Protective effect of Ligusticum chuanxiong and Angelica sinensis on endothelial cell damage induced by hydrogen peroxide

Ligusticum chuanxiong and Angelica sinensis have been widely used in traditional Chinese medicine to treat some pathological settings such as atherosclerosis and hypertension. We determined the protective effect of the extract of Ligusticum chuanxiong and Angelica sinensis (ELCAS) on human umbilical vein endothelial cells (ECV304) damage induced by hydrogen peroxide. ECV304 cells were pre-treated with ELCAS and exposed to 5 mM hydrogen peroxide. The results show that ELCAS dose- and time-dependently protected ECV304 cells against hydrogen peroxide damage and suppressed the production of reactive oxygen species (ROS). The decrement of ROS may be associated with increased activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). Western blot analysis revealed that ELCAS significantly increased the phosphorylation of ERK and promoted eNOS expression. These observations indicate that ELCAS protected ECV304 cells against hydrogen peroxide damage by enhancing the antioxidative ability, activating ERK and eNOS signaling pathway. Our data also provide new evidence of Ligusticum chuanxiong and Angelica sinensis in preventing both cardiovascular and cerebrovascular diseases.     

Lead‐induced DNA damage and cell apoptosis in human renal proximal tubular epithelial cell: Attenuation via N‐acetyl cysteine and tannic acid

This study investigates the exposure of lead‐induced reactive oxygen species (ROS) generation, DNA damage, and apoptosis and also evaluates the therapeutic intervention using antioxidants in human renal proximal tubular cells (HK‐2 cells). Following treatment of HK‐2 cells with an increasing concentration of lead nitrate (0–50 μM) for 24 h, the intracellular ROS level increased whereas the GSH level decreased significantly in a dose‐dependent manner. Comet assay results revealed that lead nitrate showed the ability to increase the levels of DNA strand breaks in HK‐2 cells. Lead exposure also induced apoptosis through caspase‐3 activation at 30 μg/mL. Pretreatment with N‐acetylcysteine (NAC) and tannic acid showed a significant ameliorating effect on lead‐induced ROS, DNA damage, and apoptosis. In conclusion, lead induces ROS, which may exacerbate the DNA damage and apoptosis via caspase‐3 activation. Additionally, supplementation of antioxidants such as NAC and tannic acid may be used as salvage therapy for lead‐induced DNA damage and apoptosis in an exposed person.     

Effect of UV-C mediated oxidative stress in leukemia cell lines and its relation to ubiquinone content

UV-C radiation is able to impair cellular functions by directly damaging DNA, and by inducing an increased formation of reactive oxygen species that leads to a condition of oxidative stress. In this study we evaluated different responses to UV insult of two leukemia cell lines, HL-60 and Raji, and the relationship with their CoQ10 content. DNA damage was monitored by means of the alkaline single cell gel electrophoresis (Comet assay); intracellular levels of ROS, mitochondrial depolarization and cell viability was measured by flow cytometry. Raji cells appeared more resistant to the UV insult; moreover, they did not show any increase in ROS content and the extent of mitochondrial depolarisation was much lower than in HL 60 cell line. Raji cells also contained significantly higher levels of CoQ10 and their ability to incorporate and to reduce exogenous CoQ10 added to the culture medium was remarkably elevated compared with HL 60.     

Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the Comet assay: a biomarker model

Methods are needed to assess exposure to genotoxins in humans and to improve understanding of dietary cancer prevention. The Comet assay was used to detect smoking-related exposures and dietary modulations in target tissues. Buccal scrapings, blood and faeces were collected from 38 healthy male volunteers (smokers and non-smokers) during a dietary intervention study with bread supplemented with prebiotics±antioxidants. GSTM1-genotype was determined with PCR. Buccal and peripheral lymphocytes were analysed for DNA damage using the Comet assay. Genotoxicity of faecal water (FW) was assayed in human colon HT29 clone 19A cells. 'Tail intensity' (TI) was used as a quantitative indicator of DNA damage in the Comet assay. Intervention with bread reduced DNA damage in lymphocytes of smokers (8.3±1.7% TI versus 10.2±4.1% TI, n=19), but not of non-smokers (8.6±2.8% TI versus 8.3±2.7% TI, n=15). Faecal water genotoxicity was reduced only in non-smokers (9.4±2.9% TI versus 18.9±13.1% TI, n=15) but not in smokers (15.5±10.7% TI versus 20.4±14.1% TI, n=13). The Comet assay was efficient in the detection of both smoking-related exposure (buccal cells) and efficacy of dietary intervention (faecal samples). Smokers and non-smokers profited differently from the intervention with prebiotic bread±antioxidants. Stratification of data by genotype enhanced specificity/sensitivity of the intervention effects and contributed important information on the role of susceptibility.     

Spread of oxidative damage and antioxidative response through cell layers of tobacco callus after UV-C treatment.

Tobacco (Nicotiana tabacum L. cv. Petit Havana) callus cultures were exposed to UV-C high dose pulse-treatment (254 nm, 50 kJ m(-2), 1 h-treatment). After 6, 24 and 48 h from the end of the treatment, calli were cut transversally in two layers and oxidative damage (malondialdehyde [MDA] and hydrogen peroxide), non-enzymatic (radical scavenging antioxidants [RSA] and polyamines) and enzymatic antioxidants (ascorbate peroxidase [APX, EC 1.11.1.11], glutathione reductase [GR, EC 1.6.4.2], catalase [CAT, EC 1.11.1.6] and guaiacol peroxidase [GPX, EC 1.11.1.7]) were evaluated. At each time-point data referred to UV-C treated calli were compared to data of untreated ones (control). Despite of a strong increase of H2O2 content, a slight cellular damage was observed in both upper and lower layers 24 and 48 h after UV-C treatment. An activation first of non-enzymatic antioxidants and then of enzymatic antioxidants was detected in UV-C treated calli. In particular, RSA and putrescine (PUT) accumulated 6 h after UV-C treatment while APX, GR and GPX enzyme activities increased 24 h after UV-C irradiation. Catalase activity did not change. UV-C-induced oxidative stress and antioxidative response were observed also in cell layers not directly exposed to UV irradiation, indicating that a stress signal was transmitted to the whole mass of callus.     

Antioxidants prevent oxidative DNA damage and cellular transformation elicited by the over-expression of c-MYC

Reactive oxygen species (ROS)-induced genomic damage may have important consequences in the initiation and progression of cancer. Deregulated expression of the proto-oncogene c-MYC is associated with intracellular oxidative stress and increased DNA damage. However, the protective role of antioxidants such as Vitamin C against MYC-induced genomic damage has not been fully investigated. In a variety of cell lines, we show that ectopic MYC over-expression results in the elevation of intracellular ROS levels and a concomitant increase in oxidative DNA damage, as assessed by levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in the genomic DNA. Loading cells with ascorbic acid (AA) relieved MYC-elicited intracellular oxidative stress and conferred genomic protection. A mitochondrially targeted Vitamin E analog, TPPB, also protected cells from MYC-elicited oxidative DNA damage, suggesting the involvement of mitochondria in increased ROS production. We found that deregulated MYC expression resulted in the attenuation of intracellular glutathione levels, which was reversed by loading cells with Vitamin C. Additionally, cells over-expressing MYC had elevated levels of intracellular superoxide, which was significantly quenched by Vitamin C or the selective superoxide quencher, Tiron. Consequently, Vitamin C and other antioxidants protected cells from MYC-induced cellular transformation. Our studies implicate a role for ROS, and superoxide in particular, in MYC-elicited oxidative DNA damage and cellular transformation, and point to a pharmacological role of antioxidants in cancer chemoprevention.     

Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the Comet assay: a biomarker model

Methods are needed to assess exposure to genotoxins in humans and to improve understanding of dietary cancer prevention. The Comet assay was used to detect smoking-related exposures and dietary modulations in target tissues. Buccal scrapings, blood and faeces were collected from 38 healthy male volunteers (smokers and non-smokers) during a dietary intervention study with bread supplemented with prebiotics±antioxidants. GSTM1-genotype was determined with PCR. Buccal and peripheral lymphocytes were analysed for DNA damage using the Comet assay. Genotoxicity of faecal water (FW) was assayed in human colon HT29 clone 19A cells. ‘Tail intensity’ (TI) was used as a quantitative indicator of DNA damage in the Comet assay. Intervention with bread reduced DNA damage in lymphocytes of smokers (8.3±1.7% TI versus 10.2±4.1% TI, n=19), but not of non-smokers (8.6±2.8% TI versus 8.3±2.7% TI, n=15). Faecal water genotoxicity was reduced only in non-smokers (9.4±2.9% TI versus 18.9±13.1% TI, n=15) but not in smokers (15.5±10.7% TI versus 20.4±14.1% TI, n=13). The Comet assay was efficient in the detection of both smoking-related exposure (buccal cells) and efficacy of dietary intervention (faecal samples). Smokers and non-smokers profited differently from the intervention with prebiotic bread±antioxidants. Stratification of data by genotype enhanced specificity/sensitivity of the intervention effects and contributed important information on the role of susceptibility.     

NER and HR pathways act sequentially to promote UV-C-induced germ cell apoptosis in Caenorhabditis elegans

Ultraviolet (UV) radiation-induced DNA damage evokes a complex network of molecular responses, which culminate in DNA repair, cell cycle arrest and apoptosis. Here, we provide an in-depth characterization of the molecular pathway that mediates UV-C-induced apoptosis of meiotic germ cells in the nematode Caenorhabditis elegans. We show that UV-C-induced DNA lesions are not directly pro-apoptotic. Rather, they must first be recognized and processed by the nucleotide excision repair (NER) pathway. Our data suggest that NER pathway activity transforms some of these lesions into other types of DNA damage, which in turn are recognized and acted upon by the homologous recombination (HR) pathway. HR pathway activity is in turn required for the recruitment of the C. elegans homolog of the yeast Rad9-Hus1-Rad1 (9-1-1) complex and activation of downstream checkpoint kinases. Blocking either the NER or HR pathway abrogates checkpoint pathway activation and UV-C-induced apoptosis. Our results show that, following UV-C, multiple DNA repair pathways can cooperate to signal to the apoptotic machinery to eliminate potentially hazardous cells.     

Overexpression of cytosolic glutathione peroxidase (GPX1) delays endothelial cell growth and increases resistance to toxic challenges

Oxidative stress results from the imbalance between reactive oxygen species (ROS) and ROS-scavenging molecules. Among them, cytosolic glutathione peroxidase (GPX1) plays a major role as it reduces a large part of intracellular ROS. Endothelial cells are a barrier for potentially aggressive molecules circulating in the blood stream and, therefore, are often under great oxidative stress. Thus, we investigated the potentially protective effects of GPX1 overexpression in the endothelial cell line, ECV304. We found that chronic GPX1 overexpression delays cell growth without affecting viability or decreasing resistance to hydrogen peroxide-induced oxidative stress. As GPX1 overexpression could drain the cellular reduced glutathione (GSH) pool, we also tested the effects of extracellular GSH supplementation on cell growth. Despite its largely referenced beneficial effects for cells, GSH was toxic for ECV304 cells in a dose-dependent manner but GSH-induced toxicity was reduced in selenium supplemented cultures and completely abolished in ECV304 overexpressing GPX1, compared to control. In summary, GPX1 overexpression delays cell growth and protects them from GSH and H(2)O(2) toxicity.     

Evaluation of DNA damage in flight personnel by Comet assay

There have been some suggestions that air-crew are at a higher-than-normal risk of developing cancer, since they are exposed to potential genotoxic factors. These include cosmic radiations, airborne pollutants such as the combustion products of jet propulsion, ozone, and electromagnetic fields. We used the Comet assay to investigate DNA damage in flight personnel with the aim of assessing potential health hazards in this occupational category. We studied 40 civil air-crew members who had been flying long-haul routes for at least 5 years, and compared them with a homogeneous control group of 40 healthy male ground staff. The Comet assay, or single-cell gel electrophoresis (SCGE), detects DNA single- and double-strand breaks (DSBs) and alkali-labile lesions in individual cells, and is a powerful and sensitive technique for detecting genetic damage induced by different genotoxic agents. Taking into consideration occupational risk and possible confounding factors, this assay showed a small increase, that did not reach statistical significance, of DNA damage in long-haul crew members compared to controls, indicating a lack of evident genotoxic effects. An association, although again not statistically significant, was found between reduced DNA damage and use of protective drugs (antioxidants).     

Metabolic radiolabeling: experimental tool or Trojan horse? (35)S-Methionine induces DNA fragmentation and p53-dependent ROS production.

Despite the general assumption that widely used radiolabeled metabolites such as [(35)S]methionine and (3)H-thymidine do not adversely affect or perturb cell function, we and others have shown that such low-energy beta-emitters can cause cell cycle arrest and apoptosis of proliferating cells. The goal of the present study was to elucidate the targets and mechanisms of [(35)S]methionine-induced cellular toxicity. Comet analyses (single-cell electrophoresis) demonstrated dose-dependent DNA fragmentation in rabbit smooth muscle cells within a time frame (1-4 h) well within that of most radiolabeling protocols, whereas fluorescence analyses using a peroxide/hydroperoxide-sensitive dye revealed production of reactive oxygen species (ROS). Although ROS generation was inhibitable by antioxidants, DNA fragmentation was not inhibited and was in fact observed even under hypoxic conditions, suggesting that beta-radiation-induced DNA damage can occur independently of ROS formation. Studies with p53(+/+) and p53(-/-) human colorectal carcinoma cells further demonstrated the dissociation of early DNA damage from ROS formation in that both cell types exhibited DNA fragmentation in response to radiolabeling whereas only the p53(+/+) cells exhibited significant increases in ROS formation, which occurred well after significant DNA damage was observed. These findings demonstrate that metabolically incorporated low-energy beta-emitters such as [(35)S]methionine and (3)H-thymidine can induce DNA damage, thereby initiating cellular responses leading to cell cycle arrest or apoptosis. The results of this study require a reevaluation using low-energy beta-emitters to follow not only experimental protocols in vivo processes, but also acceptable exposure levels of these genotoxic compounds in the workplace and environment.     

Antioxidative effects of fluvastatin and its metabolites against oxidative DNA damage in mammalian cultured cells

We investigated the effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on reactive oxygen species (ROS) and on oxidative DNA damage in vitro, as well as the effects of the main fluvastatin metabolites (M2, M3, and M4) and other inhibitors of the same enzyme, pravastatin and simvastatin. The hydroxyl radical and the superoxide anion scavenging activities of fluvastatin and its metabolites were evaluated using an electron spin resonance spectrometer. Fluvastatin and its metabolites showed superoxide anion scavenging activity in the hypoxanthine-xanthine oxidase system and a strong scavenging effect on the hydroxyl radical produced from Fenton's reaction. Protective effects of fluvastatin on ROS-induced DNA damage of CHL/IU cells were assessed using the single-cell gel electrophoresis assay. CHL/IU cells were exposed to either hydrogen peroxide or t-butylhydroperoxide. Fluvastatin and its metabolites showed protective effects on DNA damage as potent as the reference antioxidants, ascorbic acid, trolox, and probucol, though pravastatin and simvastatin did not exert clear protective effects. These observations suggest that fluvastatin and its metabolites may have radical scavenging activity and the potential to protect cells against oxidative DNA damage. Furthermore, ROS are thought to play a major role in the etiology of a wide variety of diseases such as cellular aging, inflammation, diabetes, and cancer development, so fluvastatin might reduce these risks.     

Cellular Antioxidant Properties of Human Natural Killer Enhancing Factor B

The protein, NKEF (natural killer enhancing factor), has been identified as a member of an antioxidant family of proteins capable of protecting against protein oxidation in cell-free assay systems. The mechanism of action for this family of proteins appears to involve scavenging or suppressing formation of protein thiyl radicals. In the present study we investigated the antioxidant protective properties of the NKEF-B protein overexpressed in an endothelial cell line (ECV304). Nkef-B-transfected cells displayed significantly lower levels of reactive oxygen species (ROS) compared with control or vector-transfected cells. Tert-Butylhydroperoxide-induced ROS was 15% lower in nkef-8-transfected cells and cytotoxicity was slightly, though not significantly, lower. NKEF-B had no effect on ROS induced by menadione or xanthine plus xanthine oxidase. NKEF-B overexpression resulted in slightly (≈ 10%) lower levels of cellular glutathione (GSH) and had no effect on rate or extent of GSH depletion following either diethylmaleate (DEM) or buthionine sulfoximine (BSO) treatment. Lipid peroxidation, assessed as thiobarbituric acid-reactive substances, was 40% lower in nkef-B-transfected cells compared with vector-only-transfected cells. DEM-induced lipid peroxidation was suppressed by NKEF-B at DEM concentrations of 20 μM to 1 mM. At 10 mM DEM, lipid peroxidation was unaffected by NKEF-B. NKEF-B expression also protected cells against menadione-induced inhibition of [³H]-thymidine uptake. The NKEF-B protein appears most effective in suppressing basal low-level oxidative injury such as that produced during normal metabolism. These results indicate that overexpression of the NKEF-B protein promotes resistance to oxidative stress in this endothelial cell line.     

Cyclin D1 expression and cell cycle response in DNA mismatch repair-deficient cells upon methylation and UV-C damage

We have evaluated cell survival, apoptosis, and cell cycle responses in a panel of DNA mismatch repair (MMR)-deficient colon and prostate cancer cell lines after alkylation and UV-C damage. We show that although these MMR-deficient cells tolerate alkylation damage, they are as sensitive to UV-C-induced damage as are the MMR-proficient cells. MMR-proficient cells arrest in the S-G2 phase of the cell cycle and initiate apoptosis following alkylation damage, whereas MMR-deficient cells continue proliferation. However, two prostate cancer cell lines that are MMR-deficient surprisingly arrest transiently in S-G2 after alkylation damage. Progression through G1 phase initially depends on the expression of one or more of the D-type cyclins (D1, D2, and/or D3). Analysis of cyclin D1 expression shows an initial MMR-independent decrease in the protein level after alkylation as well as UV-C damage. At later time points, however, only DNA damage-arrested cells showed decreased cyclin D1 levels irrespective of MMR status, indicating that reduced cyclin D1 could be a result of a smaller fraction of cells being in G1 phase rather than a result of an intact MMR system. Finally, we show that cyclin D1 is degraded by the proteasome in response to alkylation damage.     

Antigenotoxic properties of Cassia tea (Cassia tora L.): mechanism of action and the influence of roasting process

Antigenotoxic properties and the possible mechanisms of water extracts from Cassia tora L. (WECT) treated with different degrees of roasting (unroasted and roasted at 150 and 250 degrees C) were evaluated by the Ames Salmonella/microsome test and the Comet assay. Results indicated that WECT, especially unroasted C. tora (WEUCT), markedly suppressed the mutagenicity of 2-amino-6-methyldipyrido(1,2-a:3':2'-d)imidazole (Glu-P-1) and 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole (Trp-P-1). In the Comet assay performed on human lymphocytes, WECT exhibited significant protective effect on Trp-P-1-mediated DNA damage followed the order of unroasted (55%) > roasted at 150 degrees C (42% ) > roasted at 250 degrees C (29%). Pre-treatment of the lymphocytes with WEUCT resulted in 30% repression of DNA damage. However, no significant effect on excision-repair system was found during DNA damage expression time in post-treatment scheme (p>0.05). WEUCT showed 84% scavenging effect on oxygen free radicals generated in the activation process of mutagen detected by electron paramagentic resonance system. Two possible mechanisms were considered: (1) neutralization the reactive intermediate of Trp-P-1; and (2) protecting cells directly as an antioxidant that scavenge the oxygen radicals from the activation process of mutagen. The individual anthraquinone content in extracts of C. tora was measured by HPLC. Three anthraquinones, chrysophanol, emodin and rhein, have been detected under experimental conditions. The anthraquinone content decreased with increased roasting temperature. Each of these anthraquinones demonstrated significant antigenotoxicity against Trp-P-1 in the Comet assay. In conclusion, our data suggest that the decrease in antigenotoxic potency of roasted C. tora was related to the reduction in their anthraquinones.     

Effects of dietary antioxidants on DNA damage in lysed cells using a modified comet assay procedure

A modified version of the comet assay was employed to investigate the effect in vitro of dietary antioxidants in the subcellular environment. Human lymphocytes were isolated, embedded in agarose gel, lysed in high ionic strength solution with Triton X-100, and then incubated for 30 min with antioxidants at different concentrations. Gels were washed, and the comet assay performed on cells stressed by 5 min incubation with 45 microM hydrogen peroxide and on unstressed cells in parallel. Results showed that alpha-tocopherol was protective against oxidant stress, whereas caffeic acid did not protect, and at high concentration (100 microM) caused increased DNA damage. Results for quercetin suggested a direct damaging effect, but this did not reach statistical significance. However, at low concentration (3.1 microM), quercetin appeared protective. Thus some dietary antioxidants that have been shown previously to have a protective effect in the 'standard', whole-cell, comet assay cause DNA damage in this lysed-cell version. The cell membrane may have an important role in limiting cellular access of these 'double-edged' antioxidants. Furthermore, the absolute concentration and the presence of complementary or synergistic intracellular antioxidants may delineate the type of action of a putative antioxidant. We suggest that, used in conjunction with the standard comet assay, this lysed-cell version is useful for assessing the effect of the cell membrane and intracellular systems on susceptibility of DNA to oxidative damage, and will help determine the mechanism of protection or damage by phytochemicals.     

Antioxidative effects of fluvastatin and its metabolites against oxidative DNA damage in mammalian cultured cells

We investigated the effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on reactive oxygen species (ROS) and on oxidative DNA damage in vitro, as well as the effects of the main fluvastatin metabolites (M2, M3, and M4) and other inhibitors of the same enzyme, pravastatin and simvastatin. The hydroxyl radical and the superoxide anion scavenging activities of fluvastatin and its metabolites were evaluated using an electron spin resonance spectrometer. Fluvastatin and its metabolites showed superoxide anion scavenging activity in the hypoxanthine-xanthine oxidase system and a strong scavenging effect on the hydroxyl radical produced from Fenton's reaction. Protective effects of fluvastatin on ROS-induced DNA damage of CHL/IU cells were assessed using the single-cell gel electrophoresis assay. CHL/IU cells were exposed to either hydrogen peroxide or t-butylhydroperoxide. Fluvastatin and its metabolites showed protective effects on DNA damage as potent as the reference antioxidants, ascorbic acid, trolox, and probucol, though pravastatin and simvastatin did not exert clear protective effects. These observations suggest that fluvastatin and its metabolites may have radical scavenging activity and the potential to protect cells against oxidative DNA damage. Furthermore, ROS are thought to play a major role in the etiology of a wide variety of diseases such as cellular aging, inflammation, diabetes, and cancer development, so fluvastatin might reduce these risks.