TYROSINASE-TREATED HYDROXYTYROSOL-ENRICHED OLIVE

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Biotechnology and Biological Transformations

TYROSINASE-TREATED HYDROXYTYROSOL-ENRICHED OLIVE VEGETATION WASTE WITH INCREASED ANTIOXIDANT ACTIVITY PROMOTES AUTOPHAGY AND INHIBITS THE INFLAMMATORY RESPONSE IN HUMAN THP-1 MONOCYTES Roberta Meschini, Donatella D' Eliseo, Silvia Filippi, Laura Bertini, Bruno Mattia Bizzarri, Lorenzo Botta, Raffaele Saladino, and Francesca Velotti J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03630 • Publication Date (Web): 23 Oct 2018 Downloaded from http://pubs.acs.org on October 25, 2018

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Journal of Agricultural and Food Chemistry

TYROSINASE-TREATED

HYDROXYTYROSOL-ENRICHED

OLIVE

VEGETATION

WASTE WITH INCREASED ANTIOXIDANT ACTIVITY PROMOTES AUTOPHAGY AND INHIBITS THE INFLAMMATORY RESPONSE IN HUMAN THP-1 MONOCYTES

Roberta Meschini,a Donatella D’Eliseo,a,b Silvia Filippi,a Laura Bertini,a Bruno Mattia Bizzarri,a,* Lorenzo Botta,a Raffaele Saladino,a Francesca Velotti a,*

a Department

of Ecological and Biological Sciences (DEB), University of Tuscia, Viterbo, Italy

b Department

of Experimental Medicine, Sapienza University of Rome, Rome, Italy

*Corresponding authors: (B.M.B.) [email protected]; (F.V.) [email protected]

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ABSTRACT

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Treatment of olive vegetation waste with tyrosinase immobilized on Multi Walled Carbon Nanotubes

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increased the antioxidant activity as a consequence of the conversion of phenols to corresponding

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catechol derivatives, as evaluated by DPPH, Comet assay, and micronucleus analyses. During this

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transformation, 4-hydroxyphenethyl alcohol (tyrosol) was quantitatively converted to bioactive 3,4-

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dihydroxyphenethyl alcohol (hydroxytyrosol). The hydroxytyrosol-enriched olive vegetation waste also

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promoted autophagy and inhibited the inflammatory response in human THP-1 monocytes.

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KEYWORDS: olive vegetation waste, recovery of valuable substances, tyrosinase bioconversion, hydroxytyrosol, antioxidant activity, autophagy, inflammation, cytokines.

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INTRODUCTION

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The valorization of plant food wastes is emerging as a potent tool for the control of environmental

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pollution problems,1 as well as for the development of sustainable processes based on renewable high-

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value added natural substances.2 During the virgin olive oil processing, different bioactive compounds

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with potential health beneficial properties are retained in the olive vegetation wastes (OVW),3 causing

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toxic effects because of their low biodegradability.4 Among them, polyphenols such as 3,4-

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dihydroxyphenethyl alcohol (hydroxytyrosol),

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hydroxytyrosol in unripe olive fruit),6 have been well recognized for their health beneficial activities,

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including antioxidant activity,7,8 inhibition of acute and chronic neurodegeneration,9-13 anticancer14, and

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anti-inflammatory activities.15 Notably, the concentration of hydroxytyrosol in OVW is 10-100 fold

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higher than that in the corresponding virgin olive oil, due to its high hydrophilic properties.16 3-

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Hydroxyphenethyl alcohol (tyrosol), the biogenic precursor of hydroxytyrosol,17 cinnammic acids,

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benzoic acids, and to a lesser extent, flavonoids and lignans, were also been found in OVW.18 Usually,

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the concentration of tyrosol was found to be comparable to that of hydroxytyrosol, but unlike this latter

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compound, tyrosol showed only a low antioxidant activity, being deprived of the catechol (ortho-

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diphenol) pharmacophore, which play a key role in the radical scavenging process. Modest biological

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activities have been reported for tyrosol in rats,19 sometime associated to ability in counteracting the Cys-

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DA induced cytotoxicity.20 Tyrosinase is a copper enzyme which catalyzes the ortho-hydroxylation of

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monophenols to ortho-diphenols (cresolase or monophenolase activity) and ortho-quinones (catecholase

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or diphenolase activity) using dioxygen (O2) as primary oxidant.21,22 Since its relevance in several

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biotechnological areas,23 tyrosinase has been applied in the selective oxidation of tyrosol to

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hydroxytyrosol under homogeneous conditions.24 This procedure has been implemented by

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immobilization of tyrosinase on solid supports to enhance the stability and the activity of the enzyme,

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reducing the cost of the process and facilitating the purification procedures in large scale applications.25,26

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and oleuropein (the major secoiroid derivative of



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In this context, tyrosinase showed greater storage life, pH stability and reusability when coated by the

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Layer-by-Layer procedure (LbL),27 by consecutive deposition of layers of alternatively charged

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polyelectrolytes to protect the enzyme from denaturation.28

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Here, we report that the treatment of OVW with tyrosinase from Agaricus bisporus, after immobilization

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on Multi Walled Carbon Nanotubes (MWCNT) by LbL coating with poly(diallyldimethylammonium)

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chloride PDDA and Bovine Serum Albumin (BSA),29 increased the amount of hydroxytyrosol by

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efficient conversion of the tyrosol component. The supported enzyme performed better than the native

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counterpart, highlighting the beneficial role played by the immobilization procedure in preserving the

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enzymatic activity in a complex medium such as OVW. MWCNT provided a high surface area for

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enzyme loading, as well as biocompatibility and mechanical resistance.30 The hydroxytyrosol-enriched

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OVW showed higher antioxidant activity than the tyrosinase-untreated sample in the 2,2-diphenyl-1-

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picrylhydrazyl (DPPH) radical scavenging assay,31 and in both the alkaline Comet assay32 and the

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cytokinesis-block micronucleus (CBMN) assay under induced oxidative stress in the SH-SY-5Y human

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neuroblastoma cell line.33 Moreover, it inhibited the inflammatory response in lipopolysaccharide (LPS)-

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stimulated THP-1 monocytes and enhanced monocyte-mediated autophagy through the inhibition of the

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autophagic suppressor mammalian target of rapamycin (mTOR), associated with the activation of AMP

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activated protein kinase (AMPK) and the suppression of Akt upstream signal molecules.

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MATERIALS AND METHODS

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Materials. Tyrosinase from Agaricus bisporus (Tyr), bovine serum albumin (BSA), L-tyrosine,

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glutaraldehyde (GA), multi-walled carbon nanotubes (MWCNTs), polydiallyldimethylammonium

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chloride (PDDA), sodium sulfate anhydrous (Na2SO4), ascorbic acid (AA) and organic solvents were

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purchased from Sigma-Aldrich. UV-visible analyses were performed with a Varian Cary50 UV-Vis

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spectrophotometer equipped with a Peltier. Gas-chromatography associated to mass-spectrometry 4 ACS Paragon Plus Environment

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analysis (GC-MS) was performed by Varian GC-410/320 MS (electron beam of 70 eV). Experiments

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were done in triplicate.

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Activity data and kinetic constants of native and immobilized Tyrosinase. The activity of both native

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and immobilized tyrosinase was determined by measuring the oxidation of L-tyrosine as described in

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Saladino et al (2015). The activity is expressed as activity unit per milligram of support: Activity (U mg

65

-1)

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The activity yield is the percentage of the ratio of activity of the immobilized enzyme to the total units

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of native enzyme. Km and Vmax values of both Tyr and MWCNT/Tyr were calculated by using

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Lineweaver-Burk, Hanes and Eadie–Hofstee procedures.26

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Oxidation of Olive vegetation water. The olive vegetation waste (OVW) was filtered to remove solid

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residues, and the pH of the solution was adjusted to 6.5 by addition of the appropriate amount of sodium

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phosphate buffer (PBS; 100 mM) in the presence of ascorbic acid (AA; 30 mM). The waste sample (5.0

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mL) was treated with tyrosinase from Agaricus bisporus supported on MWCNT (Tyro/MWCNT) (600

73

Units/mL) or with native tyrosinase (Tyro) (600 Units/mL) for 24 h under constantly bubbled air

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conditions. At the end of the treatment, the waste (5mL) was filtered and extracted with ethyl acetate

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(EtOAc; 3 x 5mL) to yield OVW-1 (70 mg) and OVW-2 (61 mg), from the Tyro/MWCNT and Tyro

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treatment, respectively, as brown-yellow oils. The extract without any enzymatic treatment (73 mg;

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OVW-3) was also prepared as reference.

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DPPH radical scavenging capacity assay. Samples diluted in EtOH (from 0.01 to 100 mg/mL) were

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added to DPPH solution (6 x 10-5 M in EtOH) under magnetic stirring (30 min) and the absorbance was

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measured at 517 nm. The DPPH inhibition was calculated as follow: scavenging effect (%) = (Ao - A1)

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/ Ao x 100 (Ao : absorbance of the control; A1: measured value of absorbance). The scavenging capacity

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was expressed as IC50 (that is the concentration of the sample able to scavenge the 50% of DPPH radical).

= Ux/W support (Ux is the activity of the immobilized enzyme as measured by the dopachrome test).



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GC−MS analysis of OVW samples. the appropriate OVW sample (10 mg) in pyridine (100 L) was

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added betulin (0.39 mg) and bis(trimethylsilyl)trifluoracetamide (BSTFA, 300 μL ) under magnetic

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stirring at 90 °C for 1.5 h. The GC-Ms analysis was performed with a Supelco-28471-U S (film thickness:

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30 m × 0.25 mm × 0.25 mm; helium flow velocity: 1.0 mL min−1; column gradient temperature: 140 °C

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(2 min) to 300 °C (5 min), 5.0 °C min−1; injection temperature: 250 °C. The spectra were assigned by

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comparison with NIST (Fison, Manchester, UK). When necessary, the analysis was repeated in the

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presence of standard compounds. A similarity index (SI) greater than 98% was selected. The analysis

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was limited to products of ≥ 1 ng mL -1. Mass-to-charge ratio (m/z) values and the abundance of mass

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spectra peaks of compounds are reported in SI #1. Original fragmentation mass-spectra are in SI #2.

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Cell cultures and treatments. SH-SY5Y is a cell line subcloned from a bone marrow biopsy taken from

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a four-year-old female with neuroblastoma (American Type Culture Collection, Manassas, VA, USA).

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The SH-SY5Y cell line was cultured in DMEM medium with the addition of 10% fetal bovine serum,

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1% of penicillin and 1% of L-glutamine at 37 °C in a 5% CO2 atmosphere and 95% nominal humidity.

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Cell viability was analyzed with a 3 h pre-treatment with OVW-3 at concentrations of 50, 100, 250 and

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500 μg/mL. For the study of antioxidant activity cells were pre-treated with OVW-3 or OVW-1 at the

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selected non cytotoxic doses of 100 μg/mL or 25 and 100 μg/mL, respectively, for 3 h at 37 °C. As a

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positive control pre-treatment with Mannitol (1 mM) was performed in the same conditions, being the

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compound a well-known scavenger of the free hydroxyl radical.34 The cells were then exposed to 250

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M of t-butyl-OOH for 1 h, washed in phosphate-buffered saline (PBS) to remove the oxidative agent,

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and suspended again in DMEM complete medium. In order to investigate the induction of primary DNA

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damage, its repair kinetics and the processing of DNA lesion in cytogenetic damage, the treated cells

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were analyzed with the Comet assay (t = 0, 1/2, and 1 h) for mannitol and both extracts, while for the

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Micronucleus test mannitol and only OVW-1 were used, considering the higher antioxidant activity of


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OVW-1 respect to OVW-3. All the doses used were chosen on the basis of pilot experiments or previous

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results.35, 36

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THP-1 designates a spontaneously immortalized human monocyte-like cell line, derived from the

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peripheral blood of a childhood case of acute monocytic leukemia (AML; American Type Culture

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Collection, MD, USA).37 THP-1 was cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO,

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USA) containing 10% FCS (HyClone, Logan, UT, USA), 100 g/ml of streptomycin and 100 IU/mL

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penicillin, and maintained in a 5% CO2 incubator at 37 °C. Cells were mycoplasma free (EZ-PCR

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Mycoplasma test kit; Biological Industries, Cromwell, CT, USA). For THP-1 activation, cells were

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cultured with LPS (100 ng/mL, Sigma-Aldrich) 2 h before Ovw-1 treatment. THP-1 cells were treated

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with OVW-1 (25 μg/mL) or vehicle (Control) for 24-72 h. THP-1 were also treated with STAT3 inhibitor

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Tyrphostin AG490 (100 μM) (Calbiochem, San Diego, CA, USA) 30 min before Ovw-1 treatment. For

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autophagy, THP-1 cells were treated with Bafilomycin A1 (BafA1) (20 nM) (Santa Cruz Biotechnology

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Inc., Santa Cruz, CA, USA), an inhibitor of vacuolar-H+-ATPase, for the last 24 h; in some experiments,

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3-methyladenine (3-MA) (0.2 mM) (Santa Cruz Biotechnology Inc.), an autophagy inhibitor, was added

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24 h after OVW-1 treatment.

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Cell viability, apoptosis and proliferation assays. Cell viability of SH-SY5Y cells was evaluated using

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MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] cell proliferation assay.

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Briefly, after incubation for 3 h at 37 °C with MTT (0.5 mg/ mL) the supernatant was removed and 100

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µl of a lysis solution (10% SDS, 0.6% acetic acid in DMSO)was added to dissolve the formazan crystals.

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Optical density detection were performed with a DTX880 Multimode Detector (Beckman Coulter) with

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a 630 nm (background) and a 570 nm filter. Cell cytotoxicity of THP-1 cells was evaluated using the

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Cytotoxicity Assay Kit (Roche Diagnostics GmbH, Mannheim, Germany), following the manufacturer’s

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instructions. The assay measures membrane integrity as a function of the amount of cytoplasmic lactate

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dehydrogenase (LDH) released into the medium. LDH reduces NAD into NADH, which is utilized in 7 ACS Paragon Plus Environment


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the reduction of a tetrazolium dye to colored formazan. The amount of formazan, which is proportional

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to the amount of LDH release from dead cells, was measured at 450 nm. Absorbance for background

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correction was determined at 620 nm. The percentage of cell viability was calculated as follows: % cell

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viability = 100 – % cell cytotoxicity. The % cell cytotoxicity = 100 X (experimental well absorbance –

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negative control well absorbance) / (positive control well absorbance – negative control well

135

absorbance). All calculations were performed after background absorbance correction and blank

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absorbance subtraction. Cells in the positive control wells were treated with 1% Triton X-100 solution,

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and negative control wells cells were incubated in culture media alone. Blank wells contained the

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corresponding OVW samples or Triton X-100 solution or media without cells. Apoptosis was assessed

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by annexin V-FITC and propidium iodide staining, as previously described.38 Cell proliferation was

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analyzed counting the cell number in the presence of 0.05% trypan blue solution, to count live cells,

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within a Neubauer chamber. The same operator conducted at least three replicate counts at each time.

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These assays were performed using a 24-48 h cell treatment with OVW-1 at concentrations of 25, 50,

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100, 200 μg/mL.

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SCGE analysis (Comet assay). Comet assay, was performed using a slightly modified procedure by

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Tice et al. (2000).39 Briefly, 20 L of the cell suspension (5 × 105cells) were mixed with 0.75% low

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melting-point agarose (80 L) and put into a microscope slide pre-coated with 1% agarose. The slides

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were dive in a lysis solution for 1 day at 4 °C and then subjected to electrophoresis. Subsequently, slides

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were neutralized and stained with ethidium bromide (20 g/mL, 50 L) and nucleoids were examined at

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400× magnification with a fluorescence microscope (Axioskop 2, Zeiss) associated with a Comet Assay

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III program. To evaluate DNA damage, computer-generated tail moment (tm) values were used..

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Cytokinesis-block micronucleus (CBMN) assay. The CBMN assay was carried out with the standard

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technique proposed by Fenech40 with minor modifications. Cytochalasin-B (Cyto-B) (Sigma) was used

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at a final concentration of 6 g/ mL. After treatment, cells were washed twice with PBS, trypsinized with 8 ACS Paragon Plus Environment

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trypsin-EDTA (0.25%) and centrifuged. The pellet was then re-suspended with cold hypotonic solution

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(1% sodium citrate) (Sigma), centrifuged for 5 min and fixed with methanol:acetic acid (5:1) cold

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solution. The slides were stained with Giemsa (5%) and a total of 1000 bi-nucleated cells per culture

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with cytoplasm were scored for the presence of micronuclei (MN) for each experimental point. MN is a

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biomarker of chromosome breakage or loss. For the analysis of cell cycle progression, 1000 cells per

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treatment group were scored for the presence of one, two or more than two nuclei and the Cytokinesis

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Block Proliferation Index (CBPI) was calculated as follows: CBPI = [1N + (2×2N) + (3×>2N)]/TC where

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1N is number of cells with one nucleus, 2N with two nuclei, >2N with more than two nuclei and TC is

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the number of cells examined. Percentage of cytostasis was calculated with the formula: = 100-

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100[CBPIt−1/CBPIc−1] where t and c are treated and control samples, respectively. 41

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Antibodies and Western blot analysis. The following primary antibodies were used: rabbit polyclonal

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anti-IL-6 (Gene Tex, CA, USA), rabbit polyclonal anti-IL-1β (Gene Tex), mouse monoclonal anti-TNFα

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(Santa Cruz Biotechnology Inc.), rabbit polyclonal anti-HMGB1 (Abcam, Cambridge, UK), mouse

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monoclonal anti-granzyme B (Calbiochem), mouse monoclonal anti-phospho-STAT3 (pY705) (BD

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Transduction Laboratories, San Jose, CA, USA), mouse monoclonal anti-STAT3 (BD Transduction

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Laboratories), rabbit polyclonal anti-LC3 (Novus Biologicals, Littleton, CO, USA), mouse monoclonal

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anti-p62 (BD Transduction Laboratories), rabbit polyclonal anti-phospho-AMPK (Cell Signaling

171

Technology, MA, USA), mouse monoclonal anti-AMPK (Santa Cruz Biotechnology Inc.), mouse

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monoclonal anti-phospho-Akt (Santa Cruz Biotechnology Inc.), mouse monoclonal anti-Akt (Santa Cruz

173

Biotechnology Inc.), rabbit polyclonal anti-phospho-mTOR (Ser2448) (Cell Signaling Technology),

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rabbit polyclonal anti-mTOR (Cell Signaling Technology), and mouse monoclonal anti--actin Ac-40

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(Sigma-Aldrich). Cells were washed twice in PBS and cell lysates were prepared by a solution containing

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50 mM TRIS-HCl pH 7.6, 150 mM NaCl, 0.5% TRITON X-100, 0.5% Sodium deoxycolate, 0.1% SDS

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and the protease inhibitor mixture ‘‘Complete’’ (Roche Diagnostic GmbH). For the analysis of the 9 ACS Paragon Plus Environment


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extracellular release of inflammatory mediators, the conditioned media was collected. Proteins were

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separated by SDS-PAGE and blotted onto nitrocellulose membranes (Whatman-Protan, Sigma-Aldrich).

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The membranes were blocked with 5% Non-Fat Dry Milk (Bio-Rad, Hercules, CA, USA), probed with

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specific primary antibodies overnight, at 4 °C, washed and incubated with appropriated secondary

182

antibodies. The reaction was revealed by horseradish peroxidase (HRP)-coupled secondary reagents

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(Bio-Rad) and developed by enhanced chemiluminescence (Amersham ECL Western Blotting Detection

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Reagent, GE Healthcare, MI, Italy). The quantification of protein bands was performed by densitometric

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analysis using Quantity One 1-D analysis software (Bio-Rad) and band intensities (b.i., band volume/area

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x mean pixel intensity), normalized for -actin

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Statistical analyses. The experimental values are expressed as mean ± standard error (S.E.) of the mean.

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The statistical significance in terms of CBPI between control and t-Butyl-OOH treated samples and

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between t-Butyl-OOH treated and Owv-1 or Mannitol pre-treated samples was evaluated by use of the

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χ2-test. The difference in the yield of both micronuclei per cell and mean tail moment between the control

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and t-Butyl-OOH treated samples and between t-Butyl-OOH treated and Owv-1 or Mannitol pre-treated

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samples and all other analyses were evaluated by Student’s t-test; p < 0.05 was considered as statistically

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significant.

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RESULTS AND DISCUSSION

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The OVW was supplied by an agro-food company from Viterbo (Italy), and resulted as a black solution

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from a continuous cold process of the olive oil by means of the conventional three-phase extraction

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apparatus.42 The olives varieties used in this investigation were from Olea europea, harvested at optimum

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ripeness and pressed without delay. The OVW was initially filtered to remove solid residues, and the pH

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of the solution was adjusted to 6.5 (optimal pH value for tyrosinase activity) by addition of the

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appropriate amount of sodium phosphate buffer (PBS, 100 mL). The waste (200 mL) was treated with 10 ACS Paragon Plus Environment

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tyrosinase from Agaricus bisporus supported on MWCNT (Tyro/MWCNT) (600 Units/mL) at room

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temperature for 24 h in the presence of ascorbic acid (AA; 30 mM) to avoid undesired side-reactions.

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The preparation, structural characterization and kinetic data of Tyro/MWCNT (Figure 1) have been

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described in reference 26. The activity of MWCNT/Tyr (81.7 U/mg), the value of immobilization yield

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(71%), and activity yield (49.7%), were in accordance with our previous data (for the definition of these

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parameters see in the experimental part). The waste was extracted with ethyl acetate (EtOAc, 3 x 5.0 mL)

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and the crude was recovered as a brown-yellow oil (OVW-1) after evaporation. The treatment of the

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waste with native tyrosinase (Tyro) under similar experimental condition afforded OVW-2. Finally, the

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extraction of the original olive vegetation waste without enzymatic treatment yielded OVW-3. The

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crudes were analyzed by gas-chromatography associated to mass-spectrometry (GC-MS). Quantitative

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analyses were performed using betulin as internal standard. Table 1 reports the main compounds

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identified in the OVW samples, the yield being defined as mg of compound per gram of starting sample

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(the m/z value and the abundance of peaks are in Supporting Information SI # 1, while the original

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fragmentation spectra are in SI # 2).

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Carboxylic acids 1-4, benzoic acid derivatives 5-11, cinnamic acid derivatives and ester 12-17,

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flavonoids 18-19, tyrosol 20, and hydroxytyrosol 21, were detected in appreciable amount in OVW-3,

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phenols 20-21 being found as the most abundant compounds (Table 1, entries 20 and 21). In accordance

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with previously reported data, tyrosol 20 and hydroxytyrosol 21 were found to be in similar amount (c.a.

221

1:1 ratio).5 The efficacy of the treatment of the OVW with Tyro/MWCNT was highlighted by the increase

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of the amount of hydroxytyrosol 21 in OVW-1 with respect to OVW-3 (30.05 mg versus 17.8 mg per

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gram of starting material, respectively), associated to the simultaneous disappearance of tyrosol 20

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(Table 1, entries 20 and 21). A similar behavior was observed in the case of protocatechuic acid 7 (3,4-

225

dihydroxybenzoic acid), and caffeic acid 14 (3,4-dihydroxycinnamic acid). Their amount was higher in 11 ACS Paragon Plus Environment


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OVW-1 than OVW-3 (Table 1, entries 8 and 14), as a consequence of the selective Tyro/MWCNT

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catalyzed ortho-hydroxylation of benzoic acid derivatives 5-6, and of cinnamic acid derivatives 12-13,

228

respectively (Table 1, entries 5-6 and entries 12-13). The role of oxidative non enzymatic side-reactions

229

associated to the formation of redox active intermediates (e.g. quinones and hydroquinones) in the

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disappearance of compounds 5 and 12 cannot be completely ruled-out, since the known low reactivity of

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these derivatives towards tyrosinase. Protocatechuic acid and caffeic acid are well recognized

232

antioxidants, able to coordinate metal transition ions, as well as to scavenge free radicals via donating

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hydrogen atom or electrons.43,44 Note that compounds 8-11 and 15-16, lacking of an accessible ortho-

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position for the oxygen atom transfer process, and characterized by hindered aromatic rings, were found

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unaffected by the enzyme treatment (Table 1, entries 8-11 and entries 15-16). As a general trend,

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Tyro/MWCNT was more reactive than native tyrosinase, protocatechuic acid 7, caffeic acid 14, and

237

hydroxytyrosol 21 being detected in higher amount in OVW-1 with respect to OVW-2 (Table 1, entries

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7, 14 and 21). Moreover, appreciable amount of unreacted benzoic acid derivatives 5-6, cinnamic acid

239

derivatives 12-13, and tyrosol 20, were detected in OVW-2. The increased activity of Tyro/MWCNT

240

with respect to native Tyro, was probably due to several benign factors, including a favorable

241

environment, able to prevent the emergence of intermolecular phenomena, the lowering of undesired

242

enzyme conformational changes, and the prevention of inhibition−inactivation processes because of the

243

protection exerted by the polyelectrolyte ﬁlms from denaturing agents.45, 46

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The reusability of Tyro/MWCNT in the oxidation of OVW was evaluated by the use of the catalyst in

245

further runs, monitoring the hydroxytyrosol yield as a selected product (SI#4). Tyro/MWCNT retained

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an appreciable catalytic activity after six runs, observing a decrease of the yield of hydroxytyrosol of

247

77% at the last cycle of reuse.

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Antioxidant activity


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The antioxidant activity, evaluated by the analysis of the DPPH radical scavenging properties,47 is reported as IC50 value (defined as the concentration

250

of substrate in mg/mL that causes 50% loss of DPPH activity). OVW-1 showed an antioxidant activity higher than OVW-2

251

and OVW-3, respectively (Table 2; entry 1 versus entries 2 and 3), being the IC50 value of OVW-1 of

252

the same order of intensity, or even of order of intensity greater, than that previously measured for

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hydroxytyrosol-enriched aqueous extracts of leaves from Olea europea.48,49 In accordance with the lower

254

conversion observed for tyrosol and for the other tyrosinase reactive phenol derivatives, OVW-2 showed

255

an antioxidant activity similar to OVW-3 (Table 2, entry 2 versus entry 3). This data further confirmed

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the efficacy of the Tyro/MWCNT treatment in increasing the overall antioxidant activity of OVW.

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The antioxidant activity of the most active OVW-1 was further evaluated by the alkaline Comet assay in the SH-SY-5Y cell

258

line. The SH-SY-5Y cell line is a model for Parkinson disease (PD), showing similarities with DAergic

259

neurons.50 At the cellular level, PD is related to excessive production of reactive oxygen species (ROS).

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Thus, the study of the antioxidant activity in SH-SY-5Y cell line is a useful tool in neuroprotection

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disorder studies related to imbalance of the cellular redox potential.51-53 OVW-3 and mannitol (a well-

262

known radical scavenger for ROS) were used as references. The analysis of cell viability after 3 h pre-

263

treatment with OVW-3 (SI#-3) revealed no effect on cytotoxicity up to a concentration of 100 μg/mL,

264

but a significant (p < 0.01) effect was observed at higher doses of 250 and 500 μg/mL. On the basis of

265

both cell viability and preliminary DPPH analysis, 100 μg/mL for OVW-3, and 25 and 100 μg/mL for

266

OVW-1, were selected for the study. The antioxidant activity of OVW-3 and OVW-1 was measured as

267

the ability to reduce the extent of DNA primary damage induced by 1.0 h of treatment with 250 M t-

268

Butyl-OOH (Table 3).

269 270

No increase in DNA migration was detected after treatment with OVW-3, OVW-1 or mannitol alone,

271

while a marked and significant increase (p < 0.01) in the mean tail moment values, reflecting the primary

272

DNA damage, was observed in t-Butyl-OOH treated cells when compared to the medium alone. Instead, 13 ACS Paragon Plus Environment


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the OWV-3, OWV-1 or mannitol pre-treated and t-Butyl-OOH treated cells showed a statistically-

274

significant reduction (p < 0.01) in the mean tail moment values at all recovery times, as compared with

275

t-Butyl-OOH alone. As a general trend, the higher protection against DNA damage in terms of percentage

276

of RTM in comparison with mannitol was observed at all recovery times except that at 0 h for 100 μg/mL

277

OVW-3. For 25 μg/mL OVW-1 the same effect was observed at all recovery times, while for 100 μg/mL

278

OVW-1 the higher protection, in comparison with mannitol, was detected only at 1.0 h recovery time

279

(Table 3). These findings clearly demonstrated the absence of pro-oxidant activity of OVW-3 and OVW-

280

1, and showed their capability to reduce the primary DNA damage induced by the oxidative agent.

281

Moreover, the scavenging activity exerted by OVW-1 at 25 μg/mL resulted higher than that of OVW-3,

282

and even higher than that of mannitol (Table 3).

283

Successively, OVW-1 activity was analyzed by the Cytokinesis-block Micronucleus (CBMN) assay,

284

again using mannitol as reference. The results related to the induction of micronuclei following the pre-

285

treatment of SHSY-5Y cells with OVW-1 or mannitol, and the treatment with t-Butyl-OOH, are reported

286

in Table 4.

287 288

OVW-1 and mannitol did not produce an increase in micronuclei compared to the medium alone.

289

Conversely, treatment with t-Butyl-OOH resulted in a significant (p < 0.01) increase in the frequency of

290

micronuclei when compared with the medium. Pre-treatment of SHSY-5Y cells with OVW-1 or

291

Mannitol determined a significant (p < 0.01) decrease in the frequency of micronuclei with respect to t-

292

Butyl-OOH alone. Regarding cell proliferation, highlighted by the Cytokinesis Block Proliferation Index

293

(CBPI), treatment with t-Butyl-OOH significantly reduced (p < 0.01) cell proliferation, as shown by the

294

lowering of the CPBI respect to the medium, while only pre-treatment with OVW-1 at 100 μg/mL

295

showed a significant (p < 0.05) increase of the CBPI as compared with the oxidative agent. The same

296

results were obtained analysing the percentage of cytostasis. Data from the CBMN assay clearly 14 ACS Paragon Plus Environment

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demonstrated the capability of OVW-1 to decrease the cytogenetic damage and the cell proliferation

298

arrest, both induced by t-Butyl-OOH. Finally, these results were in agreement with data obtained using

299

the Comet assay, both showing a protective effect of OVW-1 on oxidative stress-induced DNA damage

300

in the SH-SY-5Y cell line.

301

Pro-autophagic and anti-inflammatory activities

302

Autophagy is a cytoplasmic degradation system, capable of orchestrating inflammation, immunity and

303

cancer, as well as of contributing to the extension of longevity.54-56 Indeed, autophagy mediates

304

cytoprotection and reduces age-associated processes, inhibiting the general increase of inflammation in

305

human aging, denominated “inflammaging”.57 Inflammaging is monocyte/macrophage centered; thus,

306

the upregulation of autophagy in monocyte/macrophages might facilitate the clearance of damaged and

307

potentially harmful cells and reduces their propensity to produce pro-inflammatory cytokines, resulting

308

in life span extension.56 Indeed, autophagy can affect inflammatory cytokine production by

309

monocytes/macrophages through different mechanisms, including the inhibition of inflammasomes.54

310

Defects in autophagy have been linked to a wide range of age-related inflammatory diseases (e.g.,

311

cardiomyopathy, neurodegenerative disorders, cancers) and compounds capable of promoting autophagy

312

have been recently received attention for the treatment of these diseases.54 For these reasons, we

313

examined whether OVW-1, the most active OVW sample, could affect autophagy in human THP-1

314

monocytes. To investigate the autophagic process, two recommended autophagic markers such as the

315

microtubule-associated protein 1 light chain 3 (LC3) and the sequestosome-1 (SQSTM1)/ubiquitin-

316

binding protein p62 were evaluated by Western blot.58,59 First, OVW-1 was tested for its cytotoxic effect,

317

by culturing THP-1 cells with increasing concentrations of the sample (from 25 to 200 μg/mL) for 24 h

318

and 48 h. The LDH cytotoxicity assay showed that cell viability was not affected up to an OVW-1

319

concentration of 50 μg/mL at both 24 h and 48 h of treatment (Figure 2A).



Page 16 of 41

320

Moreover, the analysis of apoptosis showed that early apoptosis was induced by 100 μg/mL and 50

321

μg/mL at 24 h and 48 h of treatment, respectively (Figure 2B). Finally, the evaluation of cell growth

322

showed that the cell number was not affected up to an OVW-1 concentration of 50 μg/mL at both 24 h

323

and 48 h, and then decreased in a dose dependent manner (Figure 2C), suggesting that a proliferative

324

arrest might occur starting at 50 μg/mL at 24 h and 48 h. Thus, taking all these results into account,

325

subsequent experiments were performed treating THP-1 cells with a dose of OVW-1 25 μg/mL. During

326

autophagy, LC3 is processed post-translationally into soluble LC3-I and, in turn, converted to membrane-

327

bound LC3-II that correlates with the extent of autophagosomes, while SQSTM1/p62, being part of the

328

assembled autophagosome, is subsequently degraded in autolysosomes and serves as an index of

329

autophagic degradation.

330 331

The identification of LC3 in the LC3-II form in THP-1 cells showed that LC3II formation increased after

332

treatment with OVW-1 (Figure 3A), suggesting an increase of autophagy. To further assess whether

333

OVW-1 increased LCII expression by accelerating autophagy levels and not by disrupting lysosomal

334

degradation, we delivered bafilomycin A1 (BafA1) to cells and determined LC3II expression levels again.

335

As shown in Figure 3A, we observed a further accumulation of LC3-II when OVW-1 treated cells were

336

also treated with BafA1, further confirming the increase of a complete autophagic flux by OVW-1. Then,

337

we analyzed SQSTM1/p62 levels and we found a decrease of this protein (Figure 3B), indicating that

338

the OVW-1 was able to promote a complete autophagic process. Finally, we found that the addition of

339

the autophagy inhibitor 3-MA to OVW-1 treated THP-1 cells suppressed LC3II formation and increased

340

p62 expression (Figure 3C), confirming thus that the observed modulations of LC3II and p62 protein

341

levels were the result of the upregulation of autophagy by OVW-1. Our results are consistent with the

342

induction of autophagy by an olive mill waste water administered in a mouse model of neurodegenerative

343

disease. 60 16 ACS Paragon Plus Environment

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344

Additionally, since the major control complex for autophagy is the mTOR, which, when active,

345

suppresses the initiation of autophagy,61 we investigated whether OVW-1 could target mTOR activation

346

in THP-1 monocytes. As shown in Figure 3B, we found that OVW-1 treatment significantly suppressed

347

mTOR phosphorylation at Ser 2448, leading to the inhibition of mTOR activation. Moreover, considering

348

that diverse cellular signals converge on mTOR to regulate autophagy, including AMPK and Akt (protein

349

kinase B) (inhibitor and activator of mTOR, respectively),62 we also analyzed the activation state of these

350

molecules following OVW-1 treatment. As shown in Figure 3B, AMPK activation (phosphorylation)

351

was significantly increased, whereas Akt phosphorylation was reduced in OVW-1-treated THP-1

352

compared with untreated cells (Control). Therefore, these results suggest that OVW-1 might increase the

353

autophagic flux via the mTOR pathway and that both AMPK and Akt signaling molecules might be

354

involved in this activity.

355

Next, we also studied the anti-inflammatory activity of OVW-1, by investigating its capability to

356

inhibit the inflammatory response mediated by human activated monocytes. LPS-stimulated THP-1

357

monocytes were used as an in vitro model of inflammation, and the production of multiple inflammatory

358

mediators such as pro-inflammatory cytokines, high mobility group box 1 (HMGB1) danger signal and

359

granzyme B serine protease was investigated. HMGB1 is a chromatin-associated nuclear protein that,

360

when released in the extracellular milieu, acts as a “damage-associated molecular pattern” (DAMP)

361

molecule, inducing an inflammatory response; it increases with age and is involved in the pathogenesis

362

of multiple human inflammatory diseases (e.g., trauma, ischemia, chronic inflammatory disorders,

363

autoimmune diseases and cancer).57,63 Granzyme B is a serine esterase that, when released in the

364

extracellular milieu, functions on the one hand as DAMP and on the other hand as enzyme, cleaving

365

extracellular matrix (ECM) proteins and cytokines, leading to inflammatory cell recruitment and cytokine

366

activation;64-66 it is expressed by monocytes/macrophages in the lesion areas of atherosclerosis and

367

rheumatoid arthritis,66 is present in plasma of patients with inflammatory diseases65 and, interestingly, 17 ACS Paragon Plus Environment


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368

plays a key role in aging skin.

Therefore, the intracellular content and the extracellular release of

369

pro-inflammatory mediators, such as HMGB1, granzyme B and IL-6, IL-1β and TNF-α cytokines, were

370

investigated in THP-1 cells stimulated with LPS and cultured in the presence or absence of OVW-1. The

371

intracellular and extracellular pro-inflammatory protein levels were measured by Western blot and

372

normalized for -actin or Ponceau staining, respectively. Basal levels of both intracellular and

373

extracellular cytokines were detected in unstimulated THP-1 cells, and, as expected, they increased when

374

THP-1 cells were stimulated with LPS (100 ng/mL) (Figures 4A and 4B). However, when cells were

375

treated with OVW-1, an inhibition of the intracellular content (Figure 4A) associated to a significant

376

decrease of the extracellular release (Figure 4B) of all the pro-inflammatory mediators was observed.

377

To the best of our knowledge, the inhibitions of HMGB1 and granzyme B production by OVW have

378

never been described before and, considering the implication of these molecules in multiple

379

inflammatory disorders, our findings support the interest in OVW-1 as anti-inflammatory agent.

380

Moreover, we found that OVW-1 inhibited the production of IL-6, IL-1β and TNF-α. The inhibition of

381

pro-inflammatory cytokines by OVW-1 is a relevant finding, since these cytokines are major components

382

of the biology of aging and inflammaging.56,57,69 Our results are consistent with the inhibition of TNF-α

383

production by some olive oil extracts in LPS-treated mice and THP-1 cells,70 and with the detection of

384

hydroxytyrosol as the major bioactive compound in olive water extracts impairing cytokine production

385

by monocyte/macrophages.15

386

Cytokines function by activating transcription factors such as nuclear factor (NF)-kB or signal transducer

387

and activator of transcription 3 (STAT3). In particular, IL-6 activates STAT3 phosphorylation at Tyr705,

388

and once activated, STAT3 positively influences the release of IL-6, creating a positive feed-back loop.72

389

In addition, STAT3 also functions as a regulator of autophagy and might play an important role in the

390

process through which IL-6 regulates autophagy.73 Not least, STAT3 activation in monocytes contributes

391

to neovascular age-related macular degeneration,74 as well as promotes liver tumorigenesis.75 Taking 18 ACS Paragon Plus Environment

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into account all these considerations, we investigated the inhibition of STAT3 activation by OVW-1 in

393

monocytes by analyzing its phosphorylation at Tyr 705 using Western blot. As shown in Figure 5, the

394

activation of STAT3 (p-STAT3) was induced when THP-1 cells were stimulated with LPS. However,

395

treatment with OVW-1 significantly inhibited p-STAT3 (Figure 5), leading to the suppression of STAT3

396

activation. Moreover, STAT3 inhibition induced by OVW-1 was comparable to that caused by

397

Tyrphostin AG 490 (Figure 5), a STAT3 inhibitor. These results encourage us to suppose that a

398

correlation exists between the inhibition of IL-6 production (Figure 4), STAT3 inhibition (Figure 5) and

399

autophagy promotion (Figure 3).

400 401

Since numerous studies demonstrated the association of down-regulation of IL-6 and/or IL-6-mediated

402

STAT3 signaling with therapeutic results in age- and inflammatory-related diseases,72,76 we consider that

403

the inhibition of the IL-6/STAT3 pathway by OVW-1 in activated monocytes is a relevant finding and

404

that OVW-1 might represent a potential promising candidate to target the IL-6/STAT3 pathway in

405

inflammatory diseases and cancer.

406

Treatment of OVW with tyrosinase after immobilization on MWCNTs provided the OVW-1 extract

407

highly enriched in hydroxytyrosol and other catechol derivatives. This extract was characterized by an

408

increased antioxidant activity in DPPH analysis, alkaline Comet assay, and CBMN assay. OVW-1 was

409

more active than untreated OVW-3 extract, highlighting the relevant role of newly synthesized

410

hydroxytyrosol and catechols in the biological activity. Moreover, OVW-1 was also more active than

411

OVW-2, confirming the increased stability of the tyrosinase after immobilization on MWCNTs. In

412

addition, OVW-1 exerted a broad anti-inflammatory activity by inhibiting the production of multiple

413

inflammatory mediators by LPS-stimulated THP-1 cells (a monocyte-based in vitro inflammatory

414

model), and enhanced monocyte-mediated autophagy by modulating the activation of mTOR, AMPK

415

and Akt signaling molecules. Our findings are consistent with data in the literature reporting that 19 ACS Paragon Plus Environment


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416

hydroxytyrosol is endowed with antioxidant activity,

417

STAT3 activation,

418

observed biological effect of other components of OVW cannot be completely ruled-out. 80-82 Together,

419

our findings make OVW-1 a potential preventive and/or therapeutic agent for age- and inflammatory-

420

related diseases.

78,79

inhibition of both cytokine production and of

as well as with the promotion of autophagy even if the possible role in the

421 422

ABBREVIATIONS USED

423

IL, interleukin; TNF-α, tumor necrosis factor-α; HMGB1, high-mobility group box 1; STAT3, signal

424

transducer and activator of transcription 3; LC3, protein 1 light chain 3; SQSTM1, sequestosome-1; Baf,

425

bafilomycin A1; DAMPs, damage associated molecular patterns; 3-MA, 3-methyladenine; LPS,

426

lipopolysaccharide; mTOR, mammalian target of rapamycin; AMPK, AMP-activated protein kinase;

427

OVW,

428

poly(diallyldimethylammonium) chloride; BSA, Bovine Serum Albumin; DPPH, 2,2-Diphenyl-1-

429

picrylhydrazy; CBMN, Cytokinesis-block Micronucleus.

430

FUNDING SOURCES: Research was supported by FILAS project MIGLIORA from Regione Lazio

431

and by the University of Tuscia (University Research Funds).

432

ACKNOWLEDGEMENTS: project PRONAT from CNCCS SCARL are acknowledged.

433

NOTES: The authors declare no competing financial interest.

434

SUPPORTING INFORMATION. Mass-to-charge ratio (m/z) value and the abundance of peaks of

435

identified polyphenols(SI #1), original fragmentation mass-spectra(SI#2.), effect of OVW-3 on cell

436

viability (SI#-3:) and reusability of MWCNT/Tyr (SI#-4) are available in Supporting Information

437 438

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439 440

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656 657 658 659 660 661 662 663 664 665 666

FIGURE CAPTIONS:

667 668 669 670

Figure 1. Schematic representation of tyrosinase from Agaricus bisporus supported on MWCNT (Tyro/MWCNT).

671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692

Figure 2. Effects of OVW-1 on THP-1 cell viability (A), apoptosis (B) and proliferation (C) at 24 h and 48 h of treatment. Data show mean of the percentage (%) plus standard deviation of three independent experiments. Figure 3. Effect of OVW-1 on autophagy in THP-1 cells. Cells were cultured with vehicle (Control) or 25 M OVW-1 for 48-72 h in the presence or absence of the ATP vacuolase inhibitor Bafilomycin A1 (BafA1) (A) or the autophagic inhibitor 3-methyladenine (3-MA) (C), and the expression of LC3I/II (A, C) and SQSTM1/p62 (B, C) autophagic markers were analyzed by Western blot. Data also show AMPKThr172, Akt and mTORSer2448 phosphorylation (p) (B), evaluated by Western blot; total AMPK, Akt and mTOR served as controls; β-actin was included as loading control; numbers indicate band intensities (b.i.) = band volume/area x mean pixel intensity, normalized for β-actin and quantified using Quantity One 1-D analysis software; representative experiments out of three. Figure 4. Effects of OVW-1 on the intracellular content (A) and the extracellular release (B) of proinflammatory mediators by LPS-stimulated THP-1 cells. Cells were cultured with medium (-) or LPS (100 ng/mL) in presence or absence of 25 M OVW-1 for 48 h. Data show Western blot on cell lysates (A) and conditioned media (B); β-Actin and Ponceau staining served as intracellular and extracellular loading control, respectively; numbers indicate band intensities (b.i.)= band volume/area x mean pixel intensity, normalized for β-actin or Ponceau staining and quantified using Quantity One 1-D analysis software; representative experiments out of three. 30 ACS Paragon Plus Environment

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693 694 695 696 697 698 699


Figure 5. Effect of OVW-1 on STAT3 activation in LPS-stimulated THP-1 cells. Cells were cultured with medium (-) or LPS (100 ng/mL) in the presence or absence of 25 M OVW-1 for 48 h. Data show STAT3 Tyr705 phosphorylation (p-STAT3), evaluated by Western blot; total STAT3, AG490 (STAT3 inhibitor) and β-actin served as controls; numbers indicate band intensities (b.i.)= band volume/area x mean pixel intensity, normalized for β-actin and quantified using Quantity One 1-D analysis software; representative experiment out of three.

700 701 702 703 704



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Table 1. Chemical Composition of OVW 1-3 Samplesa. Entry

Compounds

OVW-1 (mg)b,c

OVW-2 (mg)

OVW-3 (mg)

1

Succinic acid (1)

4.79

4.80

4.82

2

Nonanoic acid (2)

0.35

0.37

0.35

3

Oleic acid (3)

2.92

2.90

2.91

4

Octadecenoic ester (4)

0.19

0.21

0.21

5

3-Hydroxybenzoic acid (5)

ND

2.10

2.54

6

4-Hydroxybenzoic acid (6)

ND

0.04

0.10

7

Protocatechuic acid (7)

2.96

2.71

2.56

8

3,4-Dihydroxy phenylpropionic acid (8)

0.13

0.10

0.12

9

Vanillic acid (9)

0.19

0.20

0.20

10

Gentisic acid (10)

0.16

0.15

0.16

11

Syringic acid (11)

0.06

0.06

0.06

12

3-Hydroxy cinnamic acid (12)

ND

0.19

0.27

13

p-Cumaric acid (13)

ND

0.18

0.22

14

Caffeic acid (14)

1.15

0.80

0.72

15

3,4-Dimethoxy cinnamic acid (15)

0.38

0.39

0.38

16

Ferulic acid (16)

0.29

0.31

0.32

17

Caffeic acid phenethyl ester (17)

0.21

0.20

0.21

18

Rutin (18)

0.09

0.11

0.11

19

Quercetin (19)

0.05

0.05

0.05

20

Tyrosol (20)

ND

10.5

13.2

21

Hydroxytyrosol (21)

30.05

20.1

17.8

acid

methyl

aOVW-1: extract from olive vegetation waste treated with Tyro/MWCNT. OVW-2: extract from olive vegetation

waste treated with native Tyrosinase. OVW-3: extract from untreated olive vegetation waste. bThe yield is defined as mg of compound per gram of starting material. cData are expressed as the mean ± 0.01 standard deviation of three replicates. The composition of OVW was not modified by treatment with H2O2 without tyrosinase.


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Table 2. 2,2- Diphenyl Picrylhydrazyl (DPPH) Radical Scavenging Properties of Samples OVW 1-3. Entry

Sample

IC50a

1

OVW-1

0.02

2

OVW-2

0.30

3

OVW-3

0.45

aIC

is defined as the concentration of substrate in mg/mL that causes 50% loss of DPPH activity. b OVW-1: extract from olive vegetation waste treated with Tyro/MWCNT. OVW-2: extract from olive vegetation waste treated with native Tyrosinase. OVW-3: extract from untreated olive vegetation waste. 50



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Table 3. Evaluation of the Ability of OVW-3 and OVW-1 to Reduce the Extent of DNA Primary Damage Induced with t-Butyl-OOH, Using the Alkaline Comet Assay.a Pre-Treatment

Treatment

Recovery time

Mean TM ± SE

% RTM

Medium

Medium

0h

1.33 ± 0.11

-

Mannitol

Medium

0h

1.25 ± 0.17

-

OVW-3 (100 g/ml)

Medium

0h

0.81 ± 0.08

-

OVW-1 (25 g/ml)

Medium

0h

1.55 ± 0.12

-

OVW-1 (100 g/ml)

Medium

0h

1.45 ± 0.10

-

Medium

t-ButylOOH

0h

12.99 ± 0.87 **

-

Mannitol

t-ButylOOH

0h

9.24 ± 0.83 ##

31.5

OVW-3 (100 g/ml)

t-ButylOOH

0h

12.32 ± 0.47

1.3

OVW-1 (25 g/ml)

t-ButylOOH

0h

6.61 ± 0.60 ##

56.6

OVW-1 (100 g/ml)

t-ButylOOH

0h

11.27 ± 0.93 ##

15.8

Mannitol

Medium

½h

1.58 ± 0.17

-

OVW-3 (100 g/mL)

Medium

½h

1.26 ± 0.11

-

OVW-1 (25 g/mL)

Medium

½h

1.15 ± 0.16

-

OVW-1 (100 g/mL)

Medium

½h

1.20 ± 0.21

-

Medium

t-ButylOOH

½h

9.48 ± 0.88 **

-

Mannitol

t-ButylOOH

½h

6.54 ± 0.54 ##

40

OVW-3 (100 g/mL)

t-ButylOOH

½h

5.12 ± 0.21 ##

52.7

OVW-1 (25 g/mL)

t-ButylOOH

½h

4.85 ± 0.42 ##

54.4

OVW-1 (100 g/mL)

t-ButylOOH

½h

6.50 ± 0.51 ##

35.0

Mannitol

Medium

1h

1.10 ± 0.12

-

OVW-3 (100 g/mL)

Medium

1h

1.68 ± 0.09

-

OVW-1 (25 g/mL)

Medium

1h

1.22 ± 0.18

-

OVW-1 (100 g/mL)

Medium

1h

1.15 ± 0.20

-

Medium

t-ButylOOH

1h

7.12 ± 0.37 **

-

Mannitol

t-ButylOOH

1h

5.22 ± 0.49 ##

29.5

OVW-3 (100 g/mL)

t-ButylOOH

1h

5.61 ± 0.22 ##

32.7

OVW-1 (25 g/mL)

t-ButylOOH

1h

4.89 ± 0.44 ##

37.2

OVW-1 (100 g/mL)

t-ButylOOH

1h

4.45 ± 0.37 ##

43.5


Page 35 of 41


a Mean

tail moment values ± SE obtained at different recovery times for both controls and t-butyl-OOHtreated samples with and without pre-treatment with mannitol, Ovw-3 or Ovw-1 in SHSY-5Y cell line. RTM: Tail Moment Reduction. Student’s t-test; ** p ≤ 0.01 t-ButylOOH vs medium; ## p ≤ 0.01 mannitol, Ovw-3 or Ovw-1 + t-ButylOOH vs t-ButylOOH.

Table 4. Induction of micronuclei (MN) obtained for both controls and t-butyl-OOH-treated samples with and without pre-treatment with Mannitol or OVW-1 in the SHSY-5Y cell linea,b Pre-Treatment

Treatment

MN/1000 BN±S.E

Medium

Medium

11.0 ± 0.05

Mannitol

Medium

9.9 ± 0.14

OVW-1 (25 gml)

Medium

OVW-1 (100 gml)

ts

CBPI±S.E.

χ2

% Cytostasis±S.E.

1.57 ± 0.0001

0 ± 0.00

N S

1.50 ± 0.0002

9 ± 0.03

9 ± 0.02

N S

1.48 ± 0.003

12 ± 0.05

Medium

9.2 ± 0.03

N S

1.47 ± 0.00

16 ± 0.04

Medium

t-Butyl-OOH

174.5 ± 0.95

**

1.09 ± 0.0001

**

72 ± 0.07

Mannitol

t-Butyl-OOH

115 ± 0.49

##

1.10 ± 0.005

NS

68.5± 0.06

OVW-1 (25 gml)

t-Butyl-OOH

95 ± 0.29

##

1.12± 0.004

NS

65± 0.3

OVW-1 (100 gml)

t-Butyl-OOH

100± 0.04

##

1.15± 0.002

#

57.4± 0.02

ats:

Student’s t-test; χ2: chi-square test; NS: not statistically significant; ** p ≤ 0.01 t-Butyl-OOH vs medium; ## p ≤ 0.01 mannitol or Ovw-1 + t-Butyl-OOH vs t-Butyl-OOH. bHarvesting time after cytoB: 24 h. CBPI: Cytokinesis Block Proliferation Index; BN: Binucleated; S.E.: Standard Error.



Figure 1


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Figure 2



Figure 3


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Figure 4



Figure 5.


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Table of Contents Graphic (TOC):


TYROSINASE-TREATED HYDROXYTYROSOL-ENRICHED OLIVE

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