Activation of oxidative stress response by hydroxyl substituted chalcones and cyclic chalcone analogues in mitochondria

We investigated the effect of hydroxyl substituted chalcone (1a) and some chalcone analogues (1b-d) on isolated rat liver mitochondria to gain new insights into the cytotoxic mechanism of these compounds. We observed an inhibitory effect on phosphorylation and the partial uncoupling of compounds 1a and 1d. Increased radical generation and possible covalent interaction of the compounds with cellular thiols resulted in glutathione (GSH) depletion and modulation of the investigated mitochondrial activities. Disruption of interconnected mechanisms as electron transport chain and energetic metabolism, ROS production and insufficiency of antioxidant defensive system could lead to induction of cell death.     

DNA damage induced by novel demethylcantharidin-integrated platinum anticancer complexes

Oxaliplatin is a third generation platinum (Pt) drug with a diaminocyclohexane (DACH) entity, which has recently obtained worldwide approval for the clinical treatment of colon cancer, and apparently operates by a different mechanism of action to the classical cisplatin or carboplatin. Introducing a novel dual mechanism of action is one approach in designing a new platinum-based anticancer agent, whereby an appropriate ligand, such as demethylcantharidin (DMC), is released from the parent compound to exert a cytotoxic effect, in addition to that of the DNA-alkylating function of the platinum moiety. To investigate the likelihood of a novel dual mechanism of anticancer action, demethylcantharidin-integrated Pt complexes: Pt(R,R-DACH)(DMC) with the same Pt-DACH moiety as oxaliplatin, and Pt(NH(3))(2)(DMC) akin to carboplatin; were studied for their ability to induce DNA damage in HCT116 colorectal cancer cells by an alkaline comet assay. The results showed that the DMC ligand released from the novel complexes caused additional DNA lesions when compared with oxaliplatin and carboplatin. The comet assay also revealed that the DNA-damaging behavior of cisplatin is characteristically different; and this study is the first to demonstrate the ability of DMC to induce DNA lesions, thus providing sufficient evidence to explain the superior antiproliferative effect of the novel DMC-integrated complexes.     

Comparison of the effect of raw and blanched‐frozen broccoli on DNA damage in colonocytes

Consumption of cruciferous vegetables may protect against colorectal cancer. Cruciferous vegetables are rich in a number of bioactive constituents including polyphenols, vitamins and glucosinolates. Before consumption, cruciferous vegetables often undergo some form of processing that reduces their content of bioactive constituents and may determine whether they exert protective effects. The aim of this study was to compare the ability of raw and blanched‐frozen broccoli to protect colonocytes against DNA damage, improve antioxidant status and induce xenobiotic metabolizing enzymes (XME). Fifteen Landrace × Large White male pigs were divided into five age‐matched and weight‐matched sets (79 days, SD 3, and 34·7 kg, SD 3·9, respectively). Each set consisted of siblings to minimize genetic variation. Within each set, pigs received a cereal‐based diet, unsupplemented (control) or supplemented with 600 g day^?1 of raw or blanched‐frozen broccoli for 12 days. The consumption of raw broccoli caused a significant 27% increase in DNA damage in colonocytes (p = 0·03) relative to the control diet, whereas blanched‐frozen broccoli had no significant effect. Both broccoli diets had no significant effect on plasma antioxidant status or hepatic and colonic XME. This study is the first to report that the consumption of raw broccoli can damage DNA in porcine colonocytes. Copyright © 2015 John Wiley & Sons, Ltd.     

Calibration of the comet assay for the measurement of DNA damage in mammalian cells

We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. γ-Rays from ⁶⁰Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with γ- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10⁹ Da, respectively, (0.50 and 0.52 breaks/10⁶ bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.     

Calibration of the comet assay for the measurement of DNA damage in mammalian cells

We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. γ-Rays from ⁶⁰Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with γ- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10⁹ Da, respectively, (0.50 and 0.52 breaks/10⁶ bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.     

溴氰菊酯对真鲷肝胰脏组织及细胞DNA的损伤

为研究溴氰菊酯亚急性染毒对真鲷肝胰脏的毒性作用,将真鲷分为5组进行不同剂量半静置染毒.染毒25 d后取真鲷肝胰脏进行组织切片显微观察,并采用彗星试验技术对其肝胰脏细胞进行DNA损伤分析.结果表明: 在0.025、0.125、0.250和0.375 μg·L^-1暴露浓度下,真鲷肝胰脏组织出现了不同程度的淤血、细胞核浓缩、细胞坏死等病理性损伤,且染毒浓度越高组织细胞损伤越显著.与空白对照组相比,各染毒浓度组肝胰脏细胞DNA均有不同程度损伤,彗星拖尾率、彗尾DNA相对含量、Olive距等指标均与对照有显著性差异.一元回归分析表明,染毒浓度与拖尾率、彗星尾长等参数呈极显著正相关; 染毒浓度与各指标均呈线性关系,回归方程拟合度(R)极高,范围为0.909～0.996.表明溴氰菊酯对真鲷肝胰脏组织和细胞DNA均可产生不同程度损伤,且损伤程度与染毒浓度之间具有高度线性相关关系.     

Effects of pulsed electric fields on DNA of human lymphocytes

The effects of pulsed electric fields of low frequency (50 Hz) on DNA of human lymphocytes were investigated. The influence of additional external factors, such as hydrogen peroxide (H₂O₂) and γ-irradiation, as well as the repair efficiency in these lymphocytes, was also evaluated. The comet assay, a very sensitive and rapid method for detecting DNA damage at the single cells level was the method used. A significant amount of damage was observed after exposure to the electric fields, compared to the controls. After 2 h incubation at 37^°C, a proportion of damage was repaired. H₂O₂ and γ-irradiation increased the damage to lymphocytes exposed to pulsed electric fields according to the dose used, while the amount of the repair was proportional to the damage.     

Ex vivo Assessment of Lymphocyte Antioxidant Status Using the Comet Assay

Lymphocytes were isolated from volunteers before and after receiving a single supplement of vitamin C, vitamin E or β-carotene. The lymphocytes were treated with H₂O₂, and DNA strand breaks were measured by single cell gel electrophoresis (the comet assay). Significant protection against oxidative DNA damage was evident 2-4 h after vitamin C intake, and 18-24 h after consumption of the other antioxidants. Lymphocytes from smokers were more sensitive to DNA damage than those from non-smokers, and they showed at least as great a protective effect with antioxidants.     

Ex vivo Assessment of Lymphocyte Antioxidant Status Using the Comet Assay

Lymphocytes were isolated from volunteers before and after receiving a single supplement of vitamin C, vitamin E or β-carotene. The lymphocytes were treated with H₂O₂, and DNA strand breaks were measured by single cell gel electrophoresis (the comet assay). Significant protection against oxidative DNA damage was evident 2–4 h after vitamin C intake, and 18–24 h after consumption of the other antioxidants. Lymphocytes from smokers were more sensitive to DNA damage than those from non-smokers, and they showed at least as great a protective effect with antioxidants.     

紫外辐射诱导烟草原生质体中DNA损伤的彗星电泳检测

以烟草原生质体为材料,采用彗星电泳检测用0.5W·m^-2紫外线以不同时间（0、5、10、30、60和120s）诱导的烟草原生质体中DNA的损伤。结果表明,在0～10s的时间内代表DNA损伤程度的尾矩、Olive尾矩等参数与紫外线照射时间具有良好的时间依赖关系。本文建立的烟草原生质体体系采用彗星电泳技术,可以快速而灵敏地检测紫外线对植物细胞的损伤程度。     

Pulsatile shear stress increased mitochondrial membrane potential: Implication of Mn-SOD

Mitochondrial dysfunction is intimately involved in cardiovascular diseases. Mitochondrial membrane potential (ΔΨ_m) is coupled with oxidative phosphorylation to drive ATP synthesis. In this study, we examined the effect of physiological pulsatile shear stress (PSS) on ΔΨ_m and the role of Mn-SOD expression on ΔΨ_m. Confluent human aortic endothelial cells (HAEC) were exposed to PSS, and ΔΨ_m was monitored using tetramethylrhodamine methyl ester (TMRM⁺), a mitochondrial membrane potential probe. PSS significantly increased ΔΨ_m and the change in ΔΨ_m was a dynamic process. ΔΨ_m returned to baseline level after PSS for 2 h followed by static state for 4 h. Mitochondrial Mn-SOD expression and activities were also significantly up-regulated in response to PSS. Silencing Mn-SOD attenuated PSS-mediated ΔΨ_m increase while adding Mn-SOD mimetic, MnTMPyP, increased ΔΨ_m to the similar extent as induced by PSS. Our findings suggest that PSS-increased mitochondrial ΔΨ_m, in part, via Mn-SOD up-regulation.     

Measurement of Menadione-Mediated DNA Damage in Human Lymphocytes Using the Comet Assay

The model quinone compound menadione has been used to study the effects of oxidative stress in mammalian cells, and to investigate the mechanism of action of the quinone nucleus which is present in many anti-cancer drugs. We have used the alkaline single cell gel electrophoresis assay (comet assay) to investigate the effects of low doses of this compound on isolated human lymphocytes. We found that concentrations of menadime as low as 1μM were sufficient to induce strand breaks in these cells. Pre-incubation with the NAD(P)H quinone oxidoreductase inhibitor dicoumarol, enhanced the production of menadione-induced strand breaks. In contrast, the metal ion chelator 1,10-phenanthroline inhibited formation of strand breaks, although prolonged incubation with 1,10-phenanthroline in combination with menadione resulted in an increase in a population of very severely damaged nuclei. A marked variation in the response of lymphocytes from different donors to menadione, and in different samples from the same donor was also observed.     

Hydroxytyrosol lipophilic analogues: enzymatic synthesis, radical scavenging activity and DNA oxidative damage protection

The olive oil phenol hydroxytyrosol (3), as well its metabolite homovanillic alcohol (4), were subjected to chemoselective lipase-catalysed acylations, affording with good yield 10 derivatives (5-14) bearing C(2), C(3), C(4), C(10) and C(18) acyl chains at C-1. Hydroxytyrosol (3) and its lipophilic derivatives showed very good DPPH. radical scavenging activity. Compounds 3, 4 and their lipophilic analogues 5-14 were subjected to the atypical Comet test on whole blood cells: 3 and its analogues 5 and 6, with little hydrophobic character (logP相似文献   

Evaluation of mitochondrial membrane potential using a computerized device with a tetraphenylphosphonium-selective electrode

Mitochondrial membrane potential (Deltapsi(m)) plays important roles in the normal function of cells and in pathobiochemical situations. The application of ion-selective electrodes for the measurement of Deltapsi(m) is important for studying normal biological reactions and pathways and mitochondrial diseases. We constructed and optimized a computerized device for real-time monitoring of the Deltapsi(m), which included modification of tetraphenylphosphonium (TPP(+))-selective membrane that improved reproducibility of the TPP(+)-selective electrode. Application of MATLAB software increased the sensitivity of the system. We tested our improved device for membrane potential measurements of isolated mitochondria (in absolute scale of millivolts). In addition, we assessed relative changes of Deltapsi(m) (as changes in TPP(+) concentration) of digitonin-permeabilized cells (hepatocytes, control transmitochondrial cybrids, HeLa G and BSC-40) after addition of substrates, inhibitors, and uncoupler of respiratory chain. Our system can be successfully used for studies of many aspects of the regulation of mitochondrial bioenergetics and as a diagnostic tool for mitochondrial oxidative phosphorylation disorders.     

Co‐exposure to fluoride and sulfur dioxide on histological alteration and DNA damage in rat brain

Fluoride (F) and sulfur dioxide (SO₂) are the two common environmental contaminants that are associated with neurotoxicity. The present study was conducted to explore individual and combined exposure effects of F and SO₂ on histological alteration and DNA damage in rat brain. For this, male Wistar albino rats were exposed to sodium fluoride (100 mg/L NaF) and sulfur dioxide (39.3 mg/m³) individually and in combination for 8 weeks. Histological alteration in brain is evaluated by hematoxylin–eosin staining, showed shrunken neurons, darkly stained small nucleus and decreased cell numbers in F and SO₂ exposed groups. The effect of F and SO₂ on DNA damage was assessed by comet assay. The results showed an increase in ratio of tailing and tail length in F or/and SO₂ administered rats. In addition, the proportion of grade II and III were also increased in individual and combined exposed groups. Compared with the individual exposure, the proportion the grade III was significantly high in combined exposure, suggesting a synergistic effect of F and SO₂. These results indicate that the brain was more susceptible to the toxic effects of F and SO₂. And combined exposure to these pollutants can lead more pronounced toxic effects on brain.     

对二甲苯对皱纹盘鲍肝胰腺细胞DNA的损伤

为研究对二甲苯对皱纹盘鲍肝胰腺的毒性作用,设置4个浓度(0.5、1.0、1.5和2.0 mg·L^-1)和对照组,开展为期21 d的对二甲苯对皱纹盘鲍的亚慢性毒性试验,采用彗星试验技术进行皱纹盘鲍肝胰腺细胞DNA损伤分析,采用CASP分析软件对拖尾率、彗星尾长、彗尾DNA相对含量、Olive矩等损伤指标进行统计。结果表明: 与对照组相比,各染毒组皱纹盘鲍肝胰腺细胞DNA均受到损伤,且损伤程度存在显著性差异。随着染毒浓度的增加,肝胰腺细胞DNA受损程度加重,高浓度甚至可以引发细胞凋亡,呈现一定的剂量-损伤效应。中浓度对二甲苯短时间暴露即可对皱纹盘鲍肝胰腺细胞造成DNA损伤,随着暴露时间延长,细胞DNA受损程度加重,呈现一定的时间-损伤效应。但长时间暴露细胞DNA各损伤指标有所减小,这可能与细胞自身的DNA修复机制和生物体解毒系统的代谢机制有关。研究表明,对二甲苯可对皱纹盘鲍肝胰腺细胞产生氧化损伤,导致DNA断裂,高浓度的对二甲苯长时间暴露可导致其细胞凋亡。     

Comparison of the in vivo and in vitro genotoxicity of glyphosate isopropylamine salt in three different organisms

There is considerable controversy with regard to the genotoxicity of glyphosate, with some reports stating that this compound is non-toxic for fish, birds and mammals. In this work, we used the comet assay to examine the genotoxicity of glyphosate isopropylamine (0.7, 7, 70 and 700 μM) in human lymphocytes, erythrocytes of Oreochromis niloticus and staminal nuclei of Tradescantia (4430) in vitro and in vivo. Cells, nuclei and fish that had and had not been exposed to 5 mM N-nitrosodiethylamine (NDEA) were used as positive and negative controls, respectively. Significant (p < 0.01) genetic damage was observed in vivo and in vitro in all cell types and organisms tested. Human lymphocytes and Tradescantia hairs showed lower genetic damage in vivo compared to in vitro, possibly because of efficient metabolization of the herbicide. In O. niloticus erythrocytes, significant (p < 0.001) genotoxicity was observed at ≥ 7 μM, whereas in vitro, glyphosphate was genotoxic in human lymphocytes and Tradescantia hairs at ≥ 0.7 μM. These results indicate that glyphosate is genotoxic in the cells and organisms studied at concentrations of 0.7–7 μM.     

Measuring oxidative damage to DNA; HPLC and the comet assay compared

Depending on the analytical method employed estimates of background levels of base oxidation in human DNA vary over orders of magnitude. It is now realised that oxidation of guanine in vitro can result in serious overestimation of the nucleoside by HPLC (with electrochemical detection). We have modified procedures of isolation, hydrolysis and storage of DNA with the aim of eliminating this artefact. Vacuum- or freeze-drying, and dialysis, tend to encourage oxidation. We compare results obtained with HPLC and with the comet assay, which employs lesion-specific enzymes to introduce breaks in DNA at sites of oxidative damage. Although estimates of background levels of DNA oxidation using the comet assay are several-fold lower than the estimates by HPLC, both approaches have been used successfully to detect differences between human subjects or population groups that seem to relate to human disease and nutritional factors.     

Comet assay for assessment of DNA damage in greenhouse workers exposed to pesticides

Purpose: The main goal of the present study was to determine DNA damage in pesticide-exposed greenhouse workers and pesticides non-exposed controls.

Materials and methods: The DNA damage was measured by alkaline comet assay method (pH?>?13) in 41 greenhouse workers and 45 non-exposed individuals as the control. Pesticide exposure was assessed by duration of working in the greenhouse and pesticide application in the greenhouse time. DNA damage was estimated by arbitrary unit and damage frequency.

Results: Arbitrary unit and damage frequency were consistently significantly higher in greenhouse workers than those of the controls (p?=?0.001). In terms of gender in greenhouse, DNA damage of female workers was significantly higher than those in male workers (p?<?0.05). We found significant correlation between DNA damage and working hours spent. Multiple linear regression analysis showed that working hours in the greenhouse as an indication of pesticide exposure were significantly associated with the DNA damage, which can be attributed to the genotoxic potential of the pesticide mixture.

Conclusions: The comet assay is sensitive to detect the damage exposed to chronic effect of pesticides in greenhouse workers. Significant DNA damage was obtained for the exposed group, which was associated with the pesticide exposure.