A Diet Rich in Olive Oil Phenolics Reduces Oxidative
Stress in the Heart of SAMP8 Mice by Induction
of Nrf2-Dependent Gene Expression
Banu Bayram,1,2Beraat Ozcelik,2Stefanie Grimm,3Thomas Roeder,4Charlotte Schrader,1
Insa M.A. Ernst,1Anika E. Wagner,1Tilman Grune,3Jan Frank,5and Gerald Rimbach1
A Mediterranean diet rich in olive oil has been associated with health benefits in humans. It is unclear if and to
what extent olive oil phenolics may mediate these health benefits. In this study, we fed senescence-accelerated
mouse-prone 8 (SAMP8, n=11 per group) semisynthetic diets with 10% olive oil containing either high (HP) or
low amounts of olive oil phenolics (LP) for 4.5 months. Mice consuming the HP diet had significantly lower
concentrations of the oxidative damage markers thiobarbituric acid–reactive substances and protein carbonyls in
the heart, whereas proteasomal activity was similar in both groups. Nrf2-dependent gene expression may be
impaired during the aging process. Therefore, we measured Nrf2 and its target genes glutathione-S-transferase
(GST), c-glutamyl cysteine synthetase (c-GCS), nicotinamide adenine dinucleotide phosphate [NAD(P)H]:qui-
none oxidoreductase (NQO1), and paraoxonase-2 (PON2) in the hearts of these mice. Nrf2 as well as GST, c-
GCS, NQO1, and PON2 mRNA levels were significantly higher in heart tissue of the HP as compared to the LP
group. The HP-fed mice had significantly higher PON1 activity in serum compared to those receiving the LP
diet. Furthermore, HP feeding increased relative SIRT1 mRNA levels. Additional mechanistic cell culture ex-
periments were performed, and they suggest that the olive oil phenolic hydroxytyrosol present in the HP oil may
be responsible for the induction of Nrf2-dependent gene expression and the increase in PON activity. In con-
clusion, a diet rich in olive oil phenolics may prevent oxidative stress in the heart of SAMP8 mice by modulating
Nrf2-dependent gene expression.
tion of a so-called Mediterranean diet.1–3Despite the scarcity
of clinical studies investigating the underlying mechanisms,
beneficial effects of the Mediterranean diet have partly been
attributed to the use of olive oil.4–7Olive oil is a rich source of
oleic acid.8However, it has been suggested that the benefi-
cial effects of its consumption in the prevention of chronic
diseases may not only be attributed to its fatty acid compo-
sition but also to its high content of phenolics.9–11Studies in
cultured cells12–19and laboratory animals20–23have demon-
strated that the olive oil phenolics tyrosol, hydroxytyrosol,
and oleuropein exert antioxidant, antiinflammatory and gene
pidemiological studies suggest an inverse relation-
ship between coronary heart disease and the consump-
Rodent models may be used to understand the cellular
and molecular mechanisms of age-dependent degeneration
and to develop dietary interventions for healthy aging.24It
has been previously shown that biomarkers of oxidative
stress are elevated in the heart tissue of the senescence-
accelerated mouse-prone 8 (SAMP8) mouse as compared to
the senescence-accelerated mouse-resistant strain.25,26Fur-
thermore, SAMP8 mice may exhibit elevated biomarkers of
lipid27–29and protein oxidation,30–32mitochondrial dys-
function,29,33and early onset of atherogenesis,34all of which
ultimately lead to a decreased life span in this mouse strain.35
These properties render SAMP8 mice a suitable rodent
model for experimental aging research.35
The transcription factor Nrf2 is an important molecular
switch that orchestrates the gene expression of antioxidant
and phase 2 enzymes.36,37Nrf2 is a basic leucine zipper
1Institute of Human Nutrition and Food Science, Christian-Albrechts-University, Kiel, Germany.
2Department of Food Engineering, Istanbul Technical University, Istanbul, Turkey.
3Institute of Nutrition, Department of Nutritional Toxicology, Friedrich Schiller University, Jena, Germany.
4Institute of Zoology, Christian-Albrechts-University, Kiel, Germany.
5Institute of Biological Chemistry and Nutrition, University of Hohenheim, Stuttgart, Germany.
Volume 15, Number 1, 2012
ª Mary Ann Liebert, Inc.
transcription factor that binds to the antioxidant response
element (ARE) in the promoter region of many adaptive
genes, such glutathione-S-transferase (GST, EC 18.104.22.168),
c-glutamyl cysteine synthetase (c-GCS; EC 22.214.171.124), and
NAD(P)H:quinone oxidoreductase 1 (NQO1; EC 126.96.36.199).38
Under basal conditions, Nrf2 is bound to Keap1 in the cy-
tosol. Upon activation, Nrf2 is released and then translocates
into the nucleus, where it heterodimerizes, binds to the ARE,
and subsequently increases Nrf2 target gene expression.39
The PON1 gene is mainly expressed in the liver and, albeit
to a lower extent, also in the heart.40,41Paraoxonase-1
(PON1) circulates in the blood bound to high-density lipo-
protein (HDL) and prevents or delays the oxidation of
low-density lipoprotein (LDL). High PON1 activity may
be associated with a reduced cardiovascular disease risk.
Paraoxonase-2 (PON2) is expressed ubiquitously42and PON2
is primarily localized in the plasma membrane, where it
prevents cell-mediated lipid peroxidation.43PON1 has been
suggested as a longevity gene due to its modulation of car-
diovascular disease risk.44However, in a recently conducted
meta-analysis, no effects or only population-specific effects of
PON1 on human longevity were found.45
It has been shown that hydroxytyrosol induces antioxidant/
detoxificant enzymes and Nrf2 translocation in hepatocytes.46
Furthermore, hydroxytyrosol-induced cytoprotection against
oxidative injury in vascular endothelial cells via Nrf2-
dependent signal transduction pathway.23Little is known
about the role of olive oil phenolics on Nrf2-dependent gene
expression in vivo. Furthermore, activation of SIRT1 by
polyphenols may be beneficial for controlling of oxidative
stress, cellular senescence, and metabolism.47
Overall, aging seems to be associated with changes in the
oxidant/antioxidant equilibrium, impaired Nrf2 signaling
and phase 2 response, and decreased SIRT1 activity. There-
fore, the aim of the present feeding study in SAMP8 mice
was to answer the question of whether a diet rich in olive oil
phenolics may affect age-related changes in heart oxidant/
antioxidant status, Nrf2-dependent gene expression, and
Materials and Methods
Chemicals and reagents
High-performance liquid chromatography (HPLC)-grade
methanol was obtained from J.T. Baker (Deventer, The
Netherlands), and acetonitrile was obtained from Sigma
Aldrich (Steinheim, Germany). Standards of tyrosol (CAS no.
501-94-0), vanillic acid (CAS no. 121-34-6), caffeic acid (CAS
no. 331-39-5), p-coumaric acid (CAS no. 501-98-4), and ferulic
acid (CAS no. 1135-24-6) were purchased from Sigma Aldrich
(Steinheim, Germany). Oleuropein (CAS no. 32619-42-4) and
hydroxytyrosol (CAS no. 10597-60-1) were supplied by Ex-
trasynthese (Genay Cedex, France) and pinoresinol by Se-
paration Research (Turku, Finland). Reference standards of
all analyzed compounds were HPLC grade, with purity
higher than 98% (except oleuropein, >90%). Stock solutions
were prepared in methanol and stored at -20?C. HPLC
grade 60% perchloric acid was obtained from Fisher Scientific
For the protein carbonyl quantification, potassium chlo-
ride, monopotassium phosphate, sodium chloride, sodium
phosphate dibasic dihydrate, disodium phosphate, mono-
sodium phosphate and guanidine hydrochloride, citric acid,
sulfuric acid, hydrogen chloride, sodium hydroxide, bovine
serum albumin, and Roti Quant?were purchased from
Carl Roth GmbH (Karlsruhe, Germany). Tween 20, 2,4-
dinitrophenylhydrazine, biotin-conjugated rabbit immuno-
globulin G (IgG) polyclonal antibody raised against a
dinitrophenol (DNP) conjugate of keyhole limpet hemocya-
nin (anti-DNP), streptavidin, biotinylated horseradish per-
oxidase, and o-phenylenediamine were from Sigma-Aldrich
Chemie GmbH (Steinheim, Germany). Hydrogen peroxide
(H2O2) was obtained from Merck KGaA (Darmstadt, Germany).
Spectrophotometric determination of total phenols
and HPLC analysis of olive oil phenolics
The total phenolic content of the extracts was determined
according to the Folin–Ciocalteu spectrophotometric meth-
od.48Diluted Folin–Ciocalteu reagent and 20% sodium car-
bonate solution were added to the phenolic extract. The
solution was left in the dark for 45min. The absorbance of
each sample as well as of gallic acid standards was deter-
mined at 750nm. The results were expressed as mg of gallic
acid/kg of oil. HPLC analyses of olive oil phenolics were
carried out on a Jasco system (Jasco GmbH Deutschland,
Gross-Umstadt, Germany) consisting of a pump (PU-2085)
and autosampler (XLC-3059AS), and detection was carried
out on an eight-channel ESA 5600A CoulArray Detector with
integrated column oven (ESA Inc., Chelmsford, MA). Se-
paration was achieved by gradient elution with water (pH
3.1), methanol, and acetonitrile (all containing 60mM Li-
ClO4) as mobile phases on a Kinetex Luna C18 column
(100·4.6mm, 2.6lm, Phenomenex, USA), and analytes were
quantified against authentic compounds as external stan-
Ferric reducing ability of plasma and Trolox
equivalent antioxidant capacity assays
The ferric reducing ability of plasma (FRAP) assay was
conducted according to Benzie and Strain,49using ascorbic
acid as a reference. The Trolox equivalent antioxidant ca-
pacity (TEAC) assay was conducted as described by Re
et al.,50using Trolox as a reference.
Animals and study design
The animal experiment was performed according to Ger-
man animal welfare laws and regulations and with permis-
sion of the Ministry of Agriculture, Environment and Rural
Areas of the state of Schleswig-Holstein (Germany). Twenty-
two female SAMP8 mice, aged 9–10 weeks, were obtained
from Harlan Winkelmann GmbH (Borchen, Germany). The
mice were housed in groups (3–5 animals per cage) in type II
polypropylene cages equipped with soft wood bedding, a
water bottle, a mouse house, and a table tennis ball in a
climate-controlled room (temperature, 22–2?C; humidity,
55%–5%) with a 12-hr light/dark cycle.
Female mice were used because they can be housed in
groups, which we considered superior to individual housing
for an experimental period of 4.5 months. One of the SAMP8
mice died of natural causes during the experiment. Mice
were randomly divided into two groups of 11 animals each
and fed with a pelletized Western-type diet (Altromin
72BAYRAM ET AL.
Spezialfutter GmbH & Co. KG, Lage, Germany) with 0.15%
cholesterol and 20% fat, in which 10% of fat was from olive
oil containing either low (44mg gallic acid/kg oil.) or high
(532mg gallic acid/kg oil; Fratelli Ferrara s.a.s, Italy)
amounts of phenolics. Both oils contained 74%–75% oleic
acid and 11% palmitic acid as determined by gas chroma-
tography (LUFA-ITL GmbH, Kiel, Germany, DGF C VI 10a/
11a). All animals had free access to feed and water. Feed
intake was controlled daily, and body weights were mea-
sured weekly during the 4.5-month feeding trial. At the end
of the experiment, the mice were starved overnight before
anesthesia by CO2and decapitation. Blood samples were
collected in tubes and serum was separated by centrifugation
(Eppendorf 5804 R, Rotor F34-6-38, Wesseling-Berzdorf,
Germany). Tissues were excised, snap-frozen in liquid ni-
trogen, and stored at -80?C until analyzed.
Preparation of heart tissue homogenates
Heart tissue homogenates were prepared from &20mg of
tissue in 250lL of ice-cold phosphate-buffered saline (PBS;
pH 7.4) at 26,000rpm with a Miccra D-8 homogenizer (ART
Prozess- & Labortechnik GmbH & Co. KG, Mullheim, Ger-
many). Homogenates were centrifuged at 4,800·g at 4?C
for 10min (Eppendorf 5804 R, Rotor F34-6-38, Wesseling-
Berzdorf, Germany), and the supernatant was stored at
-80?C until further use.
Lipid peroxidation in heart homogenates was assayed
fluorometrically as thiobarbituric acid-reactive substances
(TBARS) with a Tecan Infinite 200 microplate reader (Tecan
Group Ltd., Crailsheim, Germany) after protein precipitation
with trichloroacetic acid (TCA) and extraction in 1-butanol.
Excitation and emission wavelengths were 520nm and
560nm, respectively. A calibration curve was prepared with
1,1,3,3-tetraethoxypropane (TEP) as an external standard.51
Quantification of protein carbonyls
Protein carbonyl content was determined in the homoge-
nized heart tissue supernatant according to the enzyme-
linked immunosorbent assay (ELISA) method of Buss et al.52
with required modifications. The detection system used an
anti-dinitrophenyl rabbit IgG-antiserum (Sigma Aldrich,
Steinheim, Germany) as the primary antibody and a mono-
clonal antirabbit IgG antibody peroxidase conjugate (Sigma
Aldrich, Steinheim, Germany) as the secondary antibody.
Color change was induced with o-phenylenediamine and
Tissue (20–40mg) was homogenized in lysis buffer
(250mM sucrose, 25mM HEPES, 10mM magnesium chlo-
ride, 1mmol/L EDTA, and 1.7mmol/L dithiothreitol [DTT])
using a homogenizer (Ultra-Turrax?) and then centrifuged at
14,000·g for 30min. The supernatant was used for deter-
mination of protein content using the Bradford assay and for
measurement of the proteasomal activity. For proteasomal
activity, samples were incubated in 225mmol/L Tris buffer
(pH 7.8), 45mmol/L potassium chloride, 7.5mmol/L mag-
nesium acetate, 7.5mmol/L magnesium chloride, and
1mmol/L DTT. For the peptidyl-glutamyl like-(b1), trypsin
like-(b2), and chymotrypsin like-(b5) activity the substrates
Z-Leu-Leu-Glu-MCA (Biochem, Boston, MA), Ac-Arg-Leu-
Arg-MCA (Biochem, Boston, MA), and N-succinyl-Leu-Leu-
Val-Tyr-MCA (Sigma Aldrich, Steinheim, Germany) were
used, respectively. MCA liberation of the substrates was
measured with a fluorescence reader at 360nm excitation
and 460nm emmission. Free MCA was used as a standard.53
PON1 arylesterase activity in serum
Arylesterase activity was measured by using phenylace-
tate as an artificial substrate for PON1. Initial rates of hy-
drolysis were determined spectrophotometrically at 270nm.
The assay mixture included 4mmol/L of phenylacetate and
1mmol/L of CaCl2 in 20mmol/L of Tris HCl, pH 8.0.
Nonenzymatic hydrolysis of phenylacetate was subtracted
from the total rate of hydrolysis. One unit of arylesterase
activity is equal to 1lmol of phenylacetate hydrolyzed per
minute per milliliter.54
RNA isolation and real-time quantitative RT-PCR
RNA was isolated from heart samples (20–30mg) using
TRIsure lysis reagent (Bioline, Luckenwalde, Germany).
Real-time quantitative PCR was performed as a one-step
procedure (SensiMix One-step Kit; Quantace, Berlin, Ger-
many) with SYBR Green detection using a Rotorgene cycler
(Corbett Life Science, Sydney, Australia). Relative messenger
RNA (mRNA) concentrations of genes were quantified by
the use of a standard curve. Target gene mRNA concentra-
tion was normalized to the mRNA concentration of the
housekeeping gene glyceraldehyde 3-phosphate dehydro-
genase (GAPDH). Primers were designed by standard tools
(Spidey, Primer3 and National Center for Biotechnology In-
formation [NCBI Blast]) and purchased from MWG (Ebers-
berg, Germany). Primer sequences for analyzed genes are
summarized in Table 1.
NIH 3T3 fibroblasts (German collection of microorganisms
and cell cultures, Braunschweig, Germany) were maintained
in Dulbecco modified Eagle medium (DMEM) containing
4.5g/L glucose, 4mmol/L L-glutamine, 1mmol/L sodium
pyruvate, 10% fetal calf serum (FCS), 100U/mL penicillin
and 100lg/mL streptomycin (PAA, Coelbe, Germany). Cells
were grown in 5% CO2at 37?C under a humidified atmo-
sphere. NIH 3T3 cells comprise a nontransformed standard
fibroblast cell line that exhibits robust growth patterns and
are easily transfected, thereby delivering reliable reporter
gene data. Furthermore, we have used this cell line because
of its murine origin to be consistent with our mouse study.
Although NIH 3T3 cells do not fully reflect the cellular me-
tabolism of cardiomyocytes, they comprise a valuable ex-
perimental tool to screen for Nrf2-inducing activity of plant
All cell culture plasticware was purchased from Sarstedt
(Nuembrecht, Germany) unless otherwise stated. For cell
culture experiments, the olive oil phenolics were dissolved in
dimethylsulfoxide (DMSO; Carl Roth, Karlsruhe, Germany),
and stock solutions were stored at -80?C until usage.
NRF2 INDUCTION BY OLIVE OIL PHENOLICS73
Vehicle controls have been performed, and the solvents did
not affect any of the parameters measured.
Cytotoxicity was determined via the Neutral Red assay.56
The Neutral Red assay is based on the pH-dependent ac-
cumulation of Neutral Red in the lysosomes of viable
cells.56NIH 3T3 cells were seeded in 24-well plates (Fisher
Scientific, Schwerte, Germany) at a density of 50,000 cells/
well, precultured for 24hr, and treated with the olive oil
phenolics for 24hr, respectively. In brief, the culture me-
dium containing the olive oil phenolics was replaced with
fresh serum-containing medium including 50lg/mL of
Neutral Red (Carl Roth). After incubation for 3hr, the
medium was removed and the cells were extracted using a
solution comprising 50:49:1 (vol/vol/vol) ethanol, water,
and glacial acetic acid. The absorbance was measured in
a plate reader (Labsystems, Helsinki, Finland) at 540nm.
For all subsequent experiments, noncytotoxic concentrations
Nrf2 reporter gene assay
NIH 3T3 cells were grown to 60%–80% confluence in 24-
well plates for 24hr. The cells were transiently transfected
with an expression vector containing a 25-bp oligonucleotide
derived from the promoter region of the gastrointestinal
glutathione peroxidase 2 comprising the conserved ARE-
motif and the reporter gene firefly luciferase (pARE_GIGPx)
kindly provided by A. Banning and R. Brigelius-Flohe ´ (DIfE,
Potsdam-Rehbruecke, Germany)57and a normalization vec-
tor phRL-TK (Promega, Mannheim, Germany) containing
the Renilla reniformis luciferase gene. Transfection was per-
formed using JetPEI transfection reagent (Polyplus transfec-
tion, Illkirch Cedex, France) according to manufacturer’s
instructions. Following 24hr of transfection, cells were
incubated with the test compounds for 24hr in serum-
containing medium. Subsequently, cells were lysed, and lu-
ciferase activity was measured using the Dual-Luciferase
reporter gene assay system (Promega) according to the
manufacturer’s protocol in a Tecan Infinite 200 microplate
reader (Tecan Group Ltd, Crailsheim, Germany).
Nrf2 western blot
For Nrf2 detection in nucleic fractions, NIH 3T3 cells were
treated with test compounds for 6hr. Subsequently, cells
were washed with ice-cold PBS, scraped off, and centrifuged.
For whole-cell extracts, the remaining cell pellet was stored
at -80?C, whereas nuclear extracts were prepared subse-
quently. Samples for western blotting were prepared as de-
scribed in Wagner et al.58A quantity of 30lg of protein of
each sample were mixed with loading buffer, incubated at
95?C for 5min and separated on a 12% sodium dodecyl
sulfate polyacrylamide gel. Subsequently, the samples were
transferred onto a polyvinylidene fluoride membrane and
blocked with 3% (wt/vol) skim milk dissolved in Tris-
buffered saline plus 0.05% (vol/vol) Tween 20 for at least
2hr and probed with the respective antibody against Nrf2
(Santa Cruz; 1:200) or TATA-binding protein (TBP; 1:200,
Santa Cruz) at 4?C overnight. Subsequently, the membranes
were incubated with a secondary antibody (1:4,000) anti-
rabbit (Bio-Rad, Munich, Germany) for 1hr, and the bands
were visualized by using enhanced chemilumescent reagent
(Thermo Scientific) in a ChemiDoc XRS system (BioRad).
Molecular weight of the protein bands was estimated using a
western C protein standard (Bio-Rad).
PON1 reporter gene assay
We selected HuH7 liver hepatoma cells for the PON1
promoter activity studies because PON1 is mainly synthe-
sized in the liver and, to a lower extent, in the heart.40,41
HuH7 liver hepatoma cells of human origin stably transfected
with a 1,000-bp fragment of the human PON1 promoter
(PON1-HuH7; originating from X. Coumoul, INSERM,
France) were cultivated in DMEM with 10% heat-inactivated
FCS, 100U/mL streptomycin, and 100mg/mL penicillin (all
from PAA, Coelbe, Germany). PON1-HuH7 cells were see-
ded at an initial density of 150,000 cells per well (24-well
plate) and incubated with the olive oil phenolics hydro-
xytyrosol, tyrosol, oleuropein, pinoresinol, caffeic acid, p-
coumaric acid, and vanillic acid and resveratrol (used as a
positive control), for 48hr as described recently.59Afterward,
the cells were washed with PBS, lysed, and subjected to lu-
ciferase activity measurement as described above.
Results are expressed as mean values with standard error
of the mean (SEM). Statistical analysis was performed using
PASW Statistics 18 (IBM, Chicago, IL). Data were analyzed
for normality of distribution (Kolmogorow–Smirnov and
Shapiro–Wik tests) and equality of variance (Levene test)
before the t-test for independent samples and, in the case of
nonparametric data, Mann–Whitney U-test. Differences were
considered significant when the p value was £0.05.
The two olive oils used in this study differed notably in
their content of the major olive oil phenolics hydroxytyrosol,
Table 1. Primers Used in Real-Time PCR Experiments
GAPDH F: GACAGGATGCAGAAGAGATTACT
GST F: TACTTTGATGGCAGGGGAAG
NQO1 F: TTCTTCTGGCCGATTCAGAGT
PON2 F: ATGGTGGCTCTGAGTTTGCT
GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; c-GCS, c-
glutamyl cysteine synthetase; GST, glutathione-S-transferase; NQO1,
nicotinamide adenine dinucleotide phosphate [NAD(P)H]:quinone
oxidoreductase; PON2, paraoxonase-2; F, forward; R, reverse.
74BAYRAM ET AL.
tyrosol, oleuropein, and pinoresionol, and this was reflected
in the corresponding FRAP and TEAC values of the two oils
(Table 2). Importantly, the HP olive oil contained 12-fold
higher concentrations of total phenolics than the LP olive oil.
However, a-tocopherol concentrations were similar in both
oils (Table 2).
Mean daily feed intake was similar in mice fed the LP
(3.13–0.06g/day) and HP (3.26–0.47g/day) diets. At the
end of the 4.5-month feeding trial, no differences in the final
body weight of HP mice (27.2–1.4g) compared to LP
(29.1–1.3g) mice were observed.
To determine whether olive oil phenolics may affect lipid
and protein oxidation, TBARS and protein carbonyl con-
centrations were determined in heart tissue (Table 3). Feed-
ing the HP diet resulted in significantly lower TBARS and
protein carbonyl concentrations in heart tissue relative to the
LP diet. Because mice fed the HP diet exhibited reduced
heart protein carbonyl concentrations, we determined whe-
ther this was related to differences in proteasomal activity.
However, proteasomal activities of the subunits b-1, b-2, and
b-5 in heart tissue remained unchanged by the different di-
etary treatments (Table 3).
The transcription factor Nrf2 plays a pivotal role in anti-
oxidant and phase 2 defense mechanisms. Impaired Nrf2
signaling may be associated with increased oxidative stress.
Because Nrf2 controls its own gene expression, Nrf2 mRNA
levels were measured. SAMP8 mice receiving the LP diet
exhibited significantly lower Nrf2 mRNA levels as compared
to mice receiving the HP diet (Fig. 1A). Nrf2 controls the
gene expression of GST, c-GCS, NQO1, and PON2. Inter-
estingly, differences in Nrf2 expression between the two
groups were also reflected in terms of differences in GST
(Fig. 1B), c-GCS (Fig. 1C), NQO1 (Fig. 1D), and PON2 (Fig.
1E) mRNA. Thus, feeding the HP diet resulted in signifi-
cantly elevated mRNA levels of genes encoding for antioxi-
dant and phase 2 enzymes in the heart of our mice. Recently,
it has been shown that there is crosstalk between SIRT1
and Nrf2 because SIRT1 was reduced in Nrf2-/-murine
fibroblasts.60In the present study, the increase in Nrf2 and
Nrf2-dependent gene expression due to the HP diet was
associated with a significant increase in heart SIRT1 mRNA
levels (Fig. 1F).
Apart from PON2 gene expression in the heart, we looked
also into serum PON1 activity levels in our mice in response
to dietary supplementation with LP and HP olive oil. Mice
receiving the LP diet exhibited significantly lower PON1
activity levels in serum as compared to those receiving the
HP diet (Fig. 2).
To determine which phenol present in olive oil may
have induced Nrf2-dependent gene expression and the
increase in PON status, additional cell culture studies in
NIH 3T3 and HuH7 cells were performed. In total, we
tested seven different phenolic compounds present in
olive oil, including hydroxytyrosol, tyrosol, oleuropein,
pinoresinol, caffeic acid, p-coumaric acid, and vanillic
acid, for their ability to affect Nrf2 and PON1 transacti-
vation. Under the conditions investigated, only hydro-
xytyrosol, but not tyrosol, oleuropein, pinoresinol, caffeic
acid, p-coumaric acid, or vanillic acid, increased Nrf2
transactivation (Table 4). Furthermore, we determined
nuclear Nrf2 protein levels in NIH 3T3 cells and found
an increase in nuclear Nrf2 in hydroxytyrosol-treated
cells (Fig. 3). Also, treatment of HuH7 cells with hydro-
xytyrosol resulted in a dose-dependent increase in PON1
transactivation (Fig. 4).
Table 2. Concentrations of Phenols and TEAC
and FRAP Values of the High-Polyphenol
and Low-Polyphenol Olive Oils
Phenolic compounds (mg/kg oil)
Total phenolics (mg
GA equivalent/kg oil)
FRAP (mmol AA
TEAC (mmol trolox
TEAC, Trolox equivalent antioxidant capacity; FRAP, ferric
reducing ability of plasma; GA, .
Table 3. Concentrations of TBARS and Protein Carbonyls As Well
As Proteasomal Activities in Heart Homogenates from SAMP8 Mice Fed
for 4.5 Months Diets with 10% High-Polyphenol or Low-Polyphenol Olive Oil
Low-polyphenol dietHigh-polyphenol dietp
TBARS (nmol/g tissue)
Protein carbonyls (nmol/g protein)
Proteasomal activity (lmol/mg$min)
Values are expressed as mean–standard error of the mean (SEM) (n=10).
aMeans are significantly different at the given p value.
TBARS, Thiobarbituric acid-reactive substances; SAMP8, senescence-accelerated mouse-prone 8.
NRF2 INDUCTION BY OLIVE OIL PHENOLICS75
In the present study, feeding a HP diet reduced bio-
markers of oxidative stress, induced Nrf2, and increased
SIRT1 gene expression in the heart of SAMP8 mice compared
to animals fed a LP diet. Mice fed the HP diet had lower
concentrations of TBARS and protein carbonyls in the heart
than mice on the LP diet (Table 3). However, proteasomal
activity was not changed. Thus, differences in heart protein
carbonyl levels were independent of proteasomal activity. In
a comparable study with SAMP8 mice, neither curcumin nor
Ginkgo biloba extract, when fed for 5 months, reduced protein
carbonyl concentrations in the heart.61
On the basis of the present data, it is suggested that dif-
ferences in the oxidant/antioxidant status between the LP
and HP groups may be partly due to differences in Nrf2-
dependent gene expression. The latter drives the expression
of genes encoding phase 2 and antioxidant enzymes. How-
ever, it is unclear which phenols present in olive oil may
have improved antioxidant status and Nrf2-dependent gene
expression in our mice. Because we found an induction of
GST, c-GCS, NQO1, and PON in response to the HP diet, we
performed additional cell culture studies with purified
compounds to investigate which particular olive polyphenol
may have induced Nrf2-dependent gene expression. Our cell
culture data in NIH 3T3 and HuH7 cells suggest that
hydroxytyrosol may have mediated the induction of Nrf2-
dependent gene expression and the increase in PON status
in our mice. Our data in NIH 3T3 cells are in accordance
with a previous study by Martı ´n et al.,46demonstrating
of Nrf2 (A), glutathione-S-transferase (GST) (B), c-glutamyl cysteine synthetase (c-GCS) (C), nicotinamide adenine dinucleotide
phosphate [NAD(P)H]:quinone oxidoreductase 1 (NQO1) (D), paraoxonase-2 (PON2) (E), and SIRT-1 (F) in hearts of senes-
cence-accelerated mouse-prone 8 (SAMP8) mice fed for 4.5 months a Western type diet with 0.15% cholesterol and 20% fat, in
which 10% of fat was from olive oil containing either low or high amount of phenolics. Mice were killed at 7 months of age.
Values are expressed as mean–standard error of the mean (SEM) (n=10). * indicates statistical significant differences between
Relative messenger RNA (mRNA) expression (normalized for glyceraldehyde 3-phosphate dehydrogenase [GAPDH])
76BAYRAM ET AL.
an induction of Nrf2 activity by hydroxtyrosol in HepG2
cells. Interestingly, in the current study, hydroxytyrosol,
but not tyrosol, resulted in an induction of Nrf2 transac-
tivation and PON1 activity. Thus, the presence of the 3-
hydroxyl group may be an important structural determinant
as far as Nrf2 and PON1 induction are concerned. In our
HuH7 cell culture studies, we used hydroxytyrosol concen-
tration up to 25lmol/L. This hydroxytyrosol concentration
is similar to those reported in human plasma after the con-
sumption of 40mL of olive oil62or 20 olives.63However, in
other human studies, hydroxytyrosol concentrations in
plasma following olive oil consumption were in the lower
In the present cell culture and mouse studies, we also
observed an induction of PON1 and PON2 due to hydro-
xytyrosol and a HP diet, respectively. PON2 prevents
cell-mediated lipid peroxidation in the heart.65Thus, the
decreased lipid peroxidation levels in the heart of our mice in
response to the HP diet may be partly mediated by PON2-
Mice fed the HP diet exhibited an improved PON1 status.
In a human study, plasma oxidized LDL concentrations were
negatively correlated with the phenol content of LDL in men
who ingested different olive oils.66Because PON1 prevents
oxidation of LDL, lower oxidized LDL concentrations might
be due to PON1 induction.
In a previous study, SIRT gene expression and protein
levels were decreased in SAMP8 mice compared to normal
oxonase-1 (PON1) activity in serum of senescence-accelerated
mouse-prone 8 (SAMP8) mice fed a Western type diet with
oil containing either low or high amount of phenolics (n=9).
*indicates statistical significant differences between groups.
Mean (–standard error of the mean [SEM]) para-
Table 4. Nrf2 Transactivation in NIH 3T3
Cells Incubated with 25lmol/L
of the Respective Olive Oil Phenolics
Test component Fold Nrf2 transactivation
Mean values (–SEM) with different superscript letters are signif-
icantly different (p<0.05).
NRF2 INDUCTION BY OLIVE OIL PHENOLICS77
aging mice.67Furthermore, a moderate induction of SIRT
retarded aging of the heart and induced resistance to oxi-
dative stress.68Thus, the decreased concentrations of bio-
markers of oxidative stress in mice fed the HP diet might be
related to the moderate SIRT1 induction observed in these
Our results suggest that a diet rich in olive oil phenolics
reduces oxidative stress in the heart of SAMP8 mice, most
likely by induction of Nrf2-dependent gene expression. The
content of hydroxytyrosol seems to be of importance for the
potential health benefits of olive oils. The present data from
studies with cultured cells and mice need to be confirmed
in humans. Ultimately, studies in other model organisms
are warranted to determine whether the induction of Nrf2-
dependent gene expression by hydroxytyrosol-rich olive oil
may also affect life span.
Overall, a Mediterranean diet rich in olive oil may be an
important cornerstone in the prevention of age-dependent
chronic diseases such as coronary heart disease. Therefore,
dietary recommendations, as far as healthy aging is con-
cerned, should promote an increased consumption of olive
oil.69Health benefits of olive oil may be partly attributed to
its content of olive oil phenolics. Thus, olive producers as
well as the food industry are encouraged to establish means
by which they may increase the phenolic content of olives
and derived products.
B.B. is supported by TUBITAK (The Scientific and Tech-
nological Research Council of Turkey). J.F. is supported by
grant no. FR 2478/4-1 from the German Research Founda-
tion (DFG) and grant no. 0315679A from the German Federal
Ministry of Education and Research (BMBF). A.E.W. is
supported by the DFG Clusters of Excellence ‘‘Inflammation
of Interfaces.’’ T.G. and G.R. are supported by the Federal
Ministry of Education and Research (BMBF) and the DFG.
C.S. is supported by a grant of the Christian-Albrechts Uni-
versity of Kiel.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Institute of Human Nutrition and Food Science
Received: August 9, 2011
Accepted: September 22, 2011
NRF2 INDUCTION BY OLIVE OIL PHENOLICS81