Protective effects of tricetinidin against oxidative stress inducers in rat kidney cells: A comparison with delphinidin and standard antioxidants

Accepted Manuscript Protective effects of tricetinidin against oxidative stress inducers in rat kidney cells: A comparison with delphinidin and standard antioxidants Ezgi Eyluel Bankoglu, Jens Broscheit, Theresa Arnaudov, Norbert Roewer, Helga Stopper PII:

S0278-6915(18)30702-6

DOI:

10.1016/j.fct.2018.09.058

Reference:

FCT 10084

To appear in:

Food and Chemical Toxicology

Received Date: 8 March 2018 Revised Date:

18 September 2018

Accepted Date: 24 September 2018

Please cite this article as: Bankoglu, E.E., Broscheit, J., Arnaudov, T., Roewer, N., Stopper, H., Protective effects of tricetinidin against oxidative stress inducers in rat kidney cells: A comparison with delphinidin and standard antioxidants, Food and Chemical Toxicology (2018), doi: https:// doi.org/10.1016/j.fct.2018.09.058. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Protective effects of Tricetinidin against oxidative stress inducers in rat kidney cells: A comparison with Delphinidin and standard antioxidants

and Helga Stopper1b*

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Institute of Pharmacology and Toxicology, University of Wuerzburg, 97078 Wuerzburg,

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Germany

Department of Anesthesia and Critical Care, University of Wuerzburg, 97080 Wuerzburg,

Germany

Correspondence: Helga Stopper

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Ezgi Eyluel Bankoglu1a, Jens Broscheit2a, Theresa Arnaudov1, Norbert Roewer2b

University of Wuerzburg, Versbacher Str. 9, 97078,

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Wuerzburg, Germany

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Institute of Pharmacology and Toxicology,

Tel.: +49 931 31 48427

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Fax: +49 931 201 48446

Email: [email protected] a: Both authors contributed equally. b: Both authors contributed equally. * Corresponding author. Keywords: Antioxidant, DNA damage, Hemeoxygenase-1, Oxidative stress and Tricetinidin

ACCEPTED MANUSCRIPT Abstract The potential protective effect of tricetinidin as novel antioxidant is investigated and compared with selected known antioxidant substances in vitro. Dihydroethidium staining was performed to detect intracellular ROS formation and the protective effect of the antioxidant

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substances in combination with the superoxide-inducer antimycin a (AMA). Glutathione level, mitochondrial membrane potential and HO-1 expression were analysed for further characterization of the cellular response. The cytokinesis block micronucleus test was applied

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to investigate the anti-genotoxic effect of the substances against insulin induced genomic damage. AMA treatment caused a significant increase in intracellular ROS formation and

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insulin treatment induced a significant micronucleus induction in NRK cells. Combination of the antioxidant substances with AMA or insulin protected from the oxidative stress and the micronucleus-induction. All analysed antioxidants showed comparable effects on GSH production and mitochondrial membrane potential. Only delphinidin and tricetinidin caused

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an increase in HO-1 expression. Tricetinidin and delphinidin might be good candidates for development as an antioxidant supplement. Further research is necessary to show possible

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Highlights

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therapeutic and preventive effects of tricetinidin and delphinidin in vivo.

Tricetinidin showed in vitro antioxidant activity that was comparable to the other wellknown antioxidants

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Tricetinidin protected the NRK cells against insulin-induced genotoxicity Tricetinidin induced an increased HO-1 expression in NRK cells

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Introduction

Oxidative stress is defined as an imbalance between the generation of reactive oxygen/nitrogen species (ROS/RNS) and antioxidant reactions. Under normal physiological conditions ROS have an important role in the regulation of physiological functions by

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activating signalling pathways. An antioxidant defence systems consisting of molecules such as glutathione, glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase and hemeoxygenase1 (HO-1) helps to preserve redox homeostasis (Ruperez et al., 2014;

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Taniyama and Griendling, 2003). However, the antioxidant defence mechanisms can be saturated and the remaining ROS may attack cellular macromolecules such as proteins and

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DNA, in the latter case leading to mutations and genomic damage. There are different forms of DNA damage ranging from point mutations on the DNA base level up to aneuploidy on the level of the whole genome (Fenech, 2000). Oxidative DNA damage has been suggested to be one of the major factors in carcinogenesis (Lunec et al., 2002). In addition, oxidative stress

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plays an important role in the pathogenesis of many chronic diseases including aging, cardiovascular disease, type 2 diabetes mellitus and obesity (Ames et al., 1993; Beckman and Koppenol, 1996).

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Antioxidants are substances that can mitigate the oxidation of cellular substrates. Antioxidant functions can lower the oxidative stress, oxidation of cellular macromolecules and cell

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damage (Pisoschi and Pop, 2015). Natural antioxidants have increasingly been used as natural food components or supplements to enrich nutrition for prevention of chronic diseases (Zhang et al., 2015b).

Anthocyanidins belong to the flavonoid family. In plants, they are bound to a sugar group and are known as anthocyanins. Cyanidin, pelargonidin and delphinidin are among the most common plant anthocyanidins (Pojer et al., 2013). There is also a rare class of anthocyanin analogues, which is called 3-deoxyanthocyanins. The sorghum grass family is the known

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ACCEPTED MANUSCRIPT main natural source of 3-deoxyanthocyanins. 3-deoxyanthocyanins are more stable than anthocyanidins against light, heat and pH changes and this makes them very useful as colorant for the food industry (Awika et al., 2004; Yang et al., 2009). Tricetinidin is one of

epigallocatechin gallate (Clifford, 2000; Harborne, 1966).

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the natural 3-deoxyanthocyanins, which is found in black tea as a degallation product of

Tricetinidin may also chemically be considered as an anthocyanidin with a structure that is very similar to the structure of delphinidin, only lacking one hydroxyl group. In contrast to

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delphinidin, the effects of tricetinidin have almost not been studied at all, only very few investigations are available, e. g. studying the potential to inhibit IgE receptor expression

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(Tamura et al., 2010). Delphinidin has been studied in the context of various diseases, mainly due to its antioxidant properties (Dayoub et al., 2013). In this study, we analysed the protective effect of the 3-deoxyanthocyanin tricetinidin in vitro and compared it to several well-known antioxidants including delphinidin, tetrahydrocurcumin, coenzyme Q10, vitamin

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E and C. Tetrahydrocurcumin is one of the intestinal metabolite of phenolic compound curcumin. Curcumin has been subject of research in the context of many different diseases (Hassani et al., 2015; Shanmugam et al., 2015; Tang, 2015) and colourless metabolite

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tetrahydrocurcumin is thought to have similar properties (Aggarwal et al., 2015; Ramakrishnan et al., 2017). Coenzyme Q10, which is also known as ubiquinone, is an

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important antioxidant that is discussed to be a promising strategy in the therapy of various diseases (Brandmeyer et al., 2014; Garrido-Maraver et al., 2014; Lu and Frank, 2007). Finally, trolox (water soluble analogue of vitamin E) and vitamin C are among the most familiar and well-investigated antioxidants. The aim of the present study was to investigate in normal kidney cells whether the oxidative stress and the DNA damage caused by different substances (antimycin a, patulin and insulin) can be abrogated by co-treatment with the novel natural antioxidant tricetinidin and whether

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ACCEPTED MANUSCRIPT the effectiveness of tricetinidin is comparable to that of well-established antioxidant

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substances like delphinidin, tetrahydrocurcumin, coenzyme Q10, vitamin E (trolox) and C.

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Materials and methods

2.1

Materials

Unless stated otherwise, chemicals were purchased from Sigma Aldrich Germany (Munich, Germany). Insulin was purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

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Dihydroethidium (DHE) was purchased from Merck Biosciences (Schwalbach, Germany). GelGreen Nucleic Acid Stain was purchased from Biotrend (Cologne, Germany). Tetramethylrhodamine, ethylester (TRME) and monochlorobimane (MCB) were supplied

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from Thermofischer scientific (Schwerte, Germany). Cell culture media and reagents were purchased from PAA Laboratories (Pasching, Austria) and Life Technologies (Darmstadt,

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Germany). Tricetinidin were obtained from Connect Chemicals (Ratingen, Germany). Delphinidin was obtained from Extrasynthese (Genay, France) and tetrahydrocurcumin was supplied by Connect Chemicals (Ratingen, Germany). Coenzyme Q10 and patulin were obtained from Cayman Chemical Company (Michigan, USA). The antibody against beta-actin

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was obtained from Cell signalling Technology Inc (Beverly, USA). The antibody against hemeoxygenase-1 (HO-1) was purchased from Abcam (Cambridge, UK). The secondary antibodies HRP conjugated goat anti-mouse and donkey anti-rabbit were obtained from Santa

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Cruz Biotechnology (Santa Cruz, CA, USA). The Clarity Western ECL substrate was

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purchased from Bio-Rad (Hercules, USA). Hyperfilm was obtained from GE Healthcare Life Sciences (Buckinghamshire, UK). Insulin was dissolved in HCl solution (0.05 M). Delphinidin, tricetinidin and tetrahydrocurcumin were dissolved in ethanol. Trolox and coenzyme Q10 was prepared in DMSO solution. Ascorbic acid was dissolved in water. 2.2 2.2.1

Methods Cell Culture and Treatment of Cells

NRK cells (epithelial rat kidney cells with proximal tubule properties) were grown in DMEM medium (4.5 g/l glucose) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 6

ACCEPTED MANUSCRIPT U/ml penicillin, and 100 µg/ml streptomycin. They were subcultured three times a week in order to keep them under exponential growth conditions. One day prior to experiments, NRK cells (DHE staining, micronucleus test and mitochondrial membrane potential: 3.5x105 cells/well in a 6-well plate, GSH measurement: 1x105 cells/well

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in a 12-well plate and western blot analysis: 1x106 cells/25cm2-flask) were inoculated in wells/flasks containing medium. On the following day, cells were pre-incubated with the test substances tricetinidin (T), delphinidin (Del), tetrahydrocurcumin (THC), coenzyme Q10

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(Q10), trolox and vitamin C (1, 3.2 and 10 µM) for 15 min at 37 oC. After 15 min preincubation, cells were treated with different stress inducers according to the investigated end-

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point (DHE staining: 25 µM AMA for 30 min, MN test: 10 nM insulin for 4 h, mitochondrial membrane potential: 100 nM insulin for 30 min and GSH measurement: 1 µM patulin for 24 h). To monitor the protective effect of tricetinidin, a clear stress response for each end-point was required. Therefore, a different stressor for each assay was selected according to the

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existing experiences. Oxidative Stress Analysis

The cells were seeded on a coverslip in a 6-well plate. They were incubated with the test

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substances and 10 µM DHE for 30 min at 37 °C in the dark. After washing and mounting,

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images were taken using a Nikon Eclipse 55i microscope at 200x magnification. For quantification, grey values of 5 images per sample (containing about 1100±366 cells) were measured with ImageJ. 2.2.3

Glutathione (GSH) Analysis by Plate Reader

Cells were incubated with the test substances for 24 h at 37 °C. At the end of the treatment time, cells were rinsed with 1 ml cold PBS. In order to gain a total cell lysate, cells were incubated with 1 ml cold lysis buffer (0.2 M Mannitol, 50 mM Sucrose and 0.01 M HEPES, pH 7.5) for 10 min on ice at dark. Cell were scraped and centrifuged at 12000 g, for 10 min at

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ACCEPTED MANUSCRIPT 4 oC. Supernatant was collected and divided into two aliquots. One of these aliquots was used to measure the protein concentration. The other one was used for the determination of GSH level. Ninety µl of sample was mixed with 10 µl of 1 mM monochlorobimane (MCB) dye in a 96-well plate. The 96-well plate was incubated at room temperature for an hour on a shaker to

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allow the reaction. Fluorescence intensity of the GSH-MCB complex was determined by a plate reader (Ex/Em, 385/490). Quantification of the GSH concentration was done according to a GSH standard curve (0, 1, 5, 10, 25, 50, 75 and 100 µM). GSH concentrations were given

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as normalized to protein amounts. Mitochondrial Membrane Potential

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The cells were seeded on a coverslip in a 6-well plate. They were incubated with the test substances for 30 min and for 15 min with 50 nM tetramethylrhodamine, ethyl ester (TMRE, Thermofischer scientific, T669) at 37 °C in the dark. After washing and mounting, images were taken by using a Nikon Eclipse 55i microscope at 400x magnification. For

measured with ImageJ. 2.2.5

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quantification, grey values of 5 images per sample (containing about 199±47 cells) were

Western Blot for HO-1 Expression Detection

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Following the treatment, the medium was discarded and cells were rinsed with cold PBS.

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RIPA buffer, which contained freshly added protease and phosphatase inhibitors, was added and cells were collected by scraping. Cells were damaged by pipetting 10 times through a 1 ml syringe and centrifuged at 14000 rpm, 4 oC for 30 min. After the centrifugation, supernatant was decanted and the protein concentration was measured. Thirty µg of protein was separated by reducing 12.5% (w/v) SDS-PAGE and then was electroblotted to PVDF membrane. Blocking of membranes was done in 5% (w/v) non-fat milk powder for 2 h at room temperature and incubated with primary antibodies HO-1 (ab68477) and Beta-actin (cell signalling, #3700) (1:5000 in 5 % non-fat milk powder) overnight at 4 oC. The next day, the

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ACCEPTED MANUSCRIPT membranes were incubated with HRP-conjugated secondary antibodies ((Anti-rabbit, cell signalling, #7074) and (goat anti-mouse, sc-2005)) at room temperature for an hour. Detection of antibody binding was performed with Clarity Western ECL substrate (Bio-Rad, 1705061) according to the manufacturer instructions. Chemiluminescent signals were detected by

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Hyperfilm (Amersham, 28906836). Micronucleus Test

The cells were incubated with the test substances for 4 h. Subsequently, the medium was

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removed and the cells were washed with PBS. Then, fresh medium containing 3 µg/ml cytochalasin B was added and cells were incubated for 24 h. After that, cells were harvested,

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brought onto glass slides by cytocentrifugation, and fixed in methanol (-20 °C) for a minimum of 2 h. These preparations were stained with GelGreen Nucleic Acid Stain (1% stock solution in water) and mounted for microscopy. Micronucleus (MN) analysis was done at a Nikon Eclipse TE 2000-E microscope with 400-fold magnification. The number of

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mononucleated (MoN), binucleated (BN) and multinucleated (MuN) cells was analysed in 1,000 cells per sample. The cytochalasin B proliferation index (CBPI) was calculated according to the formula CBPI = (1·MoN + 2·BN + 3·MuN) / (MoN + BN + MuN). The

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frequency of micronuclei was counted in 1,000 binucleated cells on each of two slides per

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sample. Overall, procedures were designed according to recommendations in the OECD guideline (TG 487) (OECD, 2010). 2.2.7

Statistics

Statistical analysis was performed using SPSS 22 software. Data are presented as mean ± SD of 3 independent experiments. Normality of the data was checked with Shapiro-Wilk test and the Mann-Whitney-U test was performed to determine the significance between individual groups. Results were considered significant with p≤0.05.

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ACCEPTED MANUSCRIPT 3 3.1

Results Oxidative Stress Analysis

The production of ROS was measured by analysing DHE fluorescence. An image example can be seen in Fig. 1A. The substances were tested at concentrations of 1 µM (Fig. 1B), 3.2

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µM (Fig. 1C), and 10 µM (Fig. 1D). At all tested concentrations, the substances alone did not cause any significant induction of ROS formation, whereas AMA showed a significant increase compared to control. When AMA was combined with the test substances at all

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concentrations, ROS formation was reduced, indicating a protective effect of all substances.

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Fig. 1. Detection of intracellular ROS formation induced by AMA (25 µM for 30 min). Representative pictures for DHE staining (A). Quantification of DHE fluorescence by measuring the mean grey value of DHE signal using image j software for 1 µM (B), 3.2

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µM (C) and 10 µM (D). Data are presented as mean fold change over control ± S.D. of 3

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independent experiments. *p≤0.05 vs. Control and #p≤0.05 vs. AMA. T: tricetinidin, Del: delphinidin, THC: tetrahydrocurcumin, Q10: coenzyme Q10, VitC: vitamin C and AMA: antimycin a.

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ACCEPTED MANUSCRIPT 3.2

GSH Level

The level of glutathione was measured by a spectrometric method to determine the antioxidant status in different treatment groups. The antioxidants were tested at 1 µM (Fig. 2A), 3.2 µM (Fig. 2B) and 10 µM (Fig. 2C). At 1 µM and 3.2 µM concentration all

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substances (except delphinidin with 1 µM) caused a significant increase in GSH level compared to control. At 10 µM tricetinidin, delphinidin, trolox and vitamin C showed elevated GSH levels, which was not significant due to high day to day variations. Patulin

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alone caused a significant increase in GSH level after 24 h treatment. When patulin was combined with the antioxidants, the upregulation by patulin was prevented slightly at 1 µM.

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At 3.2 µM Q10 and trolox induced a reduction compared to patulin treatment alone. Vitamin C did not lead to any significant difference in combination with patulin. At 10 µM

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concentration THC, Q10 and trolox led to significant reduction in GSH level, which was

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elevated by patulin stress (Fig. 2).

Fig. 2. Effect of antioxidant substances on cellular GSH level. Patulin (1 µM for 24 h) was used to induce cellular stress and increase cellular GSH level. Quantification of cellular GSH level for 1 µM (A), 3.2 µM (B) and 10 µM (C). Data are presented as mean fold change of GSH concentration (µM)/µg protein compared to control ± S.D. of 3 independent experiments. 13 *p≤0.05 vs. Control and #p≤0.05 vs. patulin. T: tricetinidin, Del: delphinidin, THC: tetrahydrocurcumin, Q10: coenzyme Q10 and VitC: vitamin C.

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Mitochondrial Membrane Potential

Mitochondria have a critical role in ROS formation and cellular damage induction. We used TMRE staining to demonstrate the effect of antioxidant substances on mitochondrial membrane potential. An example of TMRE stained cells can be seen in Fig. 3A. The

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antioxidants were tested at concentrations of 1 µM (Fig. 3B), 3.2 µM (Fig. 3C), and 10 µM (Fig. 3D). Insulin showed a significant reduction in mitochondrial membrane potential. The combination of antioxidant substances with insulin helped to recover the mitochondrial

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membrane potential and the protective effect against membrane depolarization was significant for all substances at dose 3.2 µM and 10 µM. Delphinidin also showed a significant effect at 1

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µM concentration compared to the insulin treated group. Some of the antioxidant substances (THC, Q10 and trolox) showed a slight reduction in membrane potential at 3.2 µM and 10

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µM concentrations alone.

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Fig. 3. Protective effect of antioxidant substances against depolarization of mitochondrial membrane potential by insulin (100 nM for 30 min). Representative pictures of TMRE staining (A). Quantification of TRME fluorescence by measuring the mean grey value of

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TMRE signal using image j for 1 µM (B), 3.2 µM (C) and 10 µM (D). Data are presented

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as mean fold change over control ± S.D. of 3 independent experiments. *p≤0.05 vs. Control

and

#p≤0.05

vs.

insulin.

T:

tricetinidin,

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tetrahydrocurcumin, Q10: coenzyme Q10 and VitC: vitamin C.

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delphinidin,

THC:

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HO-1 Expression

In order to distinguish the possible mechanism behind the antioxidant activity of the substances, HO-1 expression was analysed after 4 h treatment. The antioxidants (tricetinidin,

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delphinidin, THC and Q10) were tested at concentrations of 1 µM, 3.2 µM, and 10 µM for 4 h. Tricetinidin led to a dose dependent significant induction of HO-1 expression (Fig. 4A and B). Delphinidin already showed a significant increase at 1 µM concentration during the 4 h

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time frame (Fig. 4A and C). THC and Q10 did not show any significant effect at the tested

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concentrations (Fig. 4A, D and E).

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Fig. 4. Relative expression of HO-1 after 4 h treatment with antioxidant substances. Representative pictures of the bands from HO-1 and β-actin (A). Quantification of the bands for 1, 3.2 and 10 µM T (B), Del (C), THC (D) and Q10 (E). Data are presented as mean fold change of HO-1/β-actin over control ± S.D. of 3 independent experiments. *p≤0.05 vs. Control. T: tricetinidin, Del: delphinidin, THC: tetrahydrocurcumin and Q10: coenzyme Q10.

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ACCEPTED MANUSCRIPT 3.5

Micronucleus Test and Morphology Analysis

To investigate whether the antioxidants can provide protection against DNA damage, the micronucleus test was applied. Examples for micronucleus containing cells can be seen in Fig. 5A. The antioxidants were tested at concentrations of 1 µM (Fig. 5B), 3.2 µM (Fig. 5C),

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and 10 µM (Fig. 5D). The substances alone did not cause any micronucleus formation at the tested concentrations. Insulin alone caused a significant 3-fold increase of micronucleus number. When insulin was combined with the antioxidants, the insulin-induced micronucleus

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formation was abrogated and the micronucleus frequency was similar to when cells were only treated with the antioxidants. The proliferation index was not affected (data not shown) by

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treatment with the antioxidants, insulin, or the combinations, demonstrating that micronucleus formation was not influenced by altered proliferation. Overall, all test substances were protective against DNA damage. The same cell preparations were used for morphology analysis assessing the frequencies of apoptotic cells to investigate possible additional effect of

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insulin and/or the antioxidants. There was no significant alteration in apoptosis for any

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treatment (data not shown).

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Fig. 5. Protective effect of antioxidant substances against micronucleus induction by insulin (10 nM for 4 h). Representative pictures of micronucleus containing cells after staining with

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GelGreen, MN indicated by arrows (A). Quantification of micronucleus test for 1 µM (B), 3.2 µM (C) and 10 µM (D). Data are presented as mean of MN-containing cells in 1000

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binucleated cells ± S.D. of 3 independent experiments. *p≤0.05 vs. Control and #p≤0.05 vs. insulin. T: tricetinidin, Del: delphinidin, THC: tetrahydrocurcumin and Q10: coenzyme

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Q10.

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Discussion Tricetinidin showed in vitro antioxidant activity

The natural antioxidant tricetinidin, as well as delphinidin and tetrahydrocurcumin showed antioxidant and genome protective abilities in cultured normal rat kidney cells. The

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effectivities were comparable between these plant-derived antioxidants and, also with that of the well-known antioxidants delphinidin, THC, trolox (soluble vitamin E derivative), vitamin C, and Q10. The antioxidant potential of the anthocyanidin tricetinidin is not surprising due to

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the close structural similarity to delphinidin. However, its antioxidant properties have not been demonstrated so far in any biological system. In fact, tricetinidin has not been studied in

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depth at all. One of the few studies available investigated IgE receptor expression and found it to be inhibited by tricetinidin (Tamura et al., 2010). The anthocyanidin delphinidin has been reported to have antioxidative properties in other biological systems which is in agreement with our findings (Dayoub et al., 2013; Noda et al., 2002). The same is true for THC, a

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metabolite of curcumin, even though the amount of evidence is more limited for this compound (Manjunatha et al., 2013).

NRK cells are non-transformed cells and are thought to resemble normal, primary cells,

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which should be the target of any protective strategy directed against oxidative stress and

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DNA damage. Insulin has been shown to cause damage in kidney cells in vitro and in vivo, which makes kidney cells a suitable model for this investigation (Othman et al., 2013a). The mechanism of action of insulin, namely interaction with the insulin receptor and the IGF1 receptor, activation of the PI3K/Akt-pathway and ROS production via mitochondria and NADPH oxidases (Othman et al., 2014) is however not limited to kidney cells, but has also been observed in other cell types such as mammalian colon cells and human lymphocytes (Othman et al., 2013b). It has been shown that insulin can cause ROS formation which subsequently leads to DNA damage (Othman et al., 2014; Othman et al., 2013a; Othman et

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ACCEPTED MANUSCRIPT al., 2013b). These findings may potentially have important consequences due to the known associations between oxidative stress and DNA damage on the one hand and diseases, particularly cancer, on the other hand (Jackson and Bartek, 2009; Klaunig et al., 2010; Lord and Ashworth, 2012). Most recently, heart failure and pulmonary hypertension have been

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investigated in the context of oxidative DNA damage (Di Minno et al., 2016; Ranchoux et al., 2016).

Oxidative stress was quantified as DHE fluorescence that mainly reflects the formation of

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superoxide. As an inducer, AMA was chosen, which is known to inhibit mitochondrial chain complex III, causing release of superoxide-radicals from the mitochondria (Drose and Brandt,

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2008; Goncalves et al., 2016). Some of these antioxidants investigated here have been shown to reduce superoxide formation caused by other agents or conditions, e. g. THC reducing superoxide formation caused by L-NAME-induced hypertension (Nakmareong et al., 2012) or delphinidin for reducing superoxide formation caused by oxidized low-density lipoprotein

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(Xie et al., 2012). Regarding tricetinidin, the present study is the first evidence for this compound to reduce oxidative stress in a cellular system. Even the lowest tested concentration of the antioxidants was able to reduce the superoxide formation to the control level.

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Therefore, no dose dependent reaction was observed. In order to distinguish the possible differences in protective mechanism of antioxidants,

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cellular GSH level, mitochondrial membrane potential and expression of HO-1 were analysed. Glutathione is one of the most important antioxidant peptide in biological systems. To influence cellular GSH level, we used the mutagenic mycotoxin patulin, which is known to deplete cellular GSH (Pillay et al., 2015; Schumacher et al., 2005; Zhang et al., 2015a) afterwards resulting in a cellular compensatory upregulation. The combination of patulin with the antioxidant substances prevented the effect of patulin and led to a dose dependent reduction in GSH level. Q10 showed significant reduction against patulin induced

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ACCEPTED MANUSCRIPT compensatory upregulation in GSH at all concentrations. THC and trolox showed significant reduction in GSH level against patulin induced stress at 10 µM concentration. However, we do not know whether this effect is due to a scavenging of the very reactive patulin molecules by the antioxidants or an effect on cellular glutathione production. The antioxidant substances

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increased cellular level significantly at 1 and 3.2 µM alone compared to control, but not at 10 µM. This might explain the dose dependent reduction of GSH level within combination treatments. At 1 and 3.2 µM antioxidants might have induced cellular GSH synthesis and

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combination with patulin resulted in a mixture of direct and indirect protective mechanisms. The direct scavenging activity of the substances against patulin might become more visible at

GSH after antioxidant treatment alone.

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10 µM due to excess amount of available substance and non-significant changes in cellular

Mitochondria are one of the main cellular sources of ROS. Mitochondrial potential is an important marker for mitochondrial health. We used insulin to induce a reduction in

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mitochondrial membrane potential. All antioxidant substances increased the mitochondrial membrane potential in combination with insulin with no difference between the tested substances. Jin and his colleagues, (Jin et al., 2013), showed the protective effect of

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delphinidin against oxidized low-density lipoprotein induced depolarization of mitochondria in human umbilical vein endothelial cells (HUVECs). Seo et al., (Seo et al., 2013),

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demonstrated the protective effect of delphinidin pre-treatment against hypoxia induced mitochondrial membrane potential loss. There are two controversial studies about the effect of THC on mitochondrial membrane potential. One of them demonstrated the protective effect of THC against doxorubicin induced mitochondrial membrane potential in MMNK-1 cells (Somparn and Senggunprai, ScienceAsia) and the other showed a dose dependent reduction in mitochondrial membrane potential after 48 h treatment with THC (Kang et al., 2014).The effect of coenzyme Q10 on mitochondrial membrane potential has been shown in different

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ACCEPTED MANUSCRIPT publications with different experimental setups. One of them is the protective effect of Q10 against rotenone induced decrease in mitochondrial membrane potential of lymphocytes (CD4+and CD8+ T cells, CD19+ B cells) (Gollapudi and Gupta, 2016). Another study, (Kumari et al., 2016), demonstrated the protective effect of Q10 against glutamate induced Distelmaier and his colleagues,

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mitochondrial membrane potential loss in HT22 cells.

(Distelmaier et al., 2009), found that chronic trolox treatment (300 µM, 72 h) could restore the reduced mitochondrial membrane potential in complex-I-deficient patient fibroblasts.

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Vitamin C has been showed to restore the depolarization of mitochondrial membrane which was induced either by rotenone or by hydrogen peroxide in HL-60 cells (Gruss-Fischer and

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Fabian, 2002; Kc et al., 2005). In this study, we showed once more that in addition to tricetinidin, also delphinidin, THC, coenzyme Q10, trolox and vitamin C could restore the depolarization of mitochondrial membrane, which was induced by insulin. The effect of in vitro insulin treatment on mitochondrial membrane potential has already been shown in our

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previous study (Othman et al., 2016) .

Tricetinidin increased expression of antioxidant enzyme HO-1

After 4 h incubation, tricetinidin caused an increase in antioxidant enzyme HO-1 expression,

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when delphinidin showed a significant increase with 1, 3.2 and 10 µM. However, THC and

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Q10 did not show any impact on HO-1 expression at the tested concentrations and incubation time. HO-1 is a protective enzyme and contributes to reduction of oxidative stress and inflammatory response. Under normal conditions, HO-1 expression is low, but it can be stimulated by stress conditions like oxidative stress. HO-1 expression is mainly regulated at the transcriptional level (Gozzelino et al., 2010). Some of the antioxidant substances have been shown to stimulate HO-1 expression and thereby activate cellular antioxidant defence mechanisms. Milbury et al., (Milbury et al., 2007) found a significant increase in HO-1 expression in ARPE-19 cells after 4 h incubation with a bilberry extract. Sorrenti and his

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ACCEPTED MANUSCRIPT colleagues (Sorrenti et al., 2007) found that 24 h incubation of HIAE-101 cells with cyanidin led to a significant increase in HO-1 expression. Another study showed a time and dose dependent increase in HO-1 expression following an incubation with Chinese bayberry extract in β cells and primary islets and knockdown of HO-1 caused a decrease in the

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protective effect of antioxidants (Zhang et al., 2011). Like our findings, delphinidin has been found to increase the expression of HO-1 in mouse hepatocytes (Inoue et al., 2012). According to our knowledge, this was the only study which demonstrated an induction of

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HO-1 by delphinidin. In addition, there are many studies with different types of plant extracts, which might contain delphinidin. Pala et. Al, (Pala et al., 2016), demonstrated an increase in

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HO-1 expression in the liver, heart and muscle of Wistar rats after 6 weeks of Q10 supplement (300 mg/kg per day). Khodir and his colleagues (Khodir et al., 2017) investigated the protective effect of coenzyme Q10 against experimentally induced ulcerative colitis in rats. They showed a dose dependent increase in total serum antioxidant capacity after

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coenzyme Q10 supplement and a significant increase in HO-1 expression in colon tissue. Curcumin has been shown to induce HO-1 expression, whereas its metabolite THC did not have any effect on HO-1 expression (Jeong et al., 2009; Pae et al., 2007). In our test system,

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expression.

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tricetinidin and delphinidin were the only antioxidants, which lead to an increase in HO-1

Tricetinidin protected in vitro against DNA damage

DNA damage was quantified as micronucleus formation, which reflects chromosomal damage caused by missegregation of chromosomal fragments or whole chromosomes (Fenech, 2000, 2007). This endpoint is of particular importance due to the association found between micronucleus frequencies in human peripheral lymphocytes and cancer incidence (Bonassi et al., 2007) although it is not yet clear whether this association reflects genotoxic exposures or rather inherited genomic instability. Delphinidin has been found to protect against

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ACCEPTED MANUSCRIPT chemotherapy-induced micronucleus formation (Khandelwal and Abraham, 2014). On the other hand, delphinidin can also induce micronucleus formation at higher concentrations (Ferguson et al., 1985). Coenzyme Q10 has also been shown to reduce micronucleus formation by other agents, e. g. cisplatin (da Silva Machado et al., 2013). Regarding THC and

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tricetinidin, no reports about their effect on protection against micronucleus formation are available. Protective effects, if they also occur in vivo, may thus be limited to a certain dose range which will have to be assessed carefully before giving any recommendations. Still,

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natural antioxidants may be a useful component in a strategy for reduction of oxidative stress

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related diseases.

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Conclusion

Overall, we showed the protective effect of the novel anthocyanidin tricetinidin in normal rat kidney cells, which was comparable to that of the well-known antioxidant substances delphinidin, THC, Q10, trolox and vitamin C. Direct and indirect cellular antioxidant activity

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of delphinidin is quite well defined. However, the antioxidant activity of tricetinidin was shown in this paper for the first time in cellular systems. We were also able to show indirect protective effect of tricetinidin through the increase in HO-1 expression in cells. This might

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indicate a possible mechanistic difference in the protective effect of tricetinidin and delphinidin. It remains to be investigated further whether tricetinidin and delphinidin

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scavenges reactive molecules or stimulates cellular defence molecules or both. Due to its higher stability, tricetinidin might be a good candidate for development as an antioxidant supplement. Further research is necessary to show possible therapeutic and preventive effects

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of tricetinidin and delphinidin in vivo.

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ACCEPTED MANUSCRIPT Acknowledgement Technical assistance by Silvana Wunram, Jonas Czimprich and Elizabeth Stein is greatly appreciated. Competing interests statement

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The authors report no conflict of interest.

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References

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Aggarwal, B.B., Deb, L., Prasad, S., 2015. Curcumin differs from tetrahydrocurcumin for molecular targets, signaling pathways and cellular responses. Molecules 20, 185205. Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proceedings of the National Academy of Sciences of the United States of America 90, 7915-7922. Awika, J.M., Rooney, L.W., Waniska, R.D., 2004. Properties of 3-Deoxyanthocyanins from Sorghum. Journal of agricultural and food chemistry 52, 4388-4394. Beckman, J.S., Koppenol, W.H., 1996. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. The American journal of physiology 271, C1424-1437. Bonassi, S., Znaor, A., Ceppi, M., Lando, C., Chang, W.P., Holland, N., KirschVolders, M., Zeiger, E., Ban, S., Barale, R., Bigatti, M.P., Bolognesi, C., CebulskaWasilewska, A., Fabianova, E., Fucic, A., Hagmar, L., Joksic, G., Martelli, A., Migliore, L., Mirkova, E., Scarfi, M.R., Zijno, A., Norppa, H., Fenech, M., 2007. An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans. Carcinogenesis 28, 625-631. Brandmeyer, E.A., Shen, Q., Thimmesch, A.R., Pierce, J.D., 2014. Using coenzyme Q10 in clinical practice. Nursing 44, 63-66. Clifford, M.N., 2000. Anthocyanins – nature, occurrence and dietary burden. Journal of the Science of Food and Agriculture 80, 1063-1072. da Silva Machado, C., Mendonca, L.M., Venancio, V.P., Bianchi, M.L., Antunes, L.M., 2013. Coenzyme Q10 protects Pc12 cells from cisplatin-induced DNA damage and neurotoxicity. Neurotoxicology 36, 10-16. Dayoub, O., Andriantsitohaina, R., Clere, N., 2013. Pleiotropic beneficial effects of epigallocatechin gallate, quercetin and delphinidin on cardiovascular diseases associated with endothelial dysfunction. Cardiovasc Hematol Agents Med Chem 11, 249-264. Di Minno, A., Turnu, L., Porro, B., Squellerio, I., Cavalca, V., Tremoli, E., Di Minno, M.N., 2016. 8-Hydroxy-2-deoxyguanosine levels and heart failure: A systematic review and meta-analysis of the literature. Nutrition, metabolism, and cardiovascular diseases : NMCD. Distelmaier, F., Visch, H.-J., Smeitink, J.A.M., Mayatepek, E., Koopman, W.J.H., Willems, P.H.G.M., 2009. The antioxidant Trolox restores mitochondrial membrane potential and Ca(2+)-stimulated ATP production in human complex I deficiency. Journal of Molecular Medicine (Berlin, Germany) 87, 515-522. Drose, S., Brandt, U., 2008. The mechanism of mitochondrial superoxide production by the cytochrome bc1 complex. The Journal of biological chemistry 283, 2164921654. Fenech, M., 2000. The in vitro micronucleus technique. Mutat Res 455, 81-95. Fenech, M., 2007. Cytokinesis-block micronucleus cytome assay. Nature protocols 2, 1084-1104. Ferguson, L.R., van Zijl, P., Holloway, W.D., Jones, W.T., 1985. Condensed tannins induce micronuclei in cultured V79 Chinese hamster cells. Mutat Res 158, 89-95. Garrido-Maraver, J., Cordero, M.D., Oropesa-Avila, M., Vega, A.F., de la Mata, M., Pavon, A.D., Alcocer-Gomez, E., Calero, C.P., Paz, M.V., Alanis, M., de Lavera, I., Cotan, D., Sanchez-Alcazar, J.A., 2014. Clinical applications of coenzyme Q10. Front Biosci (Landmark Ed) 19, 619-633.

28

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Gollapudi, S., Gupta, S., 2016. Reversal of oxidative stress-induced apoptosis in T and B lymphocytes by Coenzyme Q10 (CoQ10). American Journal of Clinical and Experimental Immunology 5, 41-47. Goncalves, R.L.S., Bunik, V.I., Brand, M.D., 2016. Production of superoxide/hydrogen peroxide by the mitochondrial 2-oxoadipate dehydrogenase complex. Free Radical Biology and Medicine 91, 247-255. Gozzelino, R., Jeney, V., Soares, M.P., 2010. Mechanisms of cell protection by heme oxygenase-1. Annual review of pharmacology and toxicology 50, 323-354. Gruss-Fischer, T., Fabian, I., 2002. Protection by ascorbic acid from denaturation and release of cytochrome c, alteration of mitochondrial membrane potential and activation of multiple caspases induced by H2O2, in human leukemia cells. Biochemical pharmacology 63, 1325-1335. Harborne, J.B., 1966. Comparative biochemistry of flavonoids—II.: 3Desoxyanthocyanins and their systematic distribution in ferns and gesnerads. Phytochemistry 5, 589-600. Hassani, S., Sepand, M.R., Jafari, A., Jaafari, J., Rezaee, R., Zeinali, M., Tavakoli, F., Razavi-Azarkhiavi, K., 2015. Protective effects of curcumin and vitamin E against chlorpyrifos-induced lung oxidative damage. Hum Exp Toxicol 34, 668-676. Inoue, H., Maeda-Yamamoto, M., Nesumi, A., Murakami, A., 2012. Delphinidin-3-Ogalactoside protects mouse hepatocytes from (-)-epigallocatechin-3-gallate-induced cytotoxicity via up-regulation of heme oxygenase-1 and heat shock protein 70. Nutrition research (New York, N.Y.) 32, 357-364. Jackson, S.P., Bartek, J., 2009. The DNA-damage response in human biology and disease. Nature 461, 1071-1078. Jeong, S.-O., Oh, G.-S., Ha, H.-Y., Soon Koo, B., Sung Kim, H., Kim, Y.-C., Kim, E.C., Lee, K.-M., Chung, H.-T., Pae, H.-O., 2009. Dimethoxycurcumin, a Synthetic Curcumin Analogue, Induces Heme Oxygenase-1 Expression through Nrf2 Activation in RAW264.7 Macrophages. Journal of clinical biochemistry and nutrition 44, 79-84. Jin, X., Yi, L., Chen, M.-l., Chen, C.-y., Chang, H., Zhang, T., Wang, L., Zhu, J.-d., Zhang, Q.-y., Mi, M.-t., 2013. Delphinidin-3-Glucoside Protects against Oxidized LowDensity Lipoprotein-Induced Mitochondrial Dysfunction in Vascular Endothelial Cells via the Sodium-Dependent Glucose Transporter SGLT1. PloS one 8, e68617. Kang, N., Wang, M.-M., Wang, Y.-H., Zhang, Z.-N., Cao, H.-R., Lv, Y.-H., Yang, Y., Fan, P.-H., Qiu, F., Gao, X.-M., 2014. Tetrahydrocurcumin induces G2/M cell cycle arrest and apoptosis involving p38 MAPK activation in human breast cancer cells. Food and Chemical Toxicology 67, 193-200. Kc, S., Carcamo, J.M., Golde, D.W., 2005. Vitamin C enters mitochondria via facilitative glucose transporter 1 (Glut1) and confers mitochondrial protection against oxidative injury. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 19, 1657-1667. Khandelwal, N., Abraham, S.K., 2014. Protective effects of common anthocyanidins against genotoxic damage induced by chemotherapeutic drugs in mice. Planta Med 80, 1278-1283. Khodir, A.E., Atef, H., Said, E., ElKashef, H.A., Salem, H.A., 2017. Implication of Nrf2/HO-1 pathway in the coloprotective effect of coenzyme Q10 against experimentally induced ulcerative colitis. Inflammopharmacology 25, 119-135. Klaunig, J.E., Kamendulis, L.M., Hocevar, B.A., 2010. Oxidative stress and oxidative damage in carcinogenesis. Toxicol Pathol 38, 96-109. Kumari, S., Mehta, S.L., Milledge, G.Z., Huang, X., Li, H., Li, P.A., 2016. Ubisol-Q10 Prevents Glutamate-Induced Cell Death by Blocking Mitochondrial Fragmentation 29

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

and Permeability Transition Pore Opening. International Journal of Biological Sciences 12, 688-700. Lord, C.J., Ashworth, A., 2012. The DNA damage response and cancer therapy. Nature 481, 287-294. Lu, J., Frank, E.L., 2007. Measurement of coenzyme Q10 in clinical practice. Clin Chim Acta 384, 180-181. Lunec, J., Holloway, K.A., Cooke, M.S., Faux, S., Griffiths, H.R., Evans, M.D., 2002. Urinary 8-oxo-2'-deoxyguanosine: redox regulation of DNA repair in vivo? Free Radic Biol Med 33, 875-885. Manjunatha, J.R., Bettadaiah, B.K., Negi, P.S., Srinivas, P., 2013. Synthesis of quinoline derivatives of tetrahydrocurcumin and zingerone and evaluation of their antioxidant and antibacterial attributes. Food chemistry 136, 650-658. Milbury, P.E., Graf, B., Curran-Celentano, J.M., Blumberg, J.B., 2007. Bilberry (Vaccinium myrtillus) anthocyanins modulate heme oxygenase-1 and glutathione Stransferase-pi expression in ARPE-19 cells. Investigative ophthalmology & visual science 48, 2343-2349. Nakmareong, S., Kukongviriyapan, U., Pakdeechote, P., Kukongviriyapan, V., Kongyingyoes, B., Donpunha, W., Prachaney, P., Phisalaphong, C., 2012. Tetrahydrocurcumin alleviates hypertension, aortic stiffening and oxidative stress in rats with nitric oxide deficiency. Hypertens Res 35, 418-425. Noda, Y., Kaneyuki, T., Mori, A., Packer, L., 2002. Antioxidant activities of pomegranate fruit extract and its anthocyanidins: delphinidin, cyanidin, and pelargonidin. J Agric Food Chem 50, 166-171. OECD, 2010. Test No. 487: In Vitro Mammalian Cell Micronucleus Test. Othman, E.M., Hintzsche, H., Stopper, H., 2014. Signaling steps in the induction of genomic damage by insulin in colon and kidney cells. Free radical biology & medicine 68, 247-257. Othman, E.M., Kreissl, M.C., Kaiser, F.R., Arias-Loza, P.A., Stopper, H., 2013a. Insulin-mediated oxidative stress and DNA damage in LLC-PK1 pig kidney cell line, female rat primary kidney cells, and male ZDF rat kidneys in vivo. Endocrinology 154, 1434-1443. Othman, E.M., Leyh, A., Stopper, H., 2013b. Insulin mediated DNA damage in mammalian colon cells and human lymphocytes in vitro. Mutat Res. Othman, E.M., Oli, R.G., Arias-Loza, P.A., Kreissl, M.C., Stopper, H., 2016. Metformin Protects Kidney Cells From Insulin-Mediated Genotoxicity In Vitro and in Male Zucker Diabetic Fatty Rats. Endocrinology 157, 548-559. Pae, H.O., Jeong, G.S., Jeong, S.O., Kim, H.S., Kim, S.A., Kim, Y.C., Yoo, S.J., Kim, H.D., Chung, H.T., 2007. Roles of heme oxygenase-1 in curcumin-induced growth inhibition in rat smooth muscle cells. Experimental & molecular medicine 39, 267277. Pala, R., Orhan, C., Tuzcu, M., Sahin, N., Ali, S., Cinar, V., Atalay, M., Sahin, K., 2016. Coenzyme Q10 Supplementation Modulates NFκB and Nrf2 Pathways in Exercise Training. Journal of Sports Science & Medicine 15, 196-203. Pillay, Y., Phulukdaree, A., Nagiah, S., Chuturgoon, A.A., 2015. Patulin triggers NRF2-mediated survival mechanisms in kidney cells. Toxicon 99, 1-5. Pisoschi, A.M., Pop, A., 2015. The role of antioxidants in the chemistry of oxidative stress: A review. European Journal of Medicinal Chemistry 97, 55-74. Pojer, E., Mattivi, F., Johnson, D., Stockley, C.S., 2013. The Case for Anthocyanin Consumption to Promote Human Health: A Review. Comprehensive Reviews in Food Science and Food Safety 12, 483-508. 30

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Ramakrishnan, R., Elangovan, P., Pari, L., 2017. Protective Role of Tetrahydrocurcumin: an Active Polyphenolic Curcuminoid on CadmiumInducedOxidative Damage in Rats. Applied biochemistry and biotechnology, 1-19. Ranchoux, B., Meloche, J., Paulin, R., Boucherat, O., Provencher, S., Bonnet, S., 2016. DNA Damage and Pulmonary Hypertension. Int J Mol Sci 17. Ruperez, A.I., Gil, A., Aguilera, C.M., 2014. Genetics of oxidative stress in obesity. Int J Mol Sci 15, 3118-3144. Schumacher, D.M., Metzler, M., Lehmann, L., 2005. Mutagenicity of the mycotoxin patulin in cultured Chinese hamster V79 cells, and its modulation by intracellular glutathione. Archives of toxicology 79, 110-121. Seo, B.N., Ryu, J.M., Yun, S.P., Jeon, J.H., Park, S.S., Oh, K.B., Park, J.-K., Han, H.J., 2013. Delphinidin prevents hypoxia-induced mouse embryonic stem cell apoptosis through reduction of intracellular reactive oxygen species-mediated activation of JNK and NF-κB, and Akt inhibition. Apoptosis : an international journal on programmed cell death 18, 811-824. Shanmugam, M.K., Rane, G., Kanchi, M.M., Arfuso, F., Chinnathambi, A., Zayed, M.E., Alharbi, S.A., Tan, B.K., Kumar, A.P., Sethi, G., 2015. The multifaceted role of curcumin in cancer prevention and treatment. Molecules 20, 2728-2769. Sorrenti, V., Mazza, F., Campisi, A., Di Giacomo, C., Acquaviva, R., Vanella, L., Galvano, F., 2007. Heme oxygenase induction by cyanidin-3-O-β-glucoside in cultured human endothelial cells. Molecular Nutrition & Food Research 51, 580-586. Tamura, S., Yoshihira, K., Fujiwara, K., Murakami, N., 2010. New inhibitors for expression of IgE receptor on human mast cell. Bioorganic & medicinal chemistry letters 20, 2299-2302. Tang, Y., 2015. Curcumin targets multiple pathways to halt hepatic stellate cell activation: updated mechanisms in vitro and in vivo. Dig Dis Sci 60, 1554-1564. Taniyama, Y., Griendling, K.K., 2003. Reactive oxygen species in the vasculature: molecular and cellular mechanisms. Hypertension 42, 1075-1081. Xie, X., Zhao, R., Shen, G.X., 2012. Influence of delphinidin-3-glucoside on oxidized low-density lipoprotein-induced oxidative stress and apoptosis in cultured endothelial cells. J Agric Food Chem 60, 1850-1856. Yang, L., Browning, J.D., Awika, J.M., 2009. Sorghum 3-deoxyanthocyanins possess strong phase II enzyme inducer activity and cancer cell growth inhibition properties. Journal of agricultural and food chemistry 57, 1797-1804. Zhang, B., Kang, M., Xie, Q., Xu, B., Sun, C., Chen, K., Wu, Y., 2011. Anthocyanins from Chinese bayberry extract protect beta cells from oxidative stress-mediated injury via HO-1 upregulation. Journal of agricultural and food chemistry 59, 537-545. Zhang, B., Peng, X., Li, G., Xu, Y., Xia, X., Wang, Q., 2015a. Oxidative stress is involved in Patulin induced apoptosis in HEK293 cells. Toxicon 94, 1-7. Zhang, Y.-J., Gan, R.-Y., Li, S., Zhou, Y., Li, A.-N., Xu, D.-P., Li, H.-B., 2015b. Antioxidant Phytochemicals for the Prevention and Treatment of Chronic Diseases. Molecules 20, 19753.

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