks （at city level） was only 109.64±2.97. The analysis also revealed that the genotoxicity of town＇s water sources was higher than that of the city. The results demonstrated that the comet assay can be successfully applied to the genotoxicity monitoring programs of drinking water sources.

Inhibition of H2O2-induced DNA damage in single cell gel electrophoresis assay (comet assay) by castasterone isolated from leaves of centella asiatica

Brassinosteroids (BRs) are a large group of polyhydroxy steroids, which regulate numerous aspects of plant growth and development, including stem elongation, leaf bending, tracheary element differentiation, stress protection and photomorphogenesis. Recent studies indicate antigenotoxic and anticancerous activities of these compounds. The role of natural BRs in H2O2 (hydrogen peroxide) -induced DNA damage in human lymphocytes is still unknown. The present study reports the presence of Castasterone from leaves of Centella asiatica, an important medicinal herb commonly used as a memory enhancer and immunomodulator. CA50 fraction isolated from Centella asiatica was characterized as Castasterone by electrospray ionization mass spectral data with standard Castasterone. An attempt has been made to study antigenotoxic activity of the isolated Castasterone against H2O2 -induced DNA damage in human blood lymphocytes using Single cell gel electrophoresis assay (Comet Assay). Castasterone at 10–9 M concentration proved to be effective in diminishing the DNA damage by 89.42 %.

Shortening of alkaline DNA unwinding time does not interfere with detecting DNA damage to mouse and human spermatozoa in the comet assay

The comet assay was performed on mouse and human spermatozoa to examine the effect of alkaline DNA unwinding time. The spermatozoa were treated in vitrowith the DNA-damaging agents, methyl methanesulfonate （MMS） or hydrogen peroxide （Hz02）, and then embedded in agarose gel on glass sl ides. The slides were immersed in alkaline solution （〉pH 13） for 1, 5, 10 and 20 min, and then subjected to the electrophoresis under neutral conditions. In mouse spermatozoa, comet tails seen in solvent controls became brighter and longer as the alkaline DNA unwinding time increased. However, in the MMS-treated mouse spermatozoa, a smaller difference in the damage from that in the solvent control was seen with time within a dose. DNA damage induced by H2O2 could also be detected accurately after alkali treatment for 1-20 min. In human spermatozoa, DNA damage induced by MMS and H2O2 could be detected in a dose-dependent manner after alkali treatment for 1 min. The ability of the comet assay to detect DNA damage was not adversely affected by the short period （1 min） of the alkaline DNA unwinding time.

Genotoxicity of Three Avermectins on Polypedates megacephalus Tadpoles Using the Comet Assay

Avermectins are a new class of macrocyclic lactones derived from mycelia of the soil actinomycete, and are used as effective agricultural pesticides and antiparasitic agents. However, run-off from crops treated with avermectins may contaminate various bodies of water, and accumulated to certain concentrations to impact the development of aquatic animals. Here, we tested the genotoxicity of three avermectins （abamectin, ABM; ivermectin, IVM; and emamectin benzoate, EMB） on Polypedates megacephalus tadpoles by the alkaline single-cell gel electrophoresis assay. Tadpoles were treated for 48 h in the laboratory with different concentrations of these three agents, 0.006, 0.012, 0.018, 0.024, 0.030 mg/L for ABM, 0.003, 0.006, 0.009, 0.012, 0.015 mg/L for IVM and 0.04, 0.06, 0.08, 0.10, 0.12 mg/L for EMB, and then measured their DNA damage by the Comet assay tail factor %. The concentrations of resulted in highly significant increases in DNA damage of the tadpoles were found above the concentration threshold of 0.012 mg/ L ABM, 0.003 mg/L IVM and 0.06 mg/L EMB and linear correlations between the intensity of DNA damage and the concentrations of these three avermectins. Our results showed clearly that avermectins caused dose dependent DNA damage on amphibian tadpoles, and there might be a control on the misuse of avermectins.

Evaluation of the Genotoxicity of Chlorpyrifos in Common Indus Valley Toad, <i>Bufo stomaticus</i>Using Alkaline Single-Cell Gel Electrophoresis (Comet) Assay

Chlorpyrifos is a commonly used pesticide of organophosphate group, which causes toxicological effects in non-target organisms especially fish and frogs. In the present study, the genotoxicity of sublethal concentrations of chlorpyrifos was observed in the erythrocytes of common Indus valley toad, Bufo stomaticus, using the Alkaline Single-Cell Gel Electrophoresis (Comet) assay. In the first step, acute toxicity of chlorpyrifos was evaluated by exposing the tadpoles to high concentrations of the pesticide. The acute LC50 value of chlorpyrifos, calculated by Trimmed Spearman-Karber (TSK) in static bioassay, was found to be 930.0 μg/L. On the basis of acute LC50 value, the tadpoles were exposed to three sublethal concentrations (155, 233 and 465 μg/L) of chlorpyrifos for 96 h. Blood cells were collected at every 24 h interval and were subjected to the Alkaline Single-Cell Gel Electrophoresis assay. The observed DNA damage was concentration and time-dependent, and those levels of DNA damage in between the tested concentrations and times were significantly different (p < 0.05). The tadpoles exposed to different concentrations of chlorpyrifos also showed different morphological abnormalities. It was concluded that chlorpyrifos is a genotoxic pesticide causing DNA damage in Bufo stomaticus.

DNA Damage （Comet Assay） as Biomarker of Cd Exposure in Marine Seed Scallops Mizuhopecten Yessoensis Age 1 Year

Cadmium-induced DNA degradation in gill cells of the scallop Mizuhopectenyessoensis was assessed using the comet assay （single-cell gel electrophoresis）. Accumulation of highly toxic cadmium in the gill cells of bivalve is accompanied by the damage of the cell genome revealed as DNA migration in the comet assay. The main mechanisms of Cd effects on the integrity of the DNA structure are discussed.

The genotoxic effects of benzo[a]pyrene and methamidophos on black porgy evaluated by comet assay

In this study, two common pollutants (benzo[a]pyrene and methamidophos) in marine environment were tested by comet assay for their inducement of in vivo genotoxic effect to the blood cells of black porgy ( Acanthopagrus schlegeli). The fish was exposed to 2 μg/L of benzo[a]pyrene (BaP) and methamidophos, and their mixture. The assay was performed on whole blood at 2 h, 5 h, 24 h and 96 h exposure intervals. A signifi- cant increase in DNA damage was observed in each treatment with the pollutants. Additive effect of BaP and methamidophos was also found in the experiment. However, the decrease ratios of DNA damage for 5 h and 96 h exposure interals compared with 2 h and 24 h exposure ones, respectively, were noticed. This phenomenon may be explained by the function of repairing process via enzyme cytochrome P450 in the animal. Evidence of the genotoxicity of organophosphorus pesticides (OPs) and polynuclear aromatic hydrocarbons (PAHs) on marine fish are discussed in this paper.

DNA damage levels in systemic lupus erythematosus patients with low disease activity:An evaluation by comet assay

Oxidative stress has been implicated in the inflammatory process of Systemic Lupus Erythematosus (SLE), particularly by the formation of anti-DNA autoantibodies, which can lead to DNA damage. The aim of this study was to investigate, through comet assay, whether the level of DNA damage in SLE patients is different from that of healthy subjects. Twenty-five adult SLE patients with SLEDAI up to ten, and 25 healthy subjects were paired according to age, gender and Body Mass Index (BMI). Other anthropometric variables were also assessed. Comet assay was assessed as the marker of oxidative stress described as DNA Damage (DD) percentage. Waist Circumference (WC), Hip Circumference (HC) and BMI were also performed. Exclusion criteria for patients and controls comprised smoking and other chronic disorders. Level of damage index was remarkably higher in SLE patients than in controls, and no significant differences between the groups were found for age, BMI, WC and HC. No stratification concerning gender was performed, since there were just two males per group. No correlation was observed between BMI and DD (%). DD increased in SLE, which reflects the oxidant/antioxidant imbalance in these patients. These findings support an association between oxidative stress and SLE. This stronger correlation observed in patients with low disease activity may be useful in elucidating the mechanisms of disease pathogenesis.

SCGE技术检测镉对背角无齿蚌血细胞DNA的损伤

以背角无齿蚌(Anodonta woodiana woodiana)为研究对象,利用单细胞凝胶电泳技术检测不同浓度及不同染毒时间氯化镉对背角无齿蚌血细胞DNA的损伤作用。结果显示,染毒后的背角无齿蚌血细胞均出现了拖尾现象,各染毒组与对照组相比,拖尾增长明显。剂量效应组中,蚌暴露在不同浓度(0、1、10、50 mg.L-1)的氯化镉中72 h,各组蚌血细胞DNA平均迁移长度及彗尾DNA含量均明显增加,与阴性对照组比较差异显著(p<0.01),存在显著的剂量-效应关系;在时间效应组中,蚌暴露在10 mg.L-1的氯化镉中分别24、48、72、96 h,随着氯化镉染毒时间的延长,各染毒组细胞DNA平均迁移长度及彗尾DNA含量与0时间组比较差异显著(p<0.01),但时间-效应关系不明显。

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.

Response of Lymphocytes to Radiation in Untreated Breast Cancer Patients as Detected with Three Different Genetic Assays

Objective To detect the response of lymphocytes to radiation in untreated breast cancer patients with three different genetic assays. Methods Blood samples were collected from 25 untreated patients and 25 controls. Each blood sample was divided into two parts： one was irradiated by 3-Gy X-ray （irradiated sample）, the other was not irradiated （non-irradiated sample）. The radiosensitivity of lymphocytes was assessed by comet assay, cytokinesis-block micronucleus （CBMN） assay and 6-TG-resistant cells scored （TG） assay. Results The baseline values of micronucleated cell frequency （MCF） and micronucleus frequency （MNF） in the patients were significantly higher than those in the controls （P〈0.01）, and 3-Gy X-ray induced genetic damage to lymphocytes in the patients increased significantly as compared with that in the controls as detected with the three genetic assays （P〈0.01）. The proportion of radiosensitive cases in the patient group was 48% for the mean tail length （MTL）, 40% for the mean tall moment （MTM）, 40% for MCF, 44% for MNF, and 48% for mutation frequencies of the hprt gene （Mfs-hprt）, respectively, whereas the proportion of radiosensitive cases in the control group was only 8% for all the parameters. Conclusion The difference in the lymphocyte radiosensitivity between the breast cancer patients and the controls is significant. Moreover, there are wide individual variations in lymphocyte radiosensitivity of patients with breast cancer. In some cases, the radiosensitivity of the same patient may be different as detected with the different assays. It is suggested that multiple assays should be used to assess the radiosensitivity of patients with breast cancer before therapy.

藏药绿萝花彗星试验(SCGE)研究

研究藏药绿萝花的抗(致)突变作用,科学合理地利用藏药资源.根据遗传毒性研究试验组合原则和判定标准,以环磷酰胺、丝裂霉素C为阳性药物对照,采用单细胞凝胶电泳试验(彗星试验),研究不同剂量绿萝花对中国仓鼠肺成纤维细胞(CHL) DNA的影响.结果显示,环磷酰胺、丝裂霉素C具有很强的致CHL细胞SCGE上升、HDNA下降、TDNA上升、TL和TM增加(超过40μm)的作用;生药浓度为0. 224 g/mL、0. 448 g/mL、0. 896 g/mL的绿萝花在分别染毒1次、2次和3次后对CHL细胞DNA无影响,对环磷酰胺和丝裂霉素C引起的DNA损伤无保护作用.研究表明藏药绿萝花无致基因突变作用,研究结果为绿萝花临床安全用药提供了科学依据.

酚类化合物对不同组织细胞DNA损伤的研究

应用单细胞凝胶电泳技术 ,研究了酚类化合物对不同组织细胞DNA的损伤 .试验结果表明 ,酚类化合物均能引起不同组织细胞不同程度的DNA损伤 ,并呈现剂量效应关系 .其高剂量组与对照组相比 ,均有极显著性差异 (P <0 0 1 ) .不同组织细胞对同一种药物呈现出不同的敏感性 .

彗星试验中全血法与分离淋巴细胞法的比较

背景与目的 :比较在彗星试验中全血法和分离淋巴细胞法的结果差异 ,为简化彗星试验方法提供实验依据。材料与方法 :重铬酸钾和乙醇腹腔注射给药 ,取全血或分离淋巴细胞 ,应用彗星实验法比较小鼠外周血液淋巴细胞的DNA损伤情况。结果 :在全血和分离淋巴细胞条件下均能检测到两种损伤剂所导致的细胞DNA损伤 ,且作用趋势相同。 结论 :采用全血进行彗星试验更为简便 。

石油烃对栉孔扇贝血淋巴细胞DNA损伤的初步研究

为研究石油烃对海洋生物的毒性效应,将栉孔扇贝(Chlamys farreri)暴露于0.08、0.21和0.88mg·L-1石油烃中,采用单细胞凝胶电泳实验(彗星实验)技术检测不同暴露时间扇贝血淋巴细胞的DNA损伤程度,对照组中石油烃背景浓度为0.04mg·L-1。结果显示,低浓度(0.08mg·L-1)的石油烃短期(<7d)内即可导致栉孔扇贝血淋巴细胞的DNA损伤,并且随石油烃浓度的增大和暴露时间的延长,DNA损伤程度增加,石油烃浓度达0.88mg·L-1时,DNA损伤程度已非常严重。3d恢复实验后,各浓度组DNA损伤又均有不同程度的恢复。研究表明,彗星实验是检测石油烃对海洋贝类DNA损伤的一种有效手段,贝类血淋巴细胞DNA损伤有望成为石油烃污染的一种生物标志物,用于海洋污染的早期预警监测。

应用慧星试验研究细胞DNA损伤的原理与方法

对应用慧星试验（即单细胞微凝胶电泳（ＳＣＧＥ技术）研究细胞ＤＮＡ损伤的操作过程、技术原理以及实验操作过程中应注意的事项。

用单细胞凝胶电泳检测砷、汞、铅致DNA损伤的研究

应用ＳＣＧＥ方法体外检测ＮａＡｓＯ２、ＨｇＣｌ２、Ｐｂ（ＮＯ３）２对人外周血淋巴细胞造成的ＤＮＡ损伤。结果表明，ＳＣＧＥ方法非常灵敏，能检测到很低剂量的ＮａＡｓＯ２和ＨｇＣｌ２所引起人外周血淋巴细胞的ＤＮＡ断裂，而且该法还具有快速、花费少等优点。

苯并[a]芘和菲对缢蛏血细胞DNA损伤的研究

为研究苯并[a]芘和菲对缢蛏的毒性效应,将缢蛏(Sinonovacula constricta)分别暴露于浓度为0.45 mg·L-1、0.15 mg·L-1、0.05 mg·L-1苯并[a]芘溶液和0.45 mg·L-1、0.15 mg·L-1、0.05 mg·L-1菲的溶液中,采用单细胞凝胶电泳实验(彗星实验)技术检测不同暴露时间缢蛏血淋巴细胞的DNA损伤程度,对照组为清洁海水。结果显示,高浓度(0.45 mg·L-1)苯并[a]芘溶液和(0.45 mg·L-1)菲溶液在短期(7 d)内即可导致缢蛏血细胞显著的DNA损伤,并且随苯并芘[a]和菲浓度的增大和暴露时间的延长,DNA损伤程度增加。21 d恢复实验后,各浓度组DNA损伤又均有不同程度的恢复,但中高浓度组(0.45 mg·L-1和0.15 mg·L-1)与对照组仍显著性差异。两种多环芳烃物质对缢蛏血细胞的DNA损伤作用均存在较显著时间-剂量-效应关系。其中,苯并芘[a]对缢蛏血细胞的DNA损伤作用要高于菲。