Optimization of a DNA Nicking Assay to Evaluate Oenocarpus bataua and Camellia sinensis Antioxidant Capacity

Louis-Jérôme Leba,Emmanuelle Vulcain,Jean-Charles Robinson,Mona Saout,Didier Bereau,Christel Brunschwig,Karine Martial

doi:10.3390/ijms151018023

Louis-Jérôme Leba, Emmanuelle Vulcain + Show 5 more

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https://doi.org/10.3390/ijms151018023

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Abstract

This study was aimed at assessing the DNA damage protective activity of different types of extracts (aqueous, methanolic and acetonic) using an in vitro DNA nicking assay. Several parameters were optimized using the pUC18 plasmid, especially FeSO4, EDTA, solvent concentrations and incubation time. Special attention has been paid to removing the protective and damaging effect of the solvent and FeSO4 respectively, as well as to identifying the relevant positive and negative controls. For each solvent, the optimal conditions were determined: (i) for aqueous extracts, 0.33 mM of FeSO4 and 0.62 mM of EDTA were incubated for 20 min at 37 °C; (ii) for acetone extracts, 1.16% solvent were incubated for 15 min at 37 °C with 1.3 mM of FeSO4 and 2.5 mM of EDTA and (iii) for methanol extracts, 0.16% solvent, were incubated for 1.5 h at 37 °C with 0.33 mM of FeSO4 and 0.62 mM of EDTA. Using the optimized conditions, the DNA damage protective activity of aqueous, methanolic and acetonic extracts of an Amazonian palm berry (Oenocarpus bataua) and green tea (Camellia sinensis) was assessed. Aqueous and acetonic Oenocarpus bataua extracts were protective against DNA damage, whereas aqueous, methanolic and acetonic extracts of Camellia sinensis extracts induced DNA damage.

Highlights

Reactive nitrogen species (RNS) and Reactive oxygen species (ROS), such as nitric oxide (NO), peroxynitrite (ONOO−), hydrogen peroxide (H2O2), superoxide (O2−.) and hydroxyl radical (OH), are implicated in oxidative stress in cells, DNA damage [1], cancer [2,3], cells aging [4] and neurodegenerative disease like Alzheimer’s and Parkinson’s [5,6]
To evaluate the antioxidant properties of Oenocarpus bataua and Camellia sinensis, extracts were assessed through chemical assays, ferric-reducing ability of plasma (FRAP), oxygen radical absorbance capacity (ORAC) and DPPH assays to find out which mechanisms were involved
This study shows that in vitro DNA nicking assays should be performed with the appropriate controls

Summary

Introduction

Reactive nitrogen species (RNS) and Reactive oxygen species (ROS), such as nitric oxide (•NO), peroxynitrite (ONOO−), hydrogen peroxide (H2O2), superoxide (O2•−.) and hydroxyl radical (•OH), are implicated in oxidative stress in cells, DNA damage [1], cancer [2,3], cells aging [4] and neurodegenerative disease like Alzheimer’s and Parkinson’s [5,6]. During the past decades, the oxidative stress implication in numerous diseases led to considerable efforts in identifying beverages, vegetables, fruits and plants with high antioxidant properties [7,8,9]. Antioxidant compounds can inhibit the oxidation chain at different stages: either by radical-chain breaking (direct radical quenching/scavenging), or by chelating metals that act as catalyst [10]. Currently, the antioxidant capacity is evaluated via several in vitro chemical assays, which measure either the oxygen radical absorbance capacity (ORAC) (peroxide radical), the 2,2-diphenylpicrylhydrazyl (DPPH) free radical-scavenging capacity and the ferric-reducing ability of plasma (FRAP). These assays are interesting because of their complementarities and the fact that they imply a variety of parameters such as different mechanisms, substrates and radicals [11,12,13]. If in vitro chemical assays are good tools to study antioxidant capacity of natural products, in vitro DNA nicking assays are more biologically relevant and they enable a fast screening of potential in vivo antioxidant substances. DNA nicking assay has been created because damage to the genome is central to the development of disease such as degenerative diseases and cancers [6,14,15]. Among the various existing in vitro DNA nicking assays, the DNA nicking assay based on the Fenton reaction mimics the in vivo biological situation, with the production of hydroxyl free radicals from endogenous entities like intracellular iron. The Fenton reaction was described more than 100 years ago [16]. During this reaction, H2O2 is cleaved in •OH by electron transfer from iron according to the reaction: Fe2+ + H2O2 → Fe3+ +

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