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

Gironde, Camille; Rigal, Mylène; Dufour, Cécile; Furger, Christophe

doi:10.3390/antiox9060471

Cited by 16 publications

(16 citation statements)

References 52 publications

(60 reference statements)

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“…The test used derived from the "LUCS" (Light-Up Cell System) technology developed by AOP (Antioxidant Power) company ( Derick et al, 2017 ; Gironde et al, 2020 ). Neuroblastoma cells were seeded onto the 96-well plate (Corning) at 35,000 cells per well.…”

Section: Methodsmentioning

confidence: 99%

“…From 3 h the fluorescence measured on the plate is higher. Note that, as described elsewhere, at low concentration, menadione is less toxic and can become "antioxidant" (Gironde et al, 2020). To analyze the role of the association of NQO2 with UGT1A6 the same set of experiments was carried out with SH-SY5Y-NQO2-UGT1A6 cells.…”

Section: Effect Of Ugt1a6 On the Cellular Homeostasismentioning

confidence: 99%

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Association of NQO2 With UDP-Glucuronosyltransferases Reduces Menadione Toxicity in Neuroblastoma Cells

Chhour

Pério

Gayon³

et al. 2021

Front. Pharmacol.

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The balance between detoxification and toxicity is linked to enzymes of the drug metabolism Phase I (cytochrome P450 or oxidoreductases) and phase II conjugating enzymes (such as the UGTs). After the reduction of quinones, the product of the reaction, the quinols—if not conjugated—re-oxidizes spontaneously to form the substrate quinone with the concomitant production of the toxic reactive oxygen species (ROS). Herein, we documented the modulation of the toxicity of the quinone menadione on a genetically modified neuroblastoma model cell line that expresses both the quinone oxidoreductase 2 (NQO2, E.C. 1.10.5.1) alone or together with the conjugation enzyme UDP-glucuronosyltransferase (UGT1A6, E.C. 2.4.1.17), one of the two UGT isoenzymes capable to conjugate menadione. As previously shown, NQO2 enzymatic activity is concomitant to massive ROS production, as previously shown. The quantification of ROS produced by the menadione metabolism was probed by electron-paramagnetic resonance (EPR) on cell homogenates, while the production of superoxide was measured by liquid chromatography coupled to mass spectrometry (LC-MS) on intact cells. In addition, the dysregulation of the redox homeostasis upon the cell exposure to menadione was studied by fluorescence measurements. Both EPR and LCMS studies confirmed a significant increase in the ROS production in the NQO2 overexpressing cells due to the fast reduction of quinone into quinol that can re-oxidize to form superoxide radicals. However, the effect of NQO2 inhibition was drastically different between cells overexpressing only NQO2 vs. both NQO2 and UGT. Whereas NQO2 inhibition decreases the amount of superoxide in the first case by decreasing the amount of quinol formed, it increased the toxicity of menadione in the cells co-expressing both enzymes. Moreover, for the cells co-expressing QR2 and UGT the homeostasis dysregulation was lower in presence of menadione than for the its counterpart expressing only QR2. Those results confirmed that the cooperation of the two enzymes plays a fundamental role during the cells’ detoxification process. The fluorescence measurements of the variation of redox homeostasis of each cell line and the detection of a glucuronide form of menadiol in the cells co-expressing NQO2 and UGT1A6 enzymes further confirmed our findings.

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Section: Methodsmentioning

confidence: 99%

Section: Effect Of Ugt1a6 On the Cellular Homeostasismentioning

confidence: 99%

Association of NQO2 With UDP-Glucuronosyltransferases Reduces Menadione Toxicity in Neuroblastoma Cells

Chhour

Pério

Gayon³

et al. 2021

Front. Pharmacol.

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“…AOP1 antioxidant live cell assay: This unique feature led to the development of a new promising antioxidant assay called AOP1 [86]. AOP1 protocol includes a run of moderate light applications, each leading to a moderate ROS production and a moderate increase in fluorescence.…”

Section: Aop1 a New Antioxidant Live Cell Approach Based On Photoinductionmentioning

confidence: 99%

“…AOP1 is to our knowledge the first approach able to quantitatively assess quenching of ROS or free radicals directly produced by living cells. The AOP1 assay has been already applied with success to classify 15 standard antioxidants according to their efficacy concentrations (EC 50 s) [86] and to assess cellular antioxidant effects of many plant extracts including a phytocomplex of bilberry (Vaccinium myrtillus) [87]. The AOP1 approach presents many benefits over the competitive CAA approach (Table 1).…”

Section: Aop1 a New Antioxidant Live Cell Approach Based On Photoinductionmentioning

confidence: 99%

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

Furger¹

2021

Antioxidants

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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.

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“…To more easily monitor the cytoprotective effect of antioxidants, a new quantitative assay called Anti Oxidant Power 1 (AOP1) was developed, which specifically measures reactive oxygen species (ROS) and/or free radical scavenging effects inside living cells using Light Up Cell System (LUCS) technology allowing fine monitoring of ROS production inside live cells. The assay was used to compare intracellular antioxidant/prooxidant efficacy of 15 well-known antioxidants with different hydrophilic/lipophilic properties [ 11 ].…”

mentioning

confidence: 99%