Rita Csepregi

A novel luminol-based enhanced chemiluminescence antioxidant capacity microplate assay for use in different biological matrices

INTRODUCTION Reactive oxygen species (ROS) are normal metabolic products of living cells. However, a decrease of the defense mechanisms against the effects of ROS or increased ROS production maybe one important causative factor of cellular damage. A non-enzymatic scavenger system is considered to be responsible for the maintenance of total antioxidant capacity (TAC) as a protection against oxidative injuries that exist in all higher plants and in mammals as well. METHODS In our work, we optimized and validated a luminol-peroxidase-4-iodophenol-H2O2 enhanced chemiluminescence-based (ECL) TAC measurement technique. BSA was applied in the reagent to prevent peroxidase from auto-oxidation. The ECL method was suitable for plant extracts and for human blood serum as well. Our TAC technique was adapted to microplates and compared to ORAC assay using plant extracts. RESULTS AND DISCUSSION The ECL method is fast (10min) with an interassay precision of <10% as CV. TAC values of ethanolic extracts of 10 plant species did correlate (ECL vs ORAC assay data: r=0.84, 95% confidence interval, CI=0.78-0.89, P<0.001) but with systematic bias. Analysis of serum samples obtained from septic and control patients showed significantly higher TAC values in the patient group compared to those of controls (p<0.01). Moreover, we could discriminate between surviving and non-surviving patients, based on their TAC values (p<0.01). Pearsons statistics showed the strongest positive correlation with serum uric acid (r=0.73). Besides the routine laboratory parameters, our novel TAC method might give complementary information on the severity of systemic inflammation.

Examination of secondary metabolites and antioxidant capacity of Anthyllis vulneraria, Fuchsia sp., Galium mollugo and Veronica beccabunga

Anthyllis vulneraria L., Fuchsia sp., Galium mollugo L., and Veronica beccabunga L. were selected to analyse the phenolic content and the antioxidant activity by ferric ion reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC) assays. The highest polyphenol, tannin, and flavonoid contents were measured in Fuchsia species (7.40 ± 0.8, 5.62 ± 0.7 and 0.72 ± 0.1 g/100 g dry weight), while the lowest values were detected in Anthyllis vulneraria (0.68 ± 0.02, 0.17 ± 0.03 and 0.45 ± 0.01 g/100 g dry weight) and Galium mollugo (1.77 ± 0.05, 0.49 ± 0.04 and 0.16 ± 0.06 g/100 g dry weight). The leaf extract of Fuchsia sp. had the highest, while the herb of A. vulneraria had the lowest antioxidant effect measured by both methods, which is probably related to total polyphenol, tannin, and flavonoid contents.

Complex Formation of Resorufin and Resazurin with Β-Cyclodextrins: Can Cyclodextrins Interfere with a Resazurin Cell Viability Assay?

Resazurin (or Alamar Blue) is a poorly fluorescent dye. During the cellular reduction of resazurin, its highly fluorescent product resorufin is formed. Resazurin assay is a commonly applied method to investigate viability of bacterial and mammalian cells. In this study, the interaction of resazurin and resorufin with β-cyclodextrins was investigated employing spectroscopic and molecular modeling studies. Furthermore, the influence of β-cyclodextrins on resazurin-based cell viability assay was also tested. Both resazurin and resorufin form stable complexes with the examined β-cyclodextrins (2.0–3.1 × 103 and 1.3–1.8 × 103 L/mol were determined as binding constants, respectively). Cells were incubated for 30 and 120 min and treated with resazurin and/or β-cyclodextrins. Our results suggest that cyclodextrins are able to interfere with the resazurin-based cell viability assay that presumably results from the following mechanisms: (1) inhibition of the cellular uptake of resazurin and (2) enhancement of the fluorescence signal of the formed resorufin.

Green Fluorescent Protein-Based Viability Assay in a Multiparametric Configuration

Green fluorescent protein (GFP) is considered to be suitable for cell viability testing. In our study, GFP transfected A549 lung carcinoma cell line was treated with sodium fluoride (NaF), cycloheximide (CHX) and ochratoxin A (OTA). GFP fluorescence, intracellular ATP, nucleic acid and protein contents were quantified by a luminescence microplate assay developed in our laboratory. Flow cytometry was used to confirm the findings and to assess the intensity of GFP during different types of cell death. A 24 h NaF and CHX exposure caused a dramatic decrease in ATP contents (p < 0.05) compared with those of the controls. GFP fluorescence of the cells was in close correlation with total protein; however, GFP/ATP increased at NaF and decreased at CHX treatments (p < 0.05). ATP/protein and ATP/propidium iodide (PI) were largely decreased at NaF exposure in a dose-dependent manner (p < 0.05), while CHX and OTA showed markedly fewer effects. Both treatments caused apoptosis/necrosis at different rates. NaF induced mainly late apoptosis while OTA, mainly apoptosis. CHX effects varied by the incubation time with 100-fold elevation in late apoptotic cells at 24 h treatment. GFP intensity did not show a significant difference between live and apoptotic populations. Our results suggest when using GFP, a multiparametric assay is necessary for more precise interpretation of cell viability.

Interaction of 2′R-ochratoxin A with Serum Albumins: Binding Site, Effects of Site Markers, Thermodynamics, Species Differences of Albumin-binding, and Influence of Albumin on Its Toxicity in MDCK Cells

Ochratoxin A (OTA) is a nephrotoxic mycotoxin. Roasting of OTA-contaminated coffee results in the formation of 2′R-ochratoxin A (2′R-OTA), which appears in the blood of coffee drinkers. Human serum albumin (HSA) binds 2′R-OTA (and OTA) with high affinity; therefore, albumin may influence the tissue uptake and elimination of ochratoxins. We aimed to investigate the binding site of 2′R-OTA (verses OTA) in HSA and the displacing effects of site markers to explore which molecules can interfere with its albumin-binding. Affinity of 2′R-OTA toward albumins from various species (human, bovine, porcine and rat) was tested to evaluate the interspecies differences regarding 2′R-OTA-albumin interaction. Thermodynamic studies were performed to give a deeper insight into the molecular background of the complex formation. Besides fluorescence spectroscopic and modeling studies, effects of HSA, and fetal bovine serum on the cytotoxicity of 2′R-OTA and OTA were tested in MDCK kidney cell line in order to demonstrate the influence of albumin-binding on the cellular uptake of ochratoxins. Site markers displaced more effectively 2′R-OTA than OTA from HSA. Fluorescence and binding constants of 2′R-OTA-albumin and OTA-albumin complexes showed different tendencies. Albumin significantly decreased the cytotoxicity of ochratoxins. 2′R-OTA, even at sub-toxic concentrations, increased the toxic action of OTA.

A One-Step Extraction and Luminescence Assay for Quantifying Glucose and ATP Levels in Cultured HepG2 Cells

A fluorescence-based enzymatic microplate intracellular glucose assay was designed and fully validated. The method was tested in a hepatocellular cancer cell line (HepG2). Our novel one-step extraction reagent gave stable cell lysates for glucose, adenosine triphosphate (ATP), and total protein determination from the same sample. Limit of detection for glucose was 0.13 µM (26 pmol/well), which is superior to commercially available glucose assays. Both intra- and interday assay imprecision in HepG2 cultures were less than 12% coefficient of variance (CV). In cell lysates spiked with glucose, recovery at two levels varied between 83.70% and 91.81%, and both linearity and stability were acceptable. HepG2 cells treated with agents affecting glucose uptake/metabolism (phloretin, quercetin, quercetin-3′-sulfate, NaF, 3-bromopyruvate, NaN3, oligomycin A, ochratoxin A, cytochalasin B, and anti-GLUT1 antibody) showed dose-dependent changes in glucose and ATP levels without total protein (cell) loss. Finally, we performed flow cytometric glucose uptake measurement in the treated cells using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose fluorescent glucose analog. Glucose uptake did not always mirror the intracellular glucose levels, which most likely reflects the differences between the two methodologies. However, interpreting data obtained by both methods and taking ATP/protein levels at the same time, one can get information on the mode of action of the compounds.

Ethnomedicinal treatment of gastrointestinal disorders in Transylvania, Romania

Ethnomedicine using mostly plants is of pivotal importance nowadays in several Transylvanian regions in Romania. In this study (2007–2015), one Swabian-German, one Hungarian, three Csango-Hungarian and nine Szekely-Hungarian villages were selected to collect ethnomedicinal treatments for various gastrointestinal diseases. Some of the studied villages have partial or no permanent medical and pharmaceutical services. The 374 inhabitants interviewed used mostly medicinal plants based on ancient knowledge. The 78 (53 wild and 25 cultivated) plants documented have 181 local names and are used to treat ailments such as loss of appetite, bloating, stomach ache, gastric ulcer, and diarrhea, mostly in tea form. This knowledge decreases continuously because of loss of interest among young people and through frequent use of media sources and books. Although some of these plants have also been described in official medicinal sources, several data suggest the need for further fieldwork and new experimental analyses to...

Histological and antimicrobial study of Ononis arvensis L.

In this study field restharrow (Ononis arvensis) was investigated for histological and antimicrobial features. The aerial part and the root were embedded in synthetic resin and investigated following sectioning by a rotation microtome. The antimicrobial activity and minimum inhibitory concentration of the solvent fractions of the aerial part were studied against four bacterial strains and one fungus. According to histology, the root covered by rhizodermis contains contiguous vascular elements, which are surrounded by sclerenchyma cells. The epidermis cells are anisodiametric in the stem, sepal, and petal. The bundles of the stem form a Ricinus type thickening. The adaxial side of the heterogeneous leaf is covered by unbranching non-glandular and capitate glandular trichomes. The stipule, petiole, sepals and petals are isolateral having mesomorphic stomata. Pollen grains are tricolpate. The different extracts of the herb showed antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Salmonella Typhimurium, Staphylococcus aureus, and Candida albicans. Data show that the extracts of the leaf contain compounds which may be responsible for the antifungal effect, while extracts obtained from display against the tested bacteria, except Escherichia coli. Further studies are required to complete the phytochemical analysis and identify the antimicrobial compounds of extracts.