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Neuroprotective Effects of Bioavailable PolyphenolDerived Metabolites against Oxidative Stress-Induced Cytotoxicity in Human Neuroblastoma SH-SY5Y Cells Antonio Gonzalez-Sarrias, María Ángeles Núñez-Sánchez, Francisco A. Tomas-Barberan, and Juan Carlos Espín J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04538 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 21, 2016

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Neuroprotective Effects of Bioavailable Polyphenol-Derived Metabolites against

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Oxidative Stress-Induced Cytotoxicity in Human Neuroblastoma SH-SY5Y Cells

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Antonio González-Sarrías*, María Ángeles Núñez-Sánchez, Francisco A. Tomás-Barberán, Juan

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Carlos Espín.

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Research Group on Quality, Safety, and Bioactivity of Plant Foods, Dept. Food Science and Technology, CEBAS-CSIC, P.O. Box 164, 30100 Campus de Espinardo, Murcia, Spain

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*Correspondence: Dr. Antonio González-Sarrías, Research Group on Quality, Safety and

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Bioactivity of Plant Foods, Dept. Food Science and Technology, CEBAS-CSIC, P.O. Box 164,

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30100 Campus de Espinardo, Murcia, Spain. Tel: +34 968 396200; Ext: 6249, Fax: +34 968

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396213.

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E-mail: [email protected]

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ABSTRACT

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Oxidative stress is involved in cell death in neurodegenerative diseases. Dietary polyphenols can

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exert health benefits, but their direct effects on neuronal cells are debatable since most phenolics are

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metabolized and do not reach the brain as they occur in the dietary sources. Herein, we evaluate the

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effects of a panel of bioavailable polyphenols and derived metabolites at physiologically relevant

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conditions against H2O2-induced apoptosis in human neuroblastoma SH-SY5Y cells. Among the 19

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metabolites tested, 3,4-dihydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid, gallic acid,

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ellagic acid and urolithins prevented neuronal apoptosis via attenuation of ROS levels, increased

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REDOX activity and decreased oxidative stress-induced apoptosis by preventing the caspase-3

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activation via the mitochondrial apoptotic pathway in SH-SY5Y cells. This suggests that dietary

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sources containing the polyphenols precursors of these molecules such as cocoa, berries, walnuts,

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and tea could be potential functional foods to reduce oxidative stress associated with the onset and

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progress of neurodegenerative diseases.

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KEYWORDS: Gallic acid; Ellagic acid; Neuroprotective effects; Phenolic acids; Polyphenol;

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Urolithins.

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Journal of Agricultural and Food Chemistry

INTRODUCTION

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Oxidative stress is involved in neuronal cell death, which is one of the leading causes of

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neurodegenerative diseases such as Alzheimer's disease, Parkison's disease, or cerebral ischemia

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reperfusion after stroke.1 Oxidative stress is characterized by the imbalance between antioxidants

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and accumulation of reactive oxygen species (ROS) and reactive nitrogen species that can be

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spontaneously generated, such as superoxide anion and hydrogen peroxide, or made due to

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exogenous factors, such as drug exposure or radiation.2 ROS generally are found in the course of

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cell death, mainly by apoptosis caused by intracellular microenvironmental changes, and in the brain

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leading to neurodegeneration and cognitive decline.3

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There is much evidence showing that naturally occurring antioxidants such as vitamins, minerals

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and phenolic compounds present in high amounts in fruits, vegetables, and natural products

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scavenge the free radicals and their intake acts as an upstream preventive measure to

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neurodegeneration.4,5 In fact, dietary polyphenols have been reported to display potential

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neuroprotective effects as antioxidants preventing oxidation of proteins, lipid peroxidation, and

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generation of ROS as well as anti-inflammatory and anti-apoptotic properties in several in vitro

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models of toxicity and animal models of neurological disorders.6,7 In this regard, previous studies

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have indicated the neuroprotective effect of red wine, grapes, pomegranate, cocoa and tea-derived

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polyphenols as sources of flavan-3-ols, stilbenes such as resveratrol and ellagitannins (ETs) using

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animal models of neurodegenerative diseases.8-11 However, polyphenols are poorly bioavailable and

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cannot reach systemic tissues as they occur in the dietary sources. On the contrary, they are

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extensively metabolized and converted by colonic microbiota into other metabolic forms.6,12

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Therefore, the actual metabolites responsible for the in vivo neuroprotective effects of polyphenols

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are not entirely known.

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To date, only low concentrations of a few polyphenol-derived metabolites have been detected in

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the brain of animals, i.e. gallic acid (GA), ellagic acid (EA) and its gut microbial derived

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metabolites urolithins,13-15 resveratrol and its microbial metabolites,16 and also anthocyanins.17,18

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Nevertheless, many derived metabolites have been detected in the human blood stream, so that they

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should be taken into account as potential candidates to exert the neuroprotective effects attributed to

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the polyphenolic dietary precursors.19,20

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Whereas dietary polyphenols such as ETs, procyanidins and stilbenes are poorly bioavailable,

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we hypothesize here that their derived metabolites, which can target the brain, could protect

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neuronal cells from oxidative stress-induced cell death. To check this hypothesis, we aimed at

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determining the anti-apoptotic and antioxidant effects of a panel of 19 polyphenols-derived

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metabolites (Figure 1), at physiologically relevant concentrations, on H2O2-induced neurotoxicity in

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human neuroblastoma SH-SY5Y cells.

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MATERIALS AND METHODS

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Materials and Reagents. Trypan blue, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

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bromide (MTT), hydrogen peroxide solution, gallic acid (GA), ellagic acid (EA), 3,4-

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dihydroxyphenylpropionic acid (DHPPA), 3,4-dihydroxyphenylacetic acid (DHPAA) and trans-

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resveratrol (RSV) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The microbial EA-

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derived urolithins were chemically synthetized and purified by Villapharma Research S.L. (Parque

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Tecnológico de Fuente Alamo, Murcia, Spain). Urolithin D (Uro-D) and urolithin C (Uro-C) were

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purchased from Dalton Pharma Services (Toronto, Canada). RES-sulfate, RES-glucuronide and the

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microbial RES-derived metabolites dihydroresveratrol (DHR) and DHR-glucuronide were obtained

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as described in Azorín-Ortuño et al. (2011).16 Phosphate Buffered Saline (PBS) was obtained from

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Journal of Agricultural and Food Chemistry

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Fisher Scientific (USA). Dimethylsulfoxide (DMSO) was obtained from Panreac (Barcelona,

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Spain). Milli-Q system (Millipore Corp., USA) ultra-pure water was used throughout the study.

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Cell Culture. The SH-SY5Y neuroblastoma cell line was purchased from the European Collection

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of Authenticated Cell Cultures (ECACC) (Salisbury, UK). Cells were cultured as recommended by

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the ECACC in Ham's F12:EMEM (EBSS) (1:1) + 2 mM Glutamine + 1% non-essential amino acids

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+ 15% fetal bovine serum (FBS). Cells were incubated at 37 °C in a humidified atmosphere of 95 %

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air/5 % CO2. Test compounds were solubilized in DMSO (