Many compounds can interact with DNA leading to changes of DNA structure as point mutation and bases excision, which could trigger some metabolic failures, which leads to the changes in DNA structure resulting in cancer. Oxidation of nucleic acid bases belongs to the one of the mostly occurred type of DNA damaging leading to the above mentioned phenomena. The investigation of processes of DNA oxidation damage is topical and electrochemical methods include a versatile and sensitive tool for these purposes. 8-hydroxydeoxyguanosine (8-OHdG) is the most widely accepted marker of DNA damage. Oxidative damage to DNA by free radicals and exposure to ionizing radiation generate several other products within the double helix besides mentioned oxidation products of nucleic acid bases. The basic electrochemical behaviour of nucleic acids bases on various types of carbon electrodes is reviewed. Further, we address our attention on description of oxidation mechanisms and on detection of the most important products of nucleic bases oxidation. The miniaturization of detector coupled with some microfluidic devices is suggested and discussed. The main aim of this review is to report the advantages and features of the electrochemical detection of guanine oxidation product as 8-OHdG and other similarly produced molecules as markers for DNA damage.

• MeSH
• deoxyguanosin analogy a deriváty   analýza   genetika   MeSH
• DNA chemie   genetika   MeSH
• elektrochemické techniky přístrojové vybavení   metody   MeSH
• lidé MeSH
• mikrofluidní analytické techniky přístrojové vybavení   metody   MeSH
• oxidační stres MeSH
• poškození DNA * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

HPLC determination of a background level of 8 oxodG and 2-dG in rat liver after addition of copper to perfusate was developed. The reversed phase analytical column Purospher® STAR C18e with 50 mmol L–1 phosphate buffer, pH 5.5 and methanol (92:8, v/v) mobile phase was applied for the analysis. The validation of the HPLC method according to linearity, accuracy and precision was carried out. Oxidative DNA damage (expressed as concentration ratio of 8-oxodG/106 2-dG) was determined by the simultaneous measuring of 2-dG with UV detection followed by coulochemical detection of 8 oxodG. The procedure using a model of liver damage caused by intoxication with copper and ischemia / reperfusion with addition of various concentrations of CuSO4 to the perfused rat livers was tested. The aim of this study was to decide whether the toxicity of copper in liver perfusates is related to protein oxidation and oxidative DNA damage. The high contribution to the DNA damage can be related to the physical liver manipulation during harvest and reperfusion as well as to artefacts induced during the sample preparation (time-consuming sample handling during DNA isolation and extraction). The obtained results pointed out that the DNA damage occurred already during liver handling even before application of CuSO4, whereby concentration of CuSO4 higher than 0.03 mmol L–1 caused a total liver damage, which led to a complete stop of the flow of the perfusate.

• MeSH
• biologické markery MeSH
• deoxyguanosin chemie   MeSH
• elektrochemické techniky * metody   využití   MeSH
• krysa rodu rattus MeSH
• měď * toxicita   MeSH
• nemoci jater * MeSH
• oxidační stres MeSH
• poškození DNA * MeSH
• spektrofotometrie metody   využití   MeSH
• vysokoúčinná kapalinová chromatografie * metody   využití   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

BACKGROUND: Flavonoids possess a rich polypharmacological profile and their biological role is linked to their oxidation state protecting DNA from oxidative stress damage. However, their bioavailability is hampered due to their poor aqueous solubility. This can be surpassed through encapsulation to supramolecular carriers as cyclodextrin (CD). A quercetin- 2HP-β-CD complex has been formerly reported by us. However, once the flavonoid is in its 2HP-β-CD encapsulated state its oxidation potential, its decomplexation mechanism, its potential to protect DNA damage from oxidative stress remained elusive. To unveil this, an array of biophysical techniques was used. METHODS: The quercetin-2HP-β-CD complex was evaluated through solubility and dissolution experiments, electrochemical and spectroelectrochemical studies (Cyclic Voltammetry), UV-Vis spectroscopy, HPLC-ESI-MS/MS and HPLC-DAD, fluorescence spectroscopy, NMR Spectroscopy, theoretical calculations (density functional theory (DFT)) and biological evaluation of the protection offered against H2O2-induced DNA damage. RESULTS: Encapsulation of quercetin inside the supramolecule's cavity enhanced its solubility and retained its oxidation profile. Although the protective ability of the quercetin-2HP-β-CD complex against H2O2 was diminished, iron serves as a chemical stimulus to dissociate the complex and release quercetin. CONCLUSIONS: We found that in a quercetin-2HP-β-CD inclusion complex quercetin retains its oxidation profile similarly to its native state, while iron can operate as a chemical stimulus to release quercetin from its host cavity. GENERAL SIGNIFICANCE: The oxidation profile of a natural product once it is encapsulated in a supramolecular carrier was unveiled as also it was discovered that decomplexation can be triggered by a chemical stimilus.

• MeSH
• biologická dostupnost MeSH
• cyklodextriny chemie   metabolismus   MeSH
• Jurkat buňky MeSH
• lidé MeSH
• oxidace-redukce MeSH
• oxidační stres účinky léků   MeSH
• oxidancia farmakologie   MeSH
• peroxid vodíku farmakologie   MeSH
• poškození DNA účinky léků   MeSH
• quercetin chemie   metabolismus   MeSH
• železo chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

RATIONALE: Peroxidation of lipids in cellular membranes results in the release of volatile organic compounds (VOCs), including saturated aldehydes. The real-time quantification of trace VOCs produced by cancer cells during peroxidative stress presents a new challenge to non-invasive clinical diagnostics, which as described here, we have met with some success. METHODS: A combination of selected ion flow tube mass spectrometry (SIFT-MS), a technique that allows rapid, reliable quantification of VOCs in humid air and liquid headspace, and electrochemistry to generate reactive oxygen species (ROS) in vitro has been used. Thus, VOCs present in the headspace of CALU-1 cancer cell line cultures exposed to ROS have been monitored and quantified in real time using SIFT-MS. RESULTS: The CALU-1 lung cancer cells were cultured in 3D collagen to mimic in vivo tissue. Real-time SIFT-MS analyses focused on the volatile aldehydes: propanal, butanal, pentanal, hexanal, heptanal and malondialdehyde (propanedial), that are expected to be products of cellular membrane peroxidation. All six aldehydes were identified in the culture headspace, each reaching peak concentrations during the time of exposure to ROS and eventually reducing as the reactants were depleted in the culture. Pentanal and hexanal were the most abundant, reaching concentrations of a few hundred parts-per-billion by volume, ppbv, in the culture headspace. CONCLUSIONS: The results of these experiments demonstrate that peroxidation of cancer cells in vitro can be monitored and evaluated by direct real-time analysis of the volatile aldehydes produced. The combination of adopted methodology potentially has value for the study of other types of VOCs that may be produced by cellular damage.

• MeSH
• aldehydy analýza   metabolismus   MeSH
• buněčné kultury metody   MeSH
• elektrochemické techniky MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory metabolismus   MeSH
• oxidace-redukce MeSH
• oxidační stres fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Besides the well-known chemoprotective effects of polyphenols, their prooxidant activities via interactions with biomacromolecules as DNA and proteins are of the utmost importance. Current research focuses not only on natural polyphenols but also on synthetically prepared analogs with promising biological activities. In the present study, the antioxidant and prooxidant properties of a semi-synthetic flavonolignan 7-O-galloylsilybin (7-GSB) are described. The presence of the galloyl moiety significantly enhances the antioxidant capacity of 7-GSB compared to that of silybin (SB). These findings were supported by electrochemistry, DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity, total antioxidant capacity (CL-TAC) and DFT (density functional theory) calculations. A three-step oxidation mechanism of 7-GSB is proposed at pH 7.4, in which the galloyl moiety is first oxidized at Ep,1=+0.20V (vs. Ag/AgCl3M KCl) followed by oxidation of the 20-OH (Ep,2=+0.55V) and most probably 5-OH (Ep,3=+0.95V) group of SB moiety. The molecular orbital analysis and the calculation of O-H bond dissociation enthalpies (BDE) fully rationalize the electrooxidation processes. The metal (Cu(2+)) complexation of 7-GSB was studied, which appeared to involve both the galloyl moiety and the 5-OH group. The prooxidant effects of the metal-complexes were then studied according to their capacity to oxidatively induce DNA modification and cleavage. These results paved the way towards the conclusion that 7-O-galloyl substitution to SB concomitantly (i) enhances antioxidant (ROS scavenging) capacity and (ii) decreases prooxidant effect/DNA damage after Cu complexation. This multidisciplinary approach provides a comprehensive mechanistic picture of the antioxidant vs. metal-induced prooxidant effects of flavonolignans at the molecular level, under ex vivo conditions.

• MeSH
• antioxidancia chemie   metabolismus   farmakologie   MeSH
• bifenylové sloučeniny metabolismus   MeSH
• elektrochemické techniky MeSH
• luminiscenční měření MeSH
• měď metabolismus   MeSH
• pikráty metabolismus   MeSH
• poškození DNA * MeSH
• silymarin analogy a deriváty   chemie   metabolismus   farmakologie   MeSH
• simulace molekulární dynamiky MeSH
• spektrofotometrie ultrafialová MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of the study was to establish and optimize an electrochemical method for the assay of low-molecular-weight antioxidants (LMWA). We used square-wave vol¬tammetry (at 0–1.1 V) on screen-printed sensors with C working electrodes. Optimum parameters were: potential step and amplitude 5 mV and frequency 1 Hz. Ascorbic acid, trolox, N-acetylcysteine and melatonin were used as standard antioxidants to test the linearity and sensitivity of the method. Limits of detection were 0.09 mmol l–1 for ascorbic acid, 0.04 mmol l–1 for melatonin, 0.03 mmol l–1 for trolox, and 0.07 mmol l–1 for N-acetylcysteine. 20 ??????l samples were used. The suitability of the method for the assay was confirmed by the standard ferric reducing ability of plasma test. Both methods correlated well (correlation coefficient 0.9778).

• MeSH
• antioxidancia * analýza   MeSH
• chemické techniky analytické metody   přístrojové vybavení   využití   MeSH
• cystein analýza   krev   MeSH
• elektrochemické techniky * metody   přístrojové vybavení   využití   MeSH
• glutathion analýza   krev   MeSH
• krevní plazma * MeSH
• kyselina askorbová * analýza   krev   MeSH
• kyselina močová analýza   krev   MeSH
• lidé MeSH
• melatonin analýza   krev   MeSH
• metody pro přípravu analytických vzorků metody   přístrojové vybavení   MeSH
• oxidační stres MeSH
• reaktivní formy kyslíku chemie   MeSH
• tokoferoly analýza   krev   MeSH
• vitamin A analýza   krev   MeSH
• volné radikály chemie   MeSH
• Check Tag
• lidé MeSH

Guanosine derivatives are important for diagnosis of oxidative DNA damage including 8-hydroxy-2'-deoxyguanosine (8-OHdG) as one of the most abundant products of DNA oxidation. This compound is commonly determined in urine, which makes 8-OHdG a good non-invasive marker of oxidation stress. In this study, we optimized and tested the isolation of 8-OHdG from biological matrix by using paramagnetic particles with an antibody-modified surface. 8-OHdG was determined using 1-naphthol generated by alkaline phosphatase conjugated with the secondary antibody. 1-Naphthol was determined by stopped flow injection analysis (SFIA) with electrochemical detector using a glassy carbon working electrode and by stationary electrochemical detection using linear sweep voltammetry. A special modular electrochemical SFIA system which needs only 10  μL of sample including working buffer for one analysis was completely designed and successfully verified. The recoveries in different matrices and analyte concentration were estimated. Detection limit (3 S/N) was estimated as 5  pg/mL of 8-OHdG. This method promises to be very easily modified to microfluidic systems as "lab on valve". The optimized method had sufficient selectivity and thus could be used for determination of 8-OHDG in human urine and therefore for estimation of oxidative DNA damage as a result of oxidation stress in prostate cancer patients.

• MeSH
• alkalická fosfatasa MeSH
• deoxyguanosin analogy a deriváty   moč   MeSH
• elektrochemické techniky metody   MeSH
• ELISA metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• magnety MeSH
• mikrofluidní analytické techniky přístrojové vybavení   metody   MeSH
• mikrosféry MeSH
• nádory prostaty moč   MeSH
• naftoly MeSH
• oxidační stres MeSH
• protilátky MeSH
• průtoková injekční analýza MeSH
• robotika přístrojové vybavení   metody   MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH