May DNA analyses be biased by hidden oxidative damage? Voltammetric study of temperature and oxidation stress effect.

The analysis of nucleic acids is one of the fundamental parts of modern molecular biology and molecular diagnostics. The information collected predominantly depends on the condition of the genetic material. All potential damage induced by oxidative stress may affect the final results of the analysis of genetic material obtained using commonly used techniques such as polymerase chain reaction or sequencing. The aim of this work was to evaluate the effects of high temperature and pH on DNA structure in the context of the occurrence of oxidative damage, using square-wave voltammetry and two independent research protocols. We resulted in visible oxidation damage registered in acidic conditions after the thermal denaturation process (pH 4.7) with changes in the intensity of guanine and adenine signals. However, using phosphate buffer (pH 7.0) for DNA denaturation negatively affected the DNA structure, but without any oxidized derivatives present. This leads to the conclusion that oxidation occurring in the DNA melting process results in the formation of various derivatives of nucleobases, both electrochemically active and inactive. These derivatives may distort the results of molecular tests due to the possibility of forming complementary bonds with various nucleobases. For example, 8-oxoguanine can form pairs with both cytosine and adenine.

Potential negative effect of long-term exposure to nitrofurans on bacteria isolated from wastewater.

Nitrofurans are broad-spectrum bactericidal agents used in a large quantity for veterinary and human therapy. This study reports the long-term impact of two nitrofuran representatives, nitrofurantoin (NFT) and furaltadone (FTD) on the bacterial strains Sphingobacterium siyangense FTD2, Achromobacter pulmonis NFZ2, and Stenotrophomonas maltophilia FZD2, isolated from a full-scale wastewater treatment plant. Bacterial whole genome sequencing was used for preliminary strains characterization. The metabolomic, electrochemical, and culture methods were applied to understand changes in the bacterial strains after 12-month exposure to nitrofurans. The most significantly altered metabolic pathways were observed in amino acid and sugar metabolism, and aminoacyl-tRNA biosynthesis. Disrupted protein biosynthesis was measured in all strains treated with antibiotics. Prolonged exposure to NFT and FTD also triggered mutagenic effects, affected metabolic activity, and facilitated oxidative stress within the cells. Nitrofuran-induced oxidative stress was evidenced from an elevated activity of catalase and glutathione S-transferases. NFT and FTD elicited similar but not identical responses in all analyzed strains. The results obtained in this study provide new insights into the potential risks of the prolonged presence of antimicrobial compounds in the environment and contribute to a better understanding of the possible impacts of nitrofuran antibiotics on the bacterial cells.

Genoprotective effect of cornelian cherry (Cornus mas L.) phytochemicals, electrochemical and ab initio interaction study.

Cornelian cherry (Cornus mas L.) has broad and multidimensional potential in preventing civilisational diseases. Part of these diseases results from DNA oxidative mutations. Thus, the paper aimed to predict how phenolics present in C. mas may interact with dsDNA in ab initio experiment and to check the effect of different cornelian cherry extracts on DNA structure and DNA oxidation. A special research model was designed using biosensor with a carbonpaste electrode. We resulted in various effects observed for phenolics and the extracts. Flavonoids, but of vitexin interacted with declining energy of the DNA models and liability of DNA oxidation. However, for 8-oxoguaniosine the trend was the opposite. Among the evaluated extracts, water-ethanolic extracts caused decline in adenine and guanine signals after dsDNA exposition on the extract. Principal component analysis showed that alcoholic extracts of cv. Szafer and Slowianin, which were rich in apigenin and kaempferol exhibit mild genoprotective effect.

Electrochemical screening of genoprotective and antioxidative effectiveness of Origanum vulgare L. and its functionality in the prevention of neurodegenerative disorders.

A new way of electrochemical DNA sensor using as a screening tool for the determination of phytochemicals with high genoprotective functionality is proposed. The biosensor's detection layer was prepared with double stranded deoxyribonucleic acid (ds DNA) that were subjected to oxidative stress induced by •OH radicals generated by Fenton reaction. The oxidized guanine derivative, 8-oxo-7,8-dihydro-2'-deoxyguanosine, was treated as an indicator of DNA oxidative damage. This derivative may cause mutation through its ability to pair with adenine. The abnormalities of DNA structure and DNA repair system are known to be directly related to progressive neurodegeneration. The present study showed that during oxidative stress, the 2.5% oregano extract protected guanine from undergoing oxidation to 8-oxoguanine. The results revealed that this genoprotective effectiveness can make oregano a very efficient protective barrier against oxidative stress. Due to these unique properties of oregano we propose the recipe of a functional bread with its addition. It was found that the functionality of the prepared bread was not limited to antioxidative activity but also is expressed in the inhibition of cholinesterases. These findings indicate that oregano can act as an important component in the therapeutic diet recommended in neurodegenerative disorders.

Chocolate desserts with ricotta hydrolysates: In vitro study of inhibitory activity against angiotensin-converting enzyme and cholinesterase.

Food can be a source of valuable peptides with high bioactivity, which regulate the functioning of cardiovascular and nervous systems. The aim of the study was to evaluate the possibility of usage ricotta after hydrolysis to obtain innovative chocolate desserts. It was shown that the hydrolysis of whey proteins in ricotta had insignificant effect on the texture indices of the products, except gumminess, as it declined to 16% in ricotta samples and to 7% in case of chocolate dessert samples. Confirmed was that the hydrolysis of the ricotta affected the activity of prepared desserts with respect to cholinesterases and angiotensin-converting enzyme. Enzymatic hydrolysate of ricotta may be consider as a semifinished product of high functional activity, and its further application in dessert production allows to provide novel prohealth new products. PRACTICAL APPLICATION: Study results indicate new feasibilities of ricotta application as functional ingredient of new products--chocolate desserts. The results show that ricotta after the stage of enzyme hydrolysis of proteins might have noticeable effect on product functionality. A measurable benefit for the consumer is the receipt of a new product with favorable health-promoting properties, and for the entrepreneur new possibilities to expand the range of functional products. Moreover, described technology allows to use dairy byproducts for new products developments, such as chocolate desserts, due to sustainability development strategy.

The Genoprotective Role of Naringin.

Since ancient times, fruits and edible plants have played a special role in the human diet for enhancing health and maintaining youthfulness. The aim of our work was to determine the interactions between naringin, a natural ingredient of grapefruits, and DNA using an electrochemical biosensor. Electrochemical methods allow analyzing the damages occurring in the structure of nucleic acids and their interactions with xenobiotics. Our study showed that the changes in the location of electrochemical signals and their intensity resulted from the structural alterations in DNA. The signal of adenine was affected at lower concentrations of naringin, but the signal of guanine was unaffected in the same condition. The dynamics of changes occurring in the peak height and surface of adenine related to naringin concentration was also significantly lower. The complete binding of all adenine bases present in the tested double-stranded DNA solution was observed at naringin concentrations ranging from 8.5 to 10.0 µM. At larger concentrations, this active compound exerted an oxidizing effect on DNA. However, the critical concentrations of naringin were found to be more than twice as high as the dose absorbable in an average human (4 µM). The results of our work might be helpful in the construction of electrochemical sensors for testing the content of polyphenols and would allow determining their genoprotective functionality.

Detection of bar gene encoding phosphinothricin herbicide resistance in plants by electrochemical biosensor.

An electrochemical biosensor for the detection of bar gene coding phosphinothricin herbicide resistance is presented. The detection was based on hybridization reaction between the specific to bar gene 19-mer probe immobilized on the electrode surface and complementary DNA in a sample. Single-stranded DNA probe specific to bar gene was covalently attached by 5'-phosphate end to the surface of carbon paste electrode. Outer layer of a conventional CPE was provided with carboxyl groups of stearic acid. ssDNA was coupled to the electrode through ethylenediamine with the use of water-soluble 1-ethyl-3(3'-dimethylaminopropyl)-carbodiimide and N-hydroxy-sulfosuccinimide as activating reagents. Hybridization reaction at the electrode surface was detected via Co(bpy)(3)(3+), which possess a much higher affinity to the resulting DNA duplex compared to ssDNA probe. Detection limit of the sensor was 0.1 microM of target DNA fragments and its response was linear from 5 to 20 microM. Hybridization event was also detected by measuring guanine peak but this approach presented distinctly higher detection limit (1 muM) and lower reproducibility. Complete time of one measurement with the use of the biosensor including covalent attachment of ethylenediamine (linker) and ssDNA probe to the electrode, hybridization with target and interaction with electroactive indicator was about 70 min.

Application of DNA Hybridization Biosensor as a Screening Method for the Detection of Genetically Modified Food Components.

An electrochemical biosensor for the detection of genetically modified food components is presented. The biosensor was based on 21-mer single-stranded oligonucleotide (ssDNA probe) specific to either 35S promoter or nos terminator, which are frequently present in transgenic DNA cassettes. ssDNA probe was covalently attached by 5'-phosphate end to amino group of cysteamine self-assembled monolayer (SAM) on gold electrode surface with the use of activating reagents - water soluble 1-ethyl-3(3'- dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxy-sulfosuccinimide (NHS). The hybridization reaction on the electrode surface was detected via methylene blue (MB) presenting higher affinity to ssDNA probe than to DNA duplex. The electrode modification procedure was optimized using 19-mer oligoG and oligoC nucleotides. The biosensor enabled distinction between DNA samples isolated from soybean RoundupReady® (RR soybean) and non-genetically modified soybean. The frequent introduction of investigated DNA sequences in other genetically modified organisms (GMOs) give a broad perspectives for analytical application of the biosensor.