A New Role for Yeast Cells in Health and Nutrition: Antioxidant Power Assessment.

As the use of antioxidant compounds in the domains of health, nutrition and well-being is exponentially rising, there is an urgent need to quantify antioxidant power quickly and easily, ideally within living cells. We developed an Anti Oxidant Power in Yeast (AOPY) assay which allows for the quantitative measurement of the Reactive Oxygen Species (ROS) and free-radical scavenging effects of various molecules in a high-throughput compatible format. Key parameters for Saccharomyces cerevisiae were investigated, and the optimal values were determined for each of them. The cell density in the reaction mixture was fixed at 0.6; the concentration of the fluorescent biosensor (TO) was found to be optimal at 64 µM, and the strongest response was observed for exponentially growing cells. Our optimized procedure allows accurate quantification of the antioxidant effect in yeast of well-known antioxidant molecules: resveratrol, epigallocatechin gallate, quercetin and astaxanthin added in the culture medium. Moreover, using a genetically engineered carotenoid-producing yeast strain, we realized the proof of concept of the usefulness of this new assay to measure the amount of ß-carotene directly inside living cells, without the need for cell lysis and purification.

The Extent of Intracellular Accumulation of Bilirubin Determines Its Anti- or Pro-Oxidant Effect.

BACKGROUND: Severe hyperbilirubinemia can cause permanent neurological damage in particular in neonates, whereas mildly elevated serum bilirubin protects from various oxidative stress-mediated diseases. The present work aimed to establish the intracellular unconjugated bilirubin concentrations (iUCB) thresholds differentiating between anti- and pro-oxidant effects. METHODS: Hepatic (HepG2), heart endothelial (H5V), kidney tubular (HK2) and neuronal (SH-SY5Y) cell lines were exposed to increasing concentration of bilirubin. iUCB, cytotoxicity, intracellular reactive oxygen species (ROS) concentrations, and antioxidant capacity (50% efficacy concentration (EC50)) were determined. RESULTS: Exposure of SH-SY5Y to UCB concentration > 3.6 µM (iUCB of 25 ng/mg) and >15 µM in H5V and HK2 cells (iUCB of 40 ng/mg) increased intracellular ROS production (p < 0.05). EC50 of the antioxidant activity was 21 µM (iUCB between 5.4 and 21 ng/mg) in HepG2 cells, 0.68 µM (iUCB between 3.3 and 7.5 ng/mg) in SH-SY5Y cells, 2.4 µM (iUCB between 3 and 6.7 ng/mg) in HK2 cells, and 4 µM (iUCB between 4.7 and 7.5 ng/mg) in H5V cells. CONCLUSIONS: In all the cell lines studied, iUCB of around 7 ng/mg protein had antioxidant activities, while iUCB > 25 ng/mg protein resulted in a prooxidant and cytotoxic effects. UCB metabolism was found to be cell-specific resulting in different iUCB.

Ciguatoxins activate the Calcineurin signalling pathway in Yeasts: Potential for development of an alternative detection tool?

Ciguatoxins (CTXs) are lipid-soluble polyether compounds produced by dinoflagellates from the genus Gambierdiscus spp. typically found in tropical and subtropical zones. This endemic area is however rapidly expanding due to environmental perturbations, and both toxic Gambierdiscus spp. and ciguatoxic fishes have been recently identified in the North Atlantic Ocean (Madeira and Canary islands) and Mediterranean Sea. Ciguatoxins bind to Voltage Gated Sodium Channels on the membranes of sensory neurons, causing Ciguatera Fish Poisoning (CFP) in humans, a disease characterized by a complex array of gastrointestinal, neurological, neuropsychological, and cardiovascular symptoms. Although CFP is the most frequently reported non bacterial food-borne poisoning worldwide, there is still no simple and quick way of detecting CTXs in contaminated samples. In the prospect to engineer rapid and easy-to-use CTXs live cells-based tests, we have studied the effects of CTXs on the yeast Saccharomyces cerevisiae, a unicellular model which displays a remarkable conservation of cellular signalling pathways with higher eukaryotes. Taking advantage of this high level of conservation, yeast strains have been genetically modified to encode specific transcriptional reporters responding to CTXs exposure. These yeast strains were further exposed to different concentrations of either purified CTX or micro-algal extracts containing CTXs. Our data establish that CTXs are not cytotoxic to yeast cells even at concentrations as high as 1µM, and cause an increase in the level of free intracellular calcium in yeast cells. Concomitantly, a dose-dependent activation of the calcineurin signalling pathway is observed, as assessed by measuring the activity of specific transcriptional reporters in the engineered yeast strains. These findings offer promising prospects regarding the potential development of a yeast cells-based test that could supplement or, in some instances, replace current methods for the routine detection of CTXs in seafood products.

Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part I: screening strategy and pre-validation study with lipophilic marine toxins.

Human poisoning due to consumption of seafood contaminated with phycotoxins is a worldwide problem, and routine monitoring programs have been implemented in various countries to protect human consumers. Following successive episodes of unexplained shellfish toxicity since 2005 in the Arcachon Bay on the French Atlantic coast, a national research program was set up to investigate these atypical toxic events. Part of this program was devoted to fit-for-purpose cell-based assays (CBA) as complementary tools to collect toxicity data on atypical positive-mouse bioassay shellfish extracts. A collaborative study involving five laboratories was conducted. The responses of human hepatic (HepG2), human intestinal (Caco2), and mouse neuronal (Neuro2a) cell lines exposed to three known lipophilic phycotoxins-okadaic acid (OA), azaspiracid-1 (AZA1), and pectenotoxin-2 (PTX2)-were investigated. A screening strategy composed of standard operating procedures and a decision tree for dose-response modeling and assay validation were designed after a round of "trial-and-error" process. For each toxin, the shape of the concentration-response curves and the IC(50) values were determined on the three cell lines. Whereas OA induced a similar response irrespective of the cell line (complete sigmoid), PTX2 was shown to be less toxic. AZA1 induced cytotoxicity only on HepG2 and Neuro2a, but not on Caco2. Intra- and inter-laboratory coefficients of variation of cell responses were large, with mean values ranging from 35 to 54 % and from 37 to 48 %, respectively. Investigating the responses of the selected cell lines to well-known toxins is the first step supporting the use of CBA among the panel of methods for characterizing atypical shellfish toxicity. Considering these successful results, the CBA strategy will be further applied to extracts of negative, spiked, and naturally contaminated shellfish tissues.

Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part II: application to shellfish extracts spiked with lipophilic marine toxins.

Successive unexplained shellfish toxicity events have been observed in Arcachon Bay (Atlantic coast, France) since 2005. The positive mouse bioassay (MBA) revealing atypical toxicity did not match the phytoplankton observations or the liquid chromatography-tandem mass spectrometry (LC-MS/MS) investigations used to detect some known lipophilic toxins in shellfish. The use of the three cell lines (Caco2, HepG2, and Neuro2a) allows detection of azaspiracid-1 (AZA1), okadaic acid (OA), or pectenotoxin-2 (PTX2). In this study, we proposed the cell-based assays (CBA) as complementary tools for collecting toxicity data about atypical positive MBA shellfish extracts and tracking their chromatographic fractionation in order to identify toxic compound(s). The present study was intended to investigate the responses of these cell lines to shellfish extracts, which were either control or spiked with AZA1, OA, or PTX2 used as positive controls. Digestive glands of control shellfish were extracted using the procedure of the standard MBA for lipophilic toxins and then tested for their cytotoxic effects in CBA. The same screening strategy previously used with pure lipophilic toxins was conducted for determining the intra- and inter-laboratory variabilities of the responses. Cytotoxicity was induced by control shellfish extracts whatever the cell line used and regardless of the geographical origin of the extracts. Even though the control shellfish extracts demonstrated some toxic effects on the selected cell lines, the extracts spiked with the selected lipophilic toxins were significantly more toxic than the control ones. This study is a crucial step for supporting that cell-based assays can contribute to the detection of the toxic compound(s) responsible for the atypical toxicity observed in Arcachon Bay, and which could also occur at other coastal areas.

Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport.

In this work, both direct and indirect cell-based antioxidant profiles were established for 27 plant extracts and 1 algal extract. To evaluate the direct antioxidant effects, fluorescent AOP1 cell assay was utilized, which measures the ability of different samples to neutralize intracellular free radicals produced by a cell-based photo-induction process. As the intestinal barrier is the first cell line crossed by the product, dose response curves obtained from Caco-2 cells were used to establish EC50 values for 26 out of the 28 natural extracts. Among them, 11 extracts from Vitis, Hamamelis, Syzygium, Helichrysum, Ilex and Ribes genera showed remarkable EC50s in the range of 10 µg/mL. In addition to this, a synergistic effect was found when combinations of the most potent extracts (S. aromaticum, H. italicum, H. virginiana, V. vinifera) were utilized compared to extracts alone. Indirect antioxidant activities (i.e., the ability of cells to trigger antioxidant defenses) were studied using the ARE/Nrf2 luminescence reporter-gene assay in HepG2 cells, as liver is the first organ crossed by an edible ingredient once it enters in the bloodstream. Twelve extracts were subjected to an intestinal epithelial barrier passage in order to partially mimic intestinal absorption and show whether basolateral compartments could maintain direct or indirect antioxidant properties. Using postepithelial barrier samples and HepG2 cells as a target model, we demonstrate that indirect antioxidant activities are maintained for three extracts, S. aromaticum, H. virginiana and H. italicum. Our experimental work also confirms the synergistic effects of combinations of post-intestinal barrier compartments issued from apical treatment with these three extracts. By combining cell-based assays together with an intestinal absorption process, this study demonstrates the power of cell systems to address the issue of antioxidant effects in humans.

Cellular Antioxidant Effect of an Aronia Extract and Its Polyphenolic Fractions Enriched in Proanthocyanidins, Phenolic Acids, and Anthocyanins.

Oxidative stress and chronic inflammation contribute to some chronic diseases. Aronia berries are rich in polyphenols. The aim of the present study was to characterize the cellular antioxidant effect of an aronia extract to reflect the potential physiological in vivo effect. Cellular in vitro assays in three cell lines (Caco-2, HepG2, and SH-SY5Y) were used to measure the antioxidant effect of AE, in three enriched polyphenolic fractions (A1: anthocyanins and phenolic acids; A2: oligomeric proanthocyanidins; A3: polymeric proanthocyanidins), pure polyphenols and microbial metabolites. Both direct (intracellular and membrane radical scavenging, catalase-like effect) and indirect (NRF2/ARE) antioxidant effects were assessed. AE exerted an intracellular free radical scavenging activity in the three cell lines, and A2 and A3 fractions showed a higher effect in HepG2 and Caco-2 cells. AE also exhibited a catalase-like activity, with the A3 fraction having a significant higher activity. Only A1 fraction activated the NRF2/ARE pathway. Quercetin and caffeic acid are the most potent antioxidant polyphenols, whereas cyanidin and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone showed the highest antioxidant effect among polyphenol metabolites. AE rich in polyphenols possesses broad cellular antioxidant effects, and proanthocyanidins are major contributors. Polyphenol metabolites may contribute to the overall antioxidant effect of such extract in vivo.

Live Cell Assays for the Assessment of Antioxidant Activities of Plant Extracts.

Plant extracts and pharmacopoeias represent an exceptional breeding ground for the discovery of new antioxidants. Until recently, the antioxidant activity was only measured by chemical hydrogen atom transfer (HAT) and single-electron transfer (SET) cell-free assays that do not inform about the actual effect of antioxidants in living systems. By providing information about the mode of action of antioxidants at the subcellular level, recently developed live cell assays are now changing the game. The idea of this review is to present the different cell-based approaches allowing a quantitative measurement of antioxidant effects of plant extracts. Up to date, only four different approaches have reached a certain degree of standardization: (1) the catalase-like assay using H2O2 as a stressor, (2) the cell antioxidant assay (CAA) using AAPH as a stressor and DCFH-DA as a readout, (3) the AOP1 assay which uses photoinduction to monitor and control cell ROS production, and (4) the Nrf2/ARE gene reporter system. The molecular aspects of these assays are presented in detail along with their features, drawbacks, and benefits. The Nrf2/ARE gene reporter system dedicated to indirect antioxidant effect measurement currently represents the most standardized approach with high-throughput applications. AOP1, the first technology linking a fine-tuning of cell ROS production with a quantitative signal, appears to be the most promising tool for the assessment of direct cellular ROS-scavenging effects at an industrial scale.

AOP1, a New Live Cell Assay for the Direct and Quantitative Measure of Intracellular Antioxidant Effects.

Taking advantage of Light Up Cell System (LUCS) technology, which allows for fine monitoring of reactive oxygen species (ROS) production inside live cells, a new assay called Anti Oxidant Power 1 (AOP1) was developed to specifically measure ROS and/or free-radical scavenging effects inside living cells. This method is quantitative and EC50s obtained from AOP1 dose-response experiments were determined in order to classify the intracellular antioxidant efficacy of 15 well known antioxidant compounds with different hydrophilic properties. Six of them (epigallocatechin gallate, quercetin, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethoxyquin, resveratrol) gave EC50s in the range of 7-64 µM, four (Trolox, catechin, epicatechin, EUK134) in the range of 0.14 to 1 mM, and 5 (sulforaphane, astaxanthin, α- and γ-tocopherols, vitamin E acetate) showed only partial or no effect. Interestingly, effects with measurable EC50s were observed for compounds with hydrophilic properties (LogP ≤ 5.3), while all antioxidants known to act at the plasma membrane level (LogP ≥ 10.3) had partial or no effect. Sulforaphane, a hydrophilic but strict Keap1/Nrf2 pathway enhancer, did not show any effect either. Importantly, AOP1 assay captures both antioxidant and prooxidant effects. Taken together, these results led us to the conclusion that AOP1 assay measures antioxidant effect of compounds that selectively enter the cell, and act as free radical scavengers in the cytosol and/or nucleus level.

LUCS (Light-Up Cell System), a universal high throughput assay for homeostasis evaluation in live cells.

Observations of fluorescent cyanine dye behavior under illumination at 500 nm lead to a novel concept in cell biology allowing the development of a new live cell assay called LUCS, for Light-Up Cell System, measuring homeostasis in live cells. Optimization of the LUCS process resulted in a standardized, straightforward and high throughput assay with applications in toxicity assessment. The mechanisms of the LUCS process were investigated. Electron Paramagnetic Resonance experiments showed that the singlet oxygen and hydroxyl radical are involved downstream of the light effect, presumably leading to deleterious oxidative stress that massively opens access of the dye to its intracellular target. Reversible modulation of LUCS by both verapamil and proton availability indicated that plasma membrane proton/cation antiporters, possibly of the MATE drug efflux transport family, are involved. A mechanistic model is presented. Our data show that intracellular oxidation can be controlled by tuning light energy, opening applications in regulatory purposes, anti-oxidant research, chemotherapy efficacy and dynamic phototherapy strategies.

Comparative cytotoxicity induced by bulk and nanoparticulated ZnO in the fish and human hepatoma cell lines PLHC-1 and Hep G2.

The increasing presence of ZnO nanoparticles (NPs) in consumer products may be having a dramatic impact in aquatic environments. The evaluation of ZnO NP toxicity represents a great challenge. This study aimed at evaluating the cytotoxic effect of micro- and nanosized ZnO in a fish and a mammalian hepatoma cell line. A detailed characterisation of the particles in exposure media showed that ZnO NPs formed large aggregates. ZnO cytotoxicity was evaluated with a battery of in vitro assays including LUCS, a new approach based on DNA alteration measurements. In fish cells, ZnO NP aggregates contributed substantially to the cytotoxic effects whereas toxicity in the human cells appeared to be mainly produced by the dissolved fraction. ROS production did not contribute to the observed cytotoxicity. This work also showed that measuring concentrations of NPs is essential to understand the mechanisms underlying their toxicity.

Image-free assessment of protein translocation in live cells.

Protein translocation is a universal event shared by most cell signalling pathways to transmit signals between cell compartments. In recent years, the use of new fluorescence microscopy technologies combined with fluorescent probes--most often fluorescent proteins--and image analysis software has allowed the visualization and extensive analysis of such dynamic events in the context of the living cell. This review article focuses on emerging fluorescence approaches that tackle live cell protein translocation in the image-free context. Such methods are based on either protein-protein interactions or analysis of spatial diffusion of proteins by fluorescence intensity measurements. The potential benefits of intensity measurement on global cell populations versus image analysis of heterogeneous cell sample are discussed in the context of drug discovery applications.