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.

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). Pearson's 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.

Antioxidant potential, tannin and polyphenol contents of seed and pericarp of three Coffea species.

OBJECTIVE: To investigate the antioxidant activity, total phenolic and total tannin content of the pericarp and the seed of Coffea benghalensis (C. benghalensis) and Coffea liberica compared to Coffea arabica (C. arabica). METHODS: The antioxidant potential, total tannin and polyphenol contents of the immature and mature seed and pericarp of C. benghalensis and Coffea liberica were quantified and compared to C. arabica. Enhanced chemiluminescence (ECL), 2,2-diphenyl-1-picrylhydrazyl (DPPH), oxygen radical absorbance capacity, Folin-Ciocalteau method and total tannin content assays were used. RESULTS: Trolox equivalent (TE/g plant material) values obtained by ECL and DPPH methods showed loose correlation (r(2) = 0.587) while those measured by oxygen radical absorbance capacity assay were higher without correlation in each plant. A closer correlation was detected between the ECL method and the percentage antioxidant activity of the DPPH technique (r(2) = 0.610 7) in each species, however the immature pericarp of C. benghalensis showed much higher DPPH scavenging potential than was seen in the ECL assay. The immature pericarp of C. benghalensis expressed the highest tannin and polyphenol content, and a high polyphenol level was also detected in the immature seed of C. arabica. The immature pericarp of Bengal and Liberian coffees showed the largest amount of phenolic contents. CONCLUSIONS: The obtained data highlight the potential role of C. benghalensis as a new source of natural antioxidants and polyphenols compared to C. arabica.

Multiparametric luminescent cell viability assay in toxicology models: A critical evaluation.

INTRODUCTION: In cellular viability assays the sole determination of a single parameter might not give precise information on the extent of toxicity. In our study we worked out a multiparametric microplate assay based on bioluminescent ATP quantification, esterase activity-related fluorescence, nucleic acid staining and total intracellular protein measurement from the same sample in MDCK and HepG2 tissue cultures. METHODS: Dose-response analyses were done after ATP depletion by metabolic poisons (NaF, NaN3) and by ochratoxin A (OTA) mycotoxin treatments. A novel perchloric acid fixation/extraction technique was applied in order to obtain intracellular ATP levels, esterase activity, DNA content and protein data simultaneously. Esterase activity was assessed by a fluorogenic staining. Estimation of cell number was done by DAPI fluorescence. Our results were expressed as ATP/protein, calcein fluorescence/ATP, calcein fluorescence/protein and ATP/DAPI ratios. Apoptosis/necrosis rates were measured by Annexin V-propidium iodide and 7-aminoactinomycin D flow cytometric assays and effects of OTA on actin cytoskeleton were also studied by using labeled phalloidin for visualization of actin. RESULTS: We could verify that the esterase assay was not an energy driven (true viability) process. ATP/protein, calcein fluorescence/ATP, calcein fluorescence/protein ratios, DAPI fluorescence and protein levels together with morphological and apoptosis/necrosis parameters deciphered subtle changes in cell viability with good between-run precision. Dose dependent loss in cell number and decreased protein levels were observed in all cases, while disorganization of actin microfilaments was seen in OTA treated cells. The two cell lines did not respond uniformly to the same treatments. DISCUSSION: ATP/protein ratio proved to be a useful viability parameter however, the suppression and/or loss of intracellular protein could cause difficulty in interpreting ATP/protein data. We conclude that correct assessment of cellular viability should be done by measuring multiple parameters related to the specific mode of action of the tested toxic compound.

Interaction of Citrinin with Human Serum Albumin.

Citrinin (CIT) is a mycotoxin produced by several Aspergillus, Penicillium, and Monascus species. CIT occurs worldwide in different foods and drinks and causes health problems for humans and animals. Human serum albumin (HSA) is the most abundant plasma protein in human circulation. Albumin forms stable complexes with many drugs and xenobiotics; therefore, HSA commonly plays important role in the pharmacokinetics or toxicokinetics of numerous compounds. However, the interaction of CIT with HSA is poorly characterized yet. In this study, the complex formation of CIT with HSA was investigated using fluorescence spectroscopy and ultrafiltration techniques. For the deeper understanding of the interaction, thermodynamic, and molecular modeling studies were performed as well. Our results suggest that CIT forms stable complex with HSA (logK ~ 5.3) and its primary binding site is located in subdomain IIA (Sudlow's Site I). In vitro cell experiments also recommend that CIT-HSA interaction may have biological relevance. Finally, the complex formations of CIT with bovine, porcine, and rat serum albumin were investigated, in order to test the potential species differences of CIT-albumin interactions.

Interactions of zearalenone with native and chemically modified cyclodextrins and their potential utilization.

Zearalenone (ZEA) is a widespread xenoestrogenic mycotoxin produced by several Fusarium species. ZEA can cause reproductive disorders in farm animals and hyperoestrogenic syndromes in humans; therefore, development of more sensitive analytical methods (to quantify the mycotoxin) as well as strategies for prevention of its toxic impacts is highly important. In this study, the interactions of ZEA with native and chemically modified cyclodextrins (CDs) were investigated using fluorescence spectroscopy. Furthermore, in vitro experiments on liver cells were also performed to test the potential effect of CDs on toxin uptake. Our results demonstrate that ZEA forms stable complexes with CDs (logK values are approximately 3.7-4.7) resulting in the considerable elevation of its fluorescence signal. In addition, some of the CDs show ability to inhibit the cellular uptake of ZEA, suggesting their potential suitability to develop new CD-based preventive/detoxification strategies against ZEA in the future.