Effect of extremely low-frequency electromagnetic fields on antioxidant activity in the human keratinocyte cell line NCTC 2544.

Some epidemiological studies have suggested possible associations between exposure to extremely low-frequency electromagnetic fields (ELF-EMFs) and various diseases. Recently, ELF-EMF has been considered as a therapeutic agent. To support ELF-EMF use in regenerative medicine, in particular in the treatment of skin injuries, we investigated whether significant cell damage occurs after ELF-EMF exposure. Reactive oxygen species (ROS) production was evaluated in the human keratinocyte exposed for 1 H to 50 Hz ELF-EMF in a range of field strengths from 0.25 to 2 G. Significant ROS increases resulted at 0.5 and 1 G and under these flux densities ROS production, glutathione content, antioxidant defense activity, and lipid peroxidation markers were assessed for different lengths of time. Analyzed parameters of antioxidant defense and membrane integrity showed a different trend at two selected magnetic fluxes, with a greater sensitivity of the cells exposed to 0.5 G, especially after 1 H. All significant alterations observed in the first 4 H of exposure reverted to controls 24 H after suggesting that under these conditions, ELF-EMF induces a slight oxidative stress that does not overwhelm the metabolic capacity of the cells or have a cytotoxic effect.

Protective Effect of Juglans regia L. Walnut Extract Against Oxidative DNA Damage.

Walnuts (Juglans regia L.) are relevant components of the Mediterranean diet providing important macronutrients, micronutrients and other bioactive constituents including unsaturated fatty acids, proteins, fiber, vitamins, minerals, phytosterols and polyphenols. Although the walnut beneficial effects in human health are widely recognized by a lot of epidemiologic studies very little is known regarding its effect on damaged DNA. The aim of the present study was to investigate the effect of Juglans regia L. ethanolic extract from kernel on the induction of DNA strand breaks by thiol/Fe3+/O2 mixed function oxidase, tert-butyl hydroperoxide or UVC radiations in acellular and cellular models. Plasmid DNA cleavage and fast Halo assay were used to monitor oxidative damage to DNA. Both approaches showed protection of oxidatively injured DNA. These results agree with a lot of scientific proofs which recommend walnut as dietary adjunct in health promotion and prevention as well as in treatment of lifestyle-related oxidative diseases.

Effects of oxidative stress on mitochondrial content and integrity of human anastomotic colorectal dehiscence: a preliminary DNA study.

BACKGROUND: Anastomotic dehiscence is one of the most severe complications of colorectal surgery. Gaining insight into the molecular mechanisms responsible for the development of anastomotic dehiscence following colorectal surgery is important for the reduction of postoperative complications. OBJECTIVE: Based on the close relationship between surgical stress and oxidative stress, the present study aimed to determine whether a correlation exists between increased levels of reactive oxygen species and colorectal anastomotic dehiscence. METHODS: Patients who underwent surgical resection for colorectal cancer were divided into three groups: patients with anastomotic dehiscence (group 1); patients without dehiscence who underwent neoadjuvant radiochemotherapy (group 2); and patients without anastomotic dehiscence who did not undergo neoadjuvant radiochemotherapy (group 3). Quantitative polymerase chain reaction and real-time polymerase chain reaction assays were performed to measure nuclear DNA and mitochondrial DNA (mtDNA) content, and possible oxidative damage to nonmalignant colon and rectal tissues adjacent to the anastomoses. RESULTS: mtDNA content was reduced in the colon tissue of patients in groups 1 and 2. Rectal mtDNA was found to be more damaged than colonic mtDNAs in all groups. The 4977 bp common deletion was observed in the mtDNA of tissues from both the colon and rectum of all patients. DISCUSSION: Patients in groups 1 and 2 were more similar to one another than to group 3, probably due to higher levels of reactive oxygen species in the mitochondria; the greater damage found in the rectum suggests that dehiscence originates primarily from the rectal area. CONCLUSIONS: The present study of mtDNA analyses of normal human colon and rectal tissues from patients with colorectal cancer is among the first of its kind.

A high number of 'natural' mitochondrial DNA polymorphisms in a symptomatic Brugada syndrome type 1 patient.

Brugada syndrome (BrS) is a rare genetic arrhythmic disorder with a complex model of transmission. At least 20 different genes have been identified as BrS-causal or susceptibility genes. Of these, SCN5A is the most frequently mutated. Coregulation of different mutations or genetic variants, including mitochondrial DNA (mtDNA), may contribute to the clinical phenotype of the disease. In thepresent study, we analysed the mitochondrial genome of a symptomatic BrS type 1 patient to investigate a possible mitochondrial involvement recently found in the arrhytmogenic diseases. No pathogenic mutation was identified; however, a high number of singlenucleotide polymorphisms were found (n=21) and some of them were already been reported in molecular autopsy case for sudden death.The results reported here further support our hypothesis on the potential role of mtDNA polymorphisms in mitochondrial dysfunction, which may represent a risk factor for arrhythmogenic disease.

Effect of quercetin on oxidative nuclear and mitochondrial DNA damage.

Quercetin is a well-investigated antioxidant known to protect cells against oxidative nuclear DNA damage. There is no knowledge regarding its effect on oxidative mitochondrial DNA damage. In this study we investigated the effect of quercetin on oxidatively-injured DNA. Cell-free and cell studies were performed. Cell-free analyses carried out on plasmidic DNA showed that quercetin protects from all oxidative challenges used. Cellular studies were carried out on NCTC 2544 cells which were insulted with hydrogen peroxide and UVC radiations. Nuclear and mitochondrial DNAs were analysed by measuring DNA damage with a quantitative polymerase chain reaction. Quercetin supplementation showed significant genoprotective activity on mitochondrial DNA when hydroperoxide was used. The evidence of the protection afforded by quercetin suggests that this flavonoid may play an important role on mitochondrial genome stability.

Protective Role of Italian Juglans regia L. nut Ethanolic Extract in Human Keratinocytes under Oxidative and Inflammatory Stress.

OBJECTIVE: In this research, fatty acid profile and polyphenolic content of an ethanolic extract of walnut from Juglans regia L. collected in Central Italy, were characterized. The potential antioxidant and anti-inflammatory effects of the extract were investigated in the human keratinocytes cell line. METHODS: Fatty acid profile was determined by gas chromatography/mass spectrometry analysis, total phenolic content by Folin-Ciocalteu method and aluminum chloride colorimetric method was used for determination of total flavonoids. Kertatinocytes were exposed to t-butyl hydroperoxide or Tumor Necrosis Factor alfa in the absence or presence of extract. Reduced glutathione was determined by Sedlak method; lipid peroxidation was assessed by measuring thiobarbituric acid reactive substances. t-butyl hydroperoxide and Tumor Necrosis Factor alfa-induced intracellular reactive oxygen species were monitored by fluorescent probes. The expression of some genes related to the inflammatory process (IL-6, IL-8, ikB, and ICAM) were analysed by Real-time PCR. RESULTS: JRE contains a favourable fatty acid profile with low saturated fats (19%) and high-unsaturated fats (81%) with a prevalence of the omega-6 linoleic acid (48%). Also a significant amount of polyphenols was found (5,0052 mg gallic acid equivalent/gdw). Antioxidant and anti-inflammatory properties of JRE were observed on analysed cellular model. JRE antioxidants counteracted ROS production, GSH depletion and lipid peroxidation as well downregulated the expression of some genes related to the inflammatory process. Moreover, polyunsaturated fatty acids exhibited anti-inflammatory properties. CONCLUSION: The obtained results uphold walnut as dietary adjunct in health promotion and drive towards its development in drug therapy against chronic inflammatory disorders, including inflammatory skin diseases.

Structural analysis of the rDNA intergenic spacer of Tuber borchii.

The sequence and characterisation of the entire nuclear rDNA intergenic spacer (IGS) for the genus Tuber are presented. Sequence analyses showed that the organisation of the Tuber borchii rDNA IGS is typical of rDNA spacers, consisting of a central repetitive region and flanking unique sequences on either side. Direct repeats, symmetry elements, tandem repeats and possible areas of recombination were found. The putative ends of the 25S and 17S rDNA were identified. The presence of 5S rDNA in the IGS region was excluded.

Effects of a static magnetic field on cell growth and gene expression in Escherichia coli.

Escherichia coli cultures exposed to a 300mT static magnetic field (SMF) were studied in order to analyse possible induced changes in cellular growth and gene expression. Biomass was evaluated by visible-light spectrometry and gene expression analyses were carried out by use of RNA arbitrarily primed PCR. The bacterial strain XL-1Blue, cultivated in traditional and modified Luria-Bertani medium, was exposed to SMF generated by permanent neodymium magnetic disks. The results show alterations induced by SMF in terms of increased cell proliferation and changes in gene expression compared with control groups. Three cDNAs were found to be expressed only in the exposed cells, whereas one cDNA was more expressed in the controls. One clone, expressed only in the exposed cells, corresponds to a putative transposase. This is of particular interest in that it suggests that exposure to a magnetic field may stimulate transposition activity.

In vitro protective effect of Rhodiola rosea extract against hypochlorous acid-induced oxidative damage in human erythrocytes.

Rhodiola rosea L. (Crassulaceae) is a plant living at high altitudes in Europe and Asia. Its roots have long been used in the traditional medical system of these geographical areas to increase the organism resistance to physical stress; today, it has become an important component of many dietary supplements. In this study we investigate the antioxidant capacity of the R. rosea aqueous extract evaluating its ability to counteract some of the main damages induced by hypochlorous acid (HOCl), a powerful oxidant generated by activated phagocytes, to human erythrocytes. Ascorbic acid was used as a reference substance because of its physiological HOCl-scavenging ability. Our study demonstrates that R. rosea is able to significantly protect, in a dose-dependent manner, human RBC from glutathione (GSH) depletion, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inactivation and hemolysis induced by the oxidant. Furthermore, we demonstrate that R. rosea aqueous extract acts from the inside of the erythrocyte suggesting a probable involving of cell components. The protection on GSH afforded by the R. rosea extract with respect to ascorbic acid, occurred also if added 2 or 5 min. later than the oxidant, suggesting a more rapid or powerful effect.

PCR amplification and polymorphism analysis of the intergenic spacer region of ribosomal DNA in Tuber borchii.

PCR amplification of the complete intergenic spacer region (IGS) of the Tuber borchii nuclear ribosomal repeat was obtained using universal primers CNL 12 and NS1rev. In order to improve amplification yield a specific primer, T1, was selected from a partial sequence of the IGS product. IGS diversity was characterized both at the intraindividual and intraspecific level. The results obtained at the intraindividual level showed 10% varying repeats on ten screened colonies, while at the intraspecific level the IGS polymorphism was evident as difference in length amplification between mycelial strains and fruit bodies: 3.5 kb and 2 kb respectively.

Effect of surgical stress on nuclear and mitochondrial DNA from healthy sections of colon and rectum of patients with colorectal cancer.

Surgical resection at any location in the body leads to stress response with cellular and subcellular change, leading to tissue damage. The intestine is extremely sensitive to surgical stress with consequent postoperative complications. It has been suggested that the increase of reactive oxygen species as subcellular changes plays an important role in this process. This article focuses on the effect of surgical stress on nuclear and mitochondrial DNA from healthy sections of colon and rectum of patients with colorectal cancer. Mitochondrial DNA copy number, mitochondrial common deletion and nuclear and mitochondrial 8-oxo-2'-deoxyguanosine content were measured. Both the colon and rectal tissue were significantly damaged either at the nuclear or mitochondrial level. In particular, mitochondrial DNA was more damaged in rectum than in colon. The present investigation found an association between surgical stress and nuclear and mitochondrial DNA damage, suggesting that surgery may generate an increase in free radicals, which trigger a cascade of molecular changes, including alterations in DNA.

Aqueous extract from Vitis vinifera tendrils is able to enrich keratinocyte antioxidant defences.

An aqueous extract of V. vinifera L. tendrils was evaluated for its ability to enrich the antioxidant capacity of cultured cells. The long-time antioxidant capability of the extract was measured by in vitro chemical methods, and its influence on reduced glutathione levels and plasma membrane oxido reductase activity was determined in cultured human keratinocytes (NCTC 2544). Keratinocytes are cells normally exposed to oxidative stress, and for this reason adequately equipped with antioxidant defences. However, it has long been suggested that exogenous antioxidants may play an important role in minimizing the adverse effects of oxidative stress on skin.We demonstrated that V. vinifera tendril aqueous extract was able to increase, in a time- and dose-dependent manner, the reduced glutathione concentration and activity of trans plasma membrane oxido reductase as an indirect evaluation of the intracellular redox status of the cells demonstrating a relevant antioxidant activity of this phytocomplex.

Rhodiola rosea ability to enrich cellular antioxidant defences of cultured human keratinocytes.

Keratinocytes are cells strongly exposed to oxidative stress, but normally good equipped for antioxidant responses. However, it has long been suggested that exogenous antioxidants could play a useful role in minimizing the adverse skin responses associated with such oxidant species. In this work it was paid attention to the extract of Rhodiola rosea L. roots by using the phytocomplex as a whole because of the important activity of its composition and mutual distribution of its components. We have measured the protection afforded by the extract to reduced glutathione levels, glyceraldehyde-3-phosphate dehydrogenase activity, and thiobarbituric acid reactive substances levels in cultured human keratinocytes (NCTC 2544) exposed to different oxidative insults: Fe(II)/ascorbate, Fe(II)/H(2)O(2), and tert-butyl-hydroperoxide. We also have investigated the influence of the R. rosea extract on the production of intracellular reactive oxygen species and on the activity of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase). Furthermore, we have demonstrated that R. rosea extract was able to increase in a time- and dose-dependent manner the activity of the trans plasma membrane oxido reductase activity as an indirect evaluation of the intracellular redox status and this effect was already evident with small concentration of the extract and in a long time. As a result, NCTC 2544 are able to better counteract to several oxidative insults if incubated with R. rosea extract demonstrating a very good antioxidant activity of this phytocomplex.

Effects of high static magnetic field exposure on different DNAs.

The effects of magnetic fields produced by permanent magnets on different DNA sources were investigated in vivo and in vitro. Escherichia coli DNA, plasmid, and amplification products of different lengths were used as the magnetic field target. The in vivo assays did not reveal any DNA alterations following exposure, demonstrating the presence of cell dependent mechanisms, such as the repair system and the buffering action of the heat shock proteins DNA K/J (Hsp 70/40). The in vitro assays displayed interactions between the magnetic field and DNA, revealing principally that magnetic field exposure induces DNA alterations in terms of point mutations. We speculate that the magnetic field can perturb DNA stability interacting with DNA directly or potentiating the activity of oxidant radicals. This genotoxic effect of the magnetic field, however, is minimized in living organisms due to the presence of protective cellular responses.