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The FASEB Journal •Research Communication
In vivo nutrigenomic effects of virgin olive oil
polyphenols within the frame of the Mediterranean
diet: a randomized controlled trial
Valentini Konstantinidou,*
,‡
Maria-Isabel Covas,*
,1
Daniel Mun˜z-Aguayo,*
Olha Khymenets,
†
Rafael de la Torre,
†
Guillermo Saez,
§
Maria del Carmen Tormos,
§
Estefania Toledo,
储
Amelia Marti,
¶
Valentina Ruiz-Gutie´ rrez,
#
Maria Victoria Ruiz Mendez,
#
and Montserrat Fito*
*Cardiovascular Risk and Nutrition Research Group and
†
Human Pharmacology and Clinical
Neurosciences Research Group, Institut Municipal d⬘Investigacio´Me`dica (IMIM-Hospital del Mar),
Centro de Investigación Biomédica Eu R
˙ed (CIBER) de Fisiopatología de la Obesidad y Nutricio´n,
Barcelona, Spain;
‡
PhD Program in Biomedicine, Departament de Ciencies Experimentals i de la Salut,
Pompeu Fabra University, Barcelona, Spain;
§
Department of Biochemistry and Molecular Biology,
University of Valencia, Valencia, Spain;
储
Department of Preventive Medicine and Public Health and
¶
Department of Nutrition, Food Science, Physiology, and Toxicology, University of Navarra, Navarra,
Spain; and
#
Instituto de Nutrition and Lipid Metabolism, Instituto de la Grasa, Seville, Spain
ABSTRACT The aim of the study was to assess
whether benefits associated with the traditional Medi-
terranean diet (TMD) and virgin olive oil (VOO) con-
sumption could be mediated through changes in the
expression of atherosclerosis-related genes. A random-
ized, parallel, controlled clinical trial in healthy volun-
teers (nⴝ90) aged 20 to 50 yr was performed. Three-
month intervention groups were as follows: 1) TMD
with VOO (TMDⴙVOO), 2) TMD with washed virgin
olive oil (TMDⴙWOO), and 3) control with partici-
pants’ habitual diet. WOO was similar to VOO, but with
a lower polyphenol content (55 vs. 328 mg/kg, respec-
tively). TMD consumption decreased plasma oxidative
and inflammatory status and the gene expression re-
lated with both inflammation [INF-␥(INF␥), Rho
GTPase-activating protein15 (ARHGAP15), and inter-
leukin-7 receptor (IL7R)] and oxidative stress [adren-
ergic 
2
-receptor (ADRB2) and polymerase (DNA-di-
rected) (POLK)] in peripheral blood mononuclear
cells. All effects, with the exception of the decrease in
POLK expression, were particularly observed when
VOO, rich in polyphenols, was present in the TMD
dietary pattern. Our results indicate a significant role
of olive oil polyphenols in the down-regulation of
proatherogenic genes in the context of a TMD. In
addition, the benefits associated with a TMD and
olive oil polyphenol consumption on cardiovascular
risk can be mediated through nutrigenomic ef-
fects.—Konstantinidou, V., Covas, M.-I., Mun˜z-
Aguayo, D., Khymenets, O., de la Torre, R., Saez, G.,
del Carmen Tormos, M., Toledo, E., Marti, A.,
Ruiz-Gutie´rrez, V., Ruiz Mendez, M. V., Fito, M. In
vivo nutrigenomic effects of virgin olive oil polyphe-
nols within the frame of the Mediterranean diet: a
randomized controlled trial. FASEB J. 24, 000– 000
(2010). www.fasebj.org
Key Words: inflammation 䡠oxidative stress 䡠DNA damage
䡠gene expression 䡠IFN␥
In 1979, Keys ET AL.(1) provided ecological evidence
of a reduced risk for coronary heart disease (CHD)
associated with the Mediterranean diet despite its high
monounsaturated fat content. This diet, when con-
sumed in sufficient amounts, provides all of the known
essential micronutrients (i.e., vitamins and minerals),
fiber, and other plant food substances to promote
health (2). A high degree of adherence to the Mediter-
ranean diet has been associated with a reduced risk of
overall and cardiovascular mortality, cancer incidence
and mortality, and incidence of Parkinson’s disease and
Alzheimer’s disease (3, 4). The most impressive bene-
fits of this diet are, however, related to cardiovascular
morbidity and mortality (5).
Olive oil is the main source of fat in the Mediterra-
nean diet. A large body of knowledge provides evidence
of the benefits of the Mediterranean diet and the
consumption of olive oil on risk factors for CHD, in
particular, on the lipid profile, lipid and DNA oxida-
tion, insulin resistance, and inflammation (6–9). In
experimental studies, olive oil has also been shown to
be able to influence stages of carcinogenesis, cell
membrane composition, signal transduction pathways,
transcription factors, and tumor suppressor genes (10).
The beneficial effects of olive oil on cardiovascular risk
factors are now recognized, but often are attributed
1
Correspondence: Cardiovascular Risk and Nutrition Re-
search Group, Institut Municipal d’Investigacio´Me`dica
(IMIM-Hospital del Mar), Parc de Recerca Biome`dica de
Barcelona (PRBB), Carrer Dr. Aiguader, 88.08003, Barce-
lona, Spain. E-mail: mcovas@imim.es
doi: 10.1096/fj.09-148452
10892-6638/10/0024-0001 © FASEB
The FASEB Journal article fj.09-148452. Published online February 23, 2010.
only to the high levels of monounsaturated fatty acids
(MUFAs) present in olive oil (11). Olive oil, however, is
a functional food that, besides a high content of
MUFAs, contains other minor biologically active com-
ponents (12). Among them, the best studied are the
polyphenols. In human studies, olive oil polyphenols
have been shown to reduce in vivo lipid oxidative
damage (13), endothelial dysfunction (14), prothrom-
botic profile (15), and inflammatory status (16–18) in
healthy volunteers and patients with stable CHD or
hypercholesterolemia.
The exact mechanisms by which the Mediterranean
diet and olive oil exert their health effects are not yet
understood. Among these mechanisms, the gene-envi-
ronment and/or gene-diet interaction could play an
important role in the development and/or protection
of chronic degenerative diseases. At present, few data
exist on the in vivo effect of the Mediterranean diet on
human gene expression (19, 20), particularly in healthy
volunteers. Gene expression changes in human periph-
eral blood mononuclear cells (PBMNCs) after virgin
olive oil consumption have been reported (21–23).
However, no data exist concerning the in vivo nutrig-
enomic effects of olive oil polyphenols in humans. The
aim of the present study was to evaluate whether a
traditional Mediterranean diet (TMD) and the poly-
phenols present in olive oil promote changes in athero-
sclerosis-related genes in healthy volunteers.
MATERIALS AND METHODS
Study design
A randomized, parallel, controlled clinical trial with 3 dietary
interventions was performed. From October 2007 to October
2008, 99 potential participants were recruited in primary care
centers. Ninety eligible participants were community-dwelling
men and women aged 20 to 50 yr. They were considered
healthy on the basis of a physical examination and routine
biochemical and hematological laboratory determinations.
The institutional ethics committee [IMM Comitè Ètic
d’Investigacio´ Clínica–Institut Municipal d’Assistència Sani-
tària (CEIC-IMAS)] approved the protocol (2004/1827/I),
and the volunteers gave written informed consent before
initiation of the study. This trial was registered in Current
Controlled Trials, London, with the International Standard
Randomized Controlled Trial Number of ISRCTN53283428.
Volunteers were randomly assigned to 3 intervention
groups (n⫽30/group), by means of a computer-generated
random-number sequence. They received the following treat-
ments during 3 mo: group 1, TMD with virgin olive oil
(TMD⫹VOO); group 2, TMD with washed virgin olive oil
(TMD⫹WOO); and group 3, control group with their habit-
ual diet. Volunteers were advised by a dietitian to maintain
their habitual lifestyle. Exclusion criteria were the following:
intake of antioxidant supplements; intake of acetosalicylic
acid or any other drug with established antioxidative proper-
ties; high levels of physical activity (⬎3000 kcal/wk in leisure-
time physical activity); obesity [body mass index (BMI) ⬎30
kg/m
2
]; hypercholesterolemia (total cholesterol ⬎8.0 mM or
dyslipidemia therapy); diabetes (glucose ⬎126 mg/dl or
diabetes treatment); hypertension [systolic blood pressure
(SBP) ⬎140 mmHg and/or diastolic blood pressure (DBP)
⬎90 mmHg or antihypertensive treatment]; multiple aller-
gies; celiac or other intestinal diseases; any condition that
could limit the mobility of the subject, making study visits
impossible; life-threatening illnesses or other diseases or
conditions that could worsen adherence to the measurements
or treatments; vegetarianism or a need for other special diets;
and alcoholism or other drug addiction. Fasting blood and
first morning spot urine samples were collected between 8
and 10 AM at study entry and after the 3-mo intervention.
Randomization and Mediterranean diet intervention
The baseline examination included the administration of a
previously validated 137-item food frequency questionnaire
(24); the Minnesota Leisure Time Physical Activity question-
naire, which has been validated for use in Spanish men and
women (25, 26); and a 47-item general questionnaire assess-
ing lifestyle, health conditions, sociodemographic variables,
history of illness, and medication use. The same dietitian
performed the interventions with the 3 randomized groups.
On the basis of the assessment of an individual 14-point
Mediterranean diet score (8), the dietitian gave personalized
advice during a 30-min session to each participant, with
recommendations on the desired frequency of intake of
specific foods. Instructions were directed to upscaling the
TMD score, including the use of olive oil for cooking and
dressing; increased consumption of fruit, vegetables, and fish;
consumption of white meat instead of red or processed meat;
preparation of homemade sauce with tomato, garlic, onion,
aromatic herbs, and olive oil to dress vegetables, pasta, rice,
and other dishes; and, for alcohol drinkers, moderate con-
sumption of red wine. At the end of the intervention (3 mo),
all baseline procedures were repeated.
Olive oil characteristics
The WOO used in intervention group 2 was obtained from
the VOO used in intervention group 1 in the Instituto de la
Grasa (Sevilla, Spain). In brief, VOO was placed in a thermo-
static reactor, washed twice with 10% water at 70°C, and
shaken at 125 rpm. Temperature was maintained at 40°C for
20 min at 95 rpm. Oil-phase separation was performed by
centrifugation, repeating the whole procedure 5 times. This
WOO maintained the same characteristics as the VOO, with
the exception of a lower content of polyphenols (55 and 328
mg/kg, respectively). Olive oils were provided to the subjects
of both intervention groups 1 and 2 in a sufficient amount for
the entire family (15 L/volunteer) during the intervention
periods for both cooking and dressing purposes. The VOO
used was of the Hojiblanca variety from Andalucía, Spain.
The composition of the olive oils was as follows: MUFAs, 75%;
polyunsaturated fatty acids (PUFAs), 18.6%; and saturated
fatty acids, (SFAs), 6.4%. Minor components, other than
polyphenols, were ␣-tocopherol (1.47 mg/kg), -carotene
(0.43 mg/kg), and sterols (15.6 mg/kg). The contents of
squalene and terpenes were 4346 and 4026 mg/kg and 48.3
and 61.3 mg/kg for VOO and WOO, respectively. Both olive
oils were stored to avoid exposure to air, light, and high
temperature to prevent oxidation.
Oxidative damage and inflammation biomarkers
Serum glucose, total cholesterol, and triglyceride levels were
measured using standard enzymatic methods, and HDL-
cholesterol (HDL-C) was measured by an accelerator selective
detergent method (ABX-Horiba Diagnostics, Montpellier,
France), in a automated PENTRA-400 autoanalyzer (ABX-
Horiba Diagnostics). LDL-cholesterol (LDL-C) was calculated
2 Vol. 24 July 2010 KONSTANTINIDOU ET AL.The FASEB Journal 䡠www.fasebj.org
by the Friedewald (27) formula whenever triglycerides were
⬍300 mg/dl. Oxidized LDL (oxLDL) was determined in
plasma by a sandwich ELISA procedure using the murine
monoclonal antibody mAB-4E6 as a capture antibody and a
peroxidase-conjugated antibody against oxidized apolipopro-
tein B bound to the solid phase (oxLDL; Mercodia AB,
Uppsala, Sweden). Urine total F2␣-isoprostanes were deter-
mined by an immunoassay kit (Cayman Chemical, Ann
Arbor, MI, USA). The amount of 7,8-dihydro-8-oxo-2⬘-
deoxyguanosine (8-oxo-dG) in urine was measured by
HPLC with electrochemical detection. Values of isoprostanes
and 8-oxo-dG in urine were normalized against creatinine
concentration. High-sensitivity C-reactive protein (CRP) was
measured by immunoturbidometry (ABX-Horiba Diagnos-
tics). Plasma levels of IFN-␥, monocyte chemoattractant pro-
tein 1 (MCP-1), soluble P-selectin (s-P-selectin), and soluble
CD40L (sCD40L) were measured by flow cytometry (Bender
MedSystems Co. Ltd., San Diego, CA, USA). All analytical
determinations were performed in the same batch.
Evaluation of the intervention
After 3 mo, all baseline procedures were repeated. Biological
assessment of intervention compliance was performed in all
participants. Tyrosol and hydroxytyrosol, the major polyphe-
nols present in olive oil, were measured in urine by gas
chromatography-mass spectrometry (28).
Gene expression analyses
The selection of candidate genes was performed on the basis
of previous data from our group concerning atherosclerosis-
related responsive genes in peripheral blood mononuclear
cells (PBMNCs) of healthy volunteers after long-term (3 wk)
(21) and short-term (23, 29) VOO consumption, and their
biological plausibility as assessed by literature review (http://
www.ncbi.nlm.nih.gov/pubmed/). Gene expression analyses
were performed in a subsample of 56 participants (20, 16, and
20 in control, TMD⫹WOO, and TMD⫹VOO groups, respec-
tively). A liquid-liquid method to isolate total RNA from
PBMNCs was performed as described previously (21–23). The
correct quality, quantity, and purity of total RNA were as-
sessed. A total of 100 ng of tRNA in a 20-l reaction was
reverse-transcribed using the High-Capacity cDNA Reverse
Transcription Kit with RNase Inhibitor (Applied Biosystems,
Foster City, CA, USA) according to the manufacturer’s pro-
tocols. An array for gene expression analysis was performed in
duplicate using 384-well Micro Fluidic cards (TaqMan Low
Density Array by Design) for 48 genes (47⫹1 control) on the
ABI Prism 7900HT Sequence Detection System (Applied
Biosystems). The human glyceraldehyde-3-phosphate dehy-
drogenase (GADPH) gene was used as an endogenous control
to correct the differences in the amount of total cDNA added
to each reaction. Results from each run were analyzed sepa-
rately using a software-defined baseline and a C
t
threshold of
0.20. Changes in gene expression were calculated using the
relative quantification (RQ) method and applying the 2
⫺⌬⌬Ct
formula (30). Each gene expression was first normalized to
the endogenous reference gene (⌬C
t
⫽C
texp
⫺C
tref
) and
afterward to its untreated control (baseline) (⌬⌬C
t
). Two
genes, NOX1 (NADPH oxidase 1) and NOX2 (NADPH oxi-
dase 2), did not amplify. Thus, they were excluded from the
analyses. Data obtained were analyzed using SDS 2.1 software.
We used the Functional Classification Tool of the DAVID
Bioinformatics Database (31, 32) to generate a gene-to-gene
similarity matrix.
Statistical analysis
The normality of continuous variables was assessed by normal
probability plots and by means of the Shapiro-Wilk test. The
relationship between continuous variables was measured by
Spearman’s rank correlation coefficient. Non-normally dis-
tributed variables were log-transformed before application of
the ttest or general linear modeling statistics. ANOVA was
used for assessing differences between the control and the
two TMD intervention groups at baseline. Comparisons of the
3-mo changes were performed by a covariance model with
polynomial content, with age and sex as covariates. Statistical
analyses were performed as 2-group analyses with the TMD
global group (TMD⫹VOO and TMD⫹WOO) vs. the control
group and as 3-group analyses, considering the 3 types of
intervention separately (TMD⫹VOO, TMD⫹WOO, and con-
trol). An a priori defined value of P⬍0.05 was considered
statistically significant. All statistical analyses were performed
with SPSS 12.3 software (SPSS Inc., Chicago, IL, USA) for
Windows XP (Microsoft, Redmond, WA). Gene set enrich-
ment analysis was applied to the Functional Classification
Tool to determine whether an a priori defined set of genes
showed statistically significant concordant differences be-
tween the two biological states (before and after the interven-
tion). The enrichment score value was used to highlight the
most overrepresented biological annotation out of thousands
of linked terms and contents.
RESULTS
We excluded 9 of the 99 invited participants before
randomization for various reasons, and 1 participant
dropped out of the study after randomization (Fig. 1).
Table 1 shows the baseline characteristics of the 90
participants (26 men and 64 women) who entered the
study. We observed lower levels of plasma IFN-␥in the
TMD global and the TMD⫹WOO groups vs. control.
We did not observe differences in general baseline
characteristics among groups (Table 1). Table 2 shows
the changes in energy, nutrient intake, and key food
items at the end of the intervention period. An increase
in vegetable, legume, and fish consumption was ob-
served in both TMD groups. Participants’ compliance
with the supplemented olive oil was good, as reflected
by both the increase in VOO consumption and the
decrease in olive oil (nonvirgin) in the TMD⫹VOO
group, whereas the opposite effect was observed in the
TMD⫹WOO group (Table 2), and the decrease in the
urinary tyrosol and hydroxytyrosol concentrations in
the TMD⫹WOO group and the increase in the
TMD⫹VOO group (P⫽0.007, for quadratic trend)
(Fig. 2). In the two-group analyses (TMD global vs.
control), plasma glucose levels, HDL-C, F
2␣
-isopros-
tanes, IFN-␥, and CRP decreased after 3 mo of TMD
intervention (P⬍0.05;) (Table 3). In the 3-group
analyses (Table 3), total cholesterol, HDL-C, and
LDL-C decreased in the TMD⫹VOO group after 3 mo
of intervention (P⬍0.05), without changes in the total
cholesterol/HDL-C or LDL-C/HDL-C ratios. The de-
crease in plasma IFN-␥,F
2␣
-isoprostanes, and s-P-selec-
tin was significant only after the TMD⫹VOO interven-
tion (P⬍0.05) (Table 3). Similar trends and results
3NUTRIGENOMIC EFFECTS OF OLIVE OIL POLYPHENOLS
were obtained when the subpopulation involved in
gene expression analyses (n⫽56) was evaluated. When
results were disclosed by sex, in females a decrease in
IFN-␥in the control group and in CRP in the TMD
global and TMD⫹WOO groups was observed. In addi-
tion, in the TMD⫹VOO group, we observed an in-
crease in HDL-C in females and a decrease in LDL-C in
males (Supplemental Table 1).
Intragroup comparisons showed no significant differ-
ences between pre- and posttreatment values in the
evaluated gene expression in any intervention group.
Table 4 shows the intergroup comparisons of the gene
expression changes in the 2-group analyses (TMD
global vs. control) expressed as the log
2
ratio of RQ
between posttreatment and basal values. Five genes
[adrenergic 
2
-receptor (ADRB2),Rho GTPase-activat-
ing protein 15 (ARHGAP15), IFN-␥(IFN␥), IL-7 recep-
tor (IL7R), and polymerase (DNA directed)-(POLK)]
were down-regulated compared with the control group
(P⬍0.05) (Table 4). When the 3-group analyses were
performed, a decreasing linear trend from the control
to the TMD⫹VOO group (P⬍0.05) was observed (Fig. 3)
in ADRB2, ARHGAP15, IL7R, and IFN␥gene expres-
sion. The down-regulation was statistically significant in
the TMD⫹VOO group vs. control group (P⬍0.05) for
ADRB2, ARHGAP15, and IFN␥genes and had border-
line significance (P⫽0.052) in the case of IL7R gene
expression (Fig. 3). No differences in the expression of
Figure 1. Study flow diagram. Gene expression anal-
yses were performed in a subsample of 56 participants
(20, 16, and 20 in control, TMD⫹WOO, and
TMD⫹VOO groups, respectively).
TABLE 1. Volunteer baseline characteristics
Parameter Control, n⫽30 TMD global, n⫽60 TMD⫹WOO, n⫽30 TMD⫹VOO, n⫽30
Age (yr) 43 ⫾13 45 ⫾10 44 ⫾10 45 ⫾10
Men (%) 34.5 25 27 23
Weight (kg) 66 ⫾16 68 ⫾13 69 ⫾13 68 ⫾14
BMI (kg/m
2
) 25 ⫾425⫾426⫾525⫾4
SBP (mmHg) 117 ⫾12 116 ⫾15 117 ⫾14 114 ⫾16
DBP (mmHg) 69 ⫾10 72 ⫾10 72 ⫾10 72 ⫾10
Glucose (mg/dl) 85 ⫾15 84 ⫾12 84 ⫾14 84 ⫾9
Total cholesterol (mg/dl) 202 ⫾54 207 ⫾50 200 ⫾47 214 ⫾54
LDL-C (mg/dl) 127 ⫾42 131 ⫾44 124 ⫾37 138 ⫾50
HDL-C (mg/dl) 58 ⫾13 60 ⫾14 58 ⫾13 61 ⫾15
Total cholesterol/HDL-C 3.6 ⫾1.0 3.6 ⫾0.9 3.5 ⫾0.6 3.6 ⫾1.0
LDL-C/HDL-C 2.2 ⫾0.7 2.3 ⫾0.8 2.2 ⫾0.5 2.3 ⫾0.9
Triglycerides (mg/dl) 67 (52, 83) 70 (57, 103) 67 (58, 102) 70 (57, 105)
oxLDL (U/L) 66 ⫾29 63 ⫾21 62 ⫾20 64 ⫾22
F
2␣
-isoprostanes in urine
(pg/mmol of creatinine) 42 (39, 79) 67 (39, 83) 54 (41, 79) 72 (39, 85)
IFN-␥(ng/ml) 0.086 (0.009, 0.124) 0.018 (0.001, 0.073)* 0.001 (0, 0.068)* 0.027 (0, 0.086)
MCP-1 (pg/ml) 282 (203, 369) 217 (170, 307) 240 (195, 349) 174 (143, 243)
s-P-selectin (ng/ml) 935 ⫾741 743 ⫾493 768 ⫾496 710 ⫾498
s-CD40L (pg/ml) 937 (586, 2254) 1217 (602, 2354) 1389 (618, 2306) 1001 (558, 2449)
CRP (mg/dl) 0.02 (0.01, 0.09) 0.07 (0.03, 0.18) 0.11 (0.02, 0.25) 0.07 (0.03, 0.11)
8-Oxo-dG in urine (nmol/
mmol creatinine) 10.09 ⫾4.07 11.32 ⫾4.01 11.10 ⫾3.89 11.55 ⫾4.19
EEPA (kcal/d) 129 (25, 269) 130 (47, 224) 113 (49, 183) 139 (32, 229)
Values are shown as means ⫾sd for normal variables and medians (25th, 75th percentiles) for nonparametric variables. Univariate ANOVA
was used to assess differences between groups for the normal variables; Kruskal-Walls test was used for nonparametric variables. EEPA, energy
expenditure in physical activity in leisure time. *P⬍0.05 vs. control group.
4 Vol. 24 July 2010 KONSTANTINIDOU ET AL.The FASEB Journal 䡠www.fasebj.org
other evaluated genes were observed, either between
the TMD⫹VOO and TMD⫹WOO groups or between
them and the control group. Gene expression changes
were observed, particularly in the female groups (Sup-
plemental Table 2). Correlation analyses showed that
postintervention IL7R expression values (all volun-
teers) were inversely correlated with urinary tyrosol
(r⫽⫺0.273, P⫽0.044) and hydroxytyrosol (r⫽⫺0.284,
P⫽0.035) levels. In addition, changes in urinary levels
of tyrosol after TMD⫹VOO intervention were inversely
correlated with changes in the expression of IFN␥
(r⫽⫺0.390, P⫽0.006). Functional annotation cluster-
ing of all 45 genes showed that 3 of the down-regulated
genes, IFN␥, IL7R, and ADRB2, clustered to the same
functional group (functional group 3, GO:0019219,
regulation of nucleobase, nucleoside, nucleotide, and
nucleic acid metabolic process) (Table 5).
DISCUSSION
In the present study, we examined whether the adher-
ence to a TMD modulates the expression of atheroscle-
rosis-related genes and systemic oxidative stress and
inflammation markers, focusing on the effect of olive
oil polyphenols. Our results indicate that the TMD
decreased the lipid oxidative and inflammatory status.
The TMD also decreased the expression of genes
TABLE 2. Change in consumption of key foods and nutrients
Variable
Change from baseline at 3 mo 关mean (95% CI)兴
Control (n⫽29) TMD ⫹WOO (n⫽30) TMD ⫹VOO (n⫽30)
VOO (g/d) ⫺0.44 (⫺2.9 to 1.9) ⫺8.0 (⫺12.6 to ⫺3.4)
‡
22.2 (15.1 to 29.2)
‡†
Olive oil (g/d)
a
⫺0.88 (⫺5.6 to 3.9) 10.3 (4.7 to 16.0)*
,‡
⫺17.3 (⫺24.2 to ⫺10.4)*
,†,‡
Total olive oil (g/d) ⫺13.5 (⫺42.1 to 15.2) 22.9 (⫺11.5 to 57.5) 41.3 (11.9 to 70.7)
‡
Fruits (g/d) 1.0 (0.1 to 1.2) ⫺0.76 (⫺2.84 to 1.33) ⫺1.31 (⫺5.06 to 2.44)
Vegetables (g/d) 1.72 (⫺2.44 to 5.89) 4.51 (⫺1.55 to 10.57) 10.31 (4.50 to 16.12)
‡
Legumes (g/d) ⫺0.02 (⫺0.76 to 0.72) 1.36 (0.47 to 2.25)
‡
2.25 (1.24 to 3.26)
‡
Fish (g/d) 1.93 (⫺1.14 to 5.00) 3.87 (1.79 to 5.94)* 7.93 (2.89 to 12.98)
‡
Nuts (g/d) 1.5 (⫺1.5 to ⫺4.5) 1.2 (0.2⫺2.2)* 0.9 (⫺1.7 to 0)*
Dairy products (g/d) 6.28 (⫺6.59 to 19.15) 1.83 (⫺3.50 to 7.16) ⫺2.41 (⫺11.30 to 6.48)
Alcohol (g/d) 0.18 (⫺0.14 to 0.49) 0.17 (⫺0.08 to 0.5) 0.02 (⫺0.02 to 0.061)
Energy (kcal) ⫺20.08 (⫺53.66 to 13.51) 24.88 (⫺13.15 to 62.90)
‡
51.01 (20.30 to 81.73)
‡
Protein (%) 0.17 (⫺0.15 to 0.49) ⫺0.08 (⫺0.45 to 0.28) 0.033 (⫺0.25 to 0.32)
Carbohydrate (%) 0.04 (⫺0.76 to 0.83) ⫺0.57 (⫺1.38 to 0.24) ⫺0.78 (⫺1.27 to ⫺0.29)*
Fat (%) ⫺0.28 (⫺1.35 to 0.79) 0.67 (⫺0.52 to 1.86) 0.80 (0.11 to 1.48)*
MUFAs (%) ⫺0.52 (⫺1.15 to 0.12) 0.47 (⫺0.47 to 1.41) 0.96 (0.06 to 1.86)*
,‡
PUFAs (%) 0.10 (⫺0.09 to 0.30) 0.09 (⫺0.22 to 0.39) ⫺0.22 (⫺0.75 to 0.31)
SFAs (%) 0.38 (⫺0.24 to 1.00) ⫺0.43 (⫺1.0 to 0.13)
‡
⫺0.77 (⫺1.24 to ⫺0.29)*
,‡
␣-Linolenic acid (g/d) 0.01 (⫺0.06 to 0.09) 0.04 (0.01 to 0.07)* 0.05 (0.02 to 0.08)*
Marine n–3 fatty acids (g/d) 0.01 (⫺0.004 to 0.03) 0.03 (0.02 to 0.05)* 0.09 (0.02 to 0.17)*
,‡
Univariate ANOVA was used to assess differences between groups.
a
Includes WOO. *P⬍0.05 vs. baseline;
†
P⬍0.05 vs. TMD ⫹WOO;
‡
P⬍
0.05 vs. control group.
Figure 2. Changes in urinary tyrosol (A) and hydroxytyrosol (B) after the 3-mo interventions. *P⬍0.05 vs. control;
†
P⬍0.05
vs. TMD⫹WOO.
5NUTRIGENOMIC EFFECTS OF OLIVE OIL POLYPHENOLS
related to inflammation processes (IFN␥,ARHGAP15,
and IL7R), oxidative stress (ADRB2), and DNA damage
(POLK) in PBMNCs. All of the above-mentioned ef-
fects, with the exception of the decrease in POLK
expression, were particularly observed when VOO, rich
in polyphenols, was present in the TMD pattern. Our
work provides, for the first time, evidence of the in vivo
nutrigenomic effect of olive oil polyphenols down-
regulating proatherogenic genes in humans. In addi-
tion and to the best of our knowledge, the in vivo
human nutrigenomic effect of the Mediterranean diet
in healthy individuals has not been reported previously.
When results were disclosed by sex, the gene
expression changes were particularly lower in the
female groups. In a previous work, we have reported
gender differences in PBMNC gene expression, with
higher expression of SOD1 and SOD2 in healthy
males (31). In the present work, however, the low
number of males in some groups could account for
the gender differences observed. Gene expression
can be considered as a quantitative trait that is highly
heritable. We used the Functional Classification Tool
of the DAVID Bioinformatics Database (32, 33) to
generate a gene-to-gene similarity matrix. Grouping
genes based on functional similarity can help to
enhance the biological interpretation of large lists of
genes derived from high-throughput studies. It has
been shown that disease-related genes tend to inter-
act (34, 35) and display significant functional clus-
tering in the analyzed molecular network. In our
results, after 3 mo of TMD⫹VOO intervention, 3 of
the down-regulated genes (IFN␥, IL7R, and ADRB2)
were clustered to the same functional group. In a
previous exploratory approach concerning the hu-
man mononuclear cell transcriptome response after
acute and sustained VOO consumption, we observed
gene expression changes in PBMNCs of healthy
volunteers (21–23). In this work (21, 23), the Gene
Ontology analysis of the differentially expressed
genes indicated that consumption of VOO could
elicit changes in the regulation of transcription and
translation activities of human PBMNCs.
The Mediterranean diet, in which the main source of
fat is olive oil, is well known to be associated with a low
prevalence of CVD (2), cancer (36), and inflammatory
diseases (37, 38). Inflammation is heavily involved in
the onset and development of atherosclerosis (39).
Previous data from short- and long-term clinical trials
have shown an anti-inflammatory effect of the Mediter-
ranean-type diet in patients with risks for cardiovascular
disease (8, 40). In these patients, a TMD enriched with
VOO prevented the increase in cyclooxygenase-2
(COX-2) and LDL receptor-related protein (LRP1)
gene expression and reduced monocyte chemoattrac-
tant protein (MCP-1), compared with a TMD enriched
with nuts or with a low-fat diet (19). In experimental
models, the anti-inflammatory effects of polyphenols
and other olive oil minor components have been
described (41). Some of the anti-inflammatory effects
of olive oil polyphenols could be attributed to oleocan-
TABLE 3. Change in biomarkers after 3 mo of intervention
Parameter
Control, n⫽29 TMD global, n⫽60
Postintervention Change Postintervention Change
Weight (kg) 67 ⫾16 0.19 (⫺0.59 to 0.97) 68 ⫾14 ⫺0.17 (⫺0.72 to 0.37)
BMI (kg/m
2
) 25 ⫾4 0.081 (⫺0.2 to 0.36) 25 ⫾4⫺0.068 (⫺0.26 to 0.13)
SBP (mmHg) 119 ⫾15 1.40 (⫺2.60 to 5.40) 115 ⫾15 ⫺1.03 (⫺3.76 to 1.7)
DBP (mmHg) 71 ⫾10 1.67 (⫺1.23 to 4.58) 72 ⫾10 0.17 (⫺1.81 to 2.15)
Glucose (mg/dl) 82 ⫾12 ⫺2.55 (⫺5.4 to 0.31) 82 ⫾10* ⫺2.1 (⫺4.09 to ⫺0.09)
Cholesterol (mg/dl) 202 ⫾57 ⫺0.12 (⫺8.56 to 8.33) 202 ⫾46 ⫺4.85 (⫺10.75 to 1.04)
HDL-C (mg/dl) 57 ⫾13 ⫺1.82 (⫺4.34 to 0.70) 57 ⫾13* ⫺2.0 (⫺3.75 to ⫺0.29)
LDL-C (mg/dl) 129 ⫾47 2.1 (⫺4.35 to 8.56) 128 ⫾40 ⫺2.80 (⫺7.22 to 1.63)
Cholesterol/HDL-C 3.6 ⫾1.0 0.09 (⫺0.05 to 0.24) 3.6 ⫾0.8 0.04 (⫺0.06 to 0.14)
LDL-C/HDL-C 2.3 ⫾0.8 0.09 (⫺0.03 to 0.22) 2.3 ⫾0.7 0.03 (⫺0.06 to 0.12)
Triglycerides (mg/dl) 62 (49, 98) ⫺2.5 (⫺17, 17.3) 71 (59, 99) 4 (⫺14, 19)
OxLDL (U/L) 70 ⫾32 3.38 (⫺2.36 to 9.16) 64 ⫾23 2.3 (⫺1.69 to 6.19)
Isoprostanes (pg/mmol of
urine creatine) 39 (34, 65) ⫺2.8 (⫺14, 5.1) 49 (41, 66)* ⫺2.5 (⫺13.7, 6.6)
8-oxo-dG (nmol/mmol of
urine creatine) 8.9 ⫾3.8 ⫺1.1 (⫺2.5 to 0.26) 10.4 ⫾3.9 ⫺0.95 (⫺1.89 to 0.003)
IFN-␥(pg/ml) 61 (0, 113) ⫺11 (⫺52, 5) 0 (0, 46)* 0 (⫺45, 11)
MCP-1 (pg/ml) 247 (211, 317) ⫺36 (⫺119, 27) 202 (176, 305) 0.14 (⫺37, 35)
s-P-selectin (ng/ml) 696 (493, 1063) ⫺78 (⫺286, 323) 578 (346, 808)* ⫺30 (⫺383, 122)
†
s-CD40L (pg/ml) 1267 (498, 2013) ⫺228 (⫺1109, 789) 943 (587, 2437) ⫺77 (⫺1077, 804)
CPR (mg/dl) 0.04 (0.01, 0.14) 0 (⫺0.01, 0.06) 0.04 (0.02, 0.11)* ⫺0.02 (⫺0.07, 0)
†
EEPA (kcal/d) 129 (52, 226) 6.8 (⫺23.7 to 37.2) 117 (32, 206) ⫺1.8 (⫺23 to 19)
Postintervention values are presented as means ⫾sd for normal variables and as medians (25th, 75th percentiles) for non-normal variables.
Change values are presented as means (95% CI) and medians (25th, 75th percentiles) for non-normal variables. Univariate ANOVA, adjusted
by sex and age, was used to assess differences between groups for the normal variables; Kruskal-Walls test was used for nonparametric variables.
EEPA, energy expenditure in physical activity in leisure time. *P⬍0.05 vs. baseline;
†
P⬍0.05 vs. control;
‡
P⬍0.05 vs. TMD ⫹WOO.
6 Vol. 24 July 2010 KONSTANTINIDOU ET AL.The FASEB Journal 䡠www.fasebj.org
thal, an olive oil polyphenol with ibuprofen-like activity
in in vitro models (42). Besides its antioxidant and
anti-inflammatory activity, recent data suggest that hy-
droxytyrosol, a major olive oil phenolic compound,
may exert beneficial effects through stimulation of
mitochondrial biogenesis (43). The in vivo anti-inflam-
matory role of olive oil polyphenols in humans is
supported by several randomized controlled clinical
trials (16, 17, 44).
The decrease in systemic inflammatory markers
and in the expression of genes related with inflam-
matory processes observed in the present study is in
agreement with the above described previous results
concerning the protective effect of Mediterranean
diet and olive oil phenolics on inflammation. The
decrease in IFN-␥was observed both at phenotypic
and gene expression levels. IFN-␥is considered to be
a key inflammatory mediator for inducing IL-6, a
prime regulator of CRP synthesis in the liver (45). We
have previously reported a down-regulation of IFN␥
expression in PBMNCs of healthy volunteers after a
single dose of VOO (22). ARHGAP15 encodes for a
Rho GTPase-activating protein that regulates activity
of GTPases (46). Ras superfamily GTPases have been
identified as strategic molecular targets in statin-
induced T-cell immunosuppression. Statins, besides
being cholesterol-lowering drugs, also harbor strong
anti-inflammatory properties (47). Members of the
Rho GTPase family have been suggested to be medi-
ators of cardiac hypertrophy (48); however to date
little is known about their physiological roles (46).
The protein encoded by the IL7R gene is a receptor
for IL-7, which has been related to inflammatory pro-
cesses (49, 50). IL-7 has been shown to enhance the
expression of chemokines in PBMNCs (51). A recent
study has shown an up-regulation of stress-response
genes, such as IL7R and POLK, in the case of induced
carbon ion irradiation in murine tumor models (52).
POLK is a DNA repair gene that copies undamaged
DNA templates and is unique among human Y-family
DNA polymerases (53). Somatic DNA mutations, pro-
moted by DNA oxidation, are considered to be a crucial
step in carcinogenesis as well as to be involved in
atherosclerotic processes (16, 54). We did not observe
changes in the levels of 8-oxo-dG after the global TMD
interventions, although a decrease was observed after
the TMD⫹VOO intervention. However, the results of
the EUROLIVE study, an intervention study performed
in 200 healthy males with 3 types of similar olive oils,
but with differences in their phenolic content, showed
that daily consumption of 25 ml of olive oil for 3 wk
reduced DNA oxidation, irrespective of the olive oil
polyphenol content (55). In agreement with the
EUROLIVE results, the down-regulation of POLK gene
expression observed in our study was associated with
the TMD intervention, but not with the olive oil poly-
phenol content. All these data suggest a protective role
for the MUFAs or other minor components of the olive
oil on DNA oxidation and damage.
TABLE 3. Continued
TMD ⫹WOO, n⫽30 TMD ⫹VOO, n⫽30
Postintervention Change Postintervention Change
69 ⫾14 ⫺0.25 (⫺1.03 to 0.53) 67 ⫾14 ⫺0.1 (⫺0.86 to 0.67)
26 ⫾5⫺0.1 (⫺0.38 to 0.18) 25 ⫾4⫺0.04 (⫺0.31 to 0.24)
116 ⫾14 ⫺1.63 (⫺5.51 to 2.24) 114 ⫾15 ⫺0.4 (⫺4.31 to 3.45)
71 ⫾9⫺0.8 (⫺3.6 to 2.0) 73 ⫾10 1.12 (⫺1.69 to 3.93)
82 ⫾11 ⫺1.76 (⫺4.58 to 1.06) 82 ⫾9⫺2.43 (⫺5.3 to 0.44)
200 ⫾48 ⫺0.2 (⫺8.1 to 7.7) 205 ⫾45* ⫺10.5 (⫺19.1 to ⫺1.84)
57 ⫾12 ⫺1.12 (⫺3.5 to 1.3) 58 ⫾15* ⫺3.14 (⫺5.54 to ⫺0.53)
126 ⫾40 1.4 (⫺4.6 to 7.4) 131 ⫾41* ⫺7.5 (⫺13.8 to ⫺1.2)
†‡
3.5 ⫾0.7 0.06 (⫺0.08 to 0.20) 3.6 ⫾0.8 0.02 (⫺0.13 to 0.17)
2.2 ⫾0.6 0.06 (⫺0.06 to 0.18) 2.3 ⫾0.8 ⫺0.003 (⫺0.13 to 0.12)
73 (58, 100) 4.5 (⫺17.3, 18.5) 68 (60, 97) 4 (⫺10, 19)
65 ⫾22 2.4 (⫺3.04 to 7.9) 63 ⫾24 2.1 (⫺3.68 to 7.83)
52 (43, 66) ⫺1.6 (⫺10.5, 7.4) 47 (35, 75)* ⫺4.3 (⫺18.2, 6.6)
10.7 ⫾3.5 ⫺0.41 (⫺1.75 to 0.93) 10.1 ⫾4.4* ⫺1.48 (⫺2.82 to ⫺0.15)
16 (0, 51) 0 (⫺47, 33) 0 (0, 39)* ⫺2.5 (⫺47, 0)
253 (175, 328) 8 (⫺60, 49) 194 (176, 250) ⫺6(⫺25, 29)
664 (368, 965) 19 (⫺375, 147) 549 (248, 634)* ⫺63 (⫺434, 75)
1256 (706, 2773) 123 (⫺875, 915) 923 (455, 2467) ⫺81 (⫺1435, 602)
0.0 (0.02, 0.12)* ⫺0.03 (⫺0.1, 0) 0.03 (0.02, 0.11)* ⫺0.02 (⫺0.06, 0)
†
113 (61, 206) 6.7 (⫺23 to 37) 120 (23, 226) ⫺10 (⫺40 to 20)
7NUTRIGENOMIC EFFECTS OF OLIVE OIL POLYPHENOLS
TABLE 4. Change in expression of atherosclerosis-related genes after 3 mo of intervention
Gene symbol Gene name Control, n⫽20
TMD-global,
n⫽36 Pvalue
Cholesterol, lipid
transport, and
metabolism
ABCA1 ATP-binding cassette, subfamily A, member 1 0.320 ⫾0.231 0.051 ⫾0.159 0.334
ABCG1 ATP-binding cassette, subfamily G, member 1 0.146 ⫾0.127 0.064 ⫾0.092 0.608
ANXA1 Annexin A1 0.259 ⫾0.229 ⫺0.444 ⫾0.161 0.160
ARHGAP15 Rho GTPase activating protein 15 0.448 ⫾0.175 ⫺0.040 ⫾0.126 0.043
ARHGAP19 Rho GTPase activating protein 19 0.400 ⫾0.151 0.134 ⫾0.112 0.166
ARHGEF6 Rac/Cdc42 guanine nucleotide exchange factor 6 0.460 ⫾0.144 0.157 ⫾0.106 0.099
CD36 CD36 molecule (thrombospondin receptor) 0.197 ⫾0.170 ⫺0.009 ⫾0.126 0.342
CETP Cholesteryl ester transfer protein, plasma ⫺0.262 ⫾0.331 ⫺0.058 ⫾0.257 0.631
MSR1 Macrophage scavenger receptor 1 0.542 ⫾0.222 0.253 ⫾0.157 0.301
PLA2G4B Phospholipase A2, group IVB 0.148 ⫾0.156 0.082 ⫾0.109 0.735
SCARB1 Scavenger receptor class B, member 1 ⫺0.025 ⫾0.078 0.085 ⫾0.056 0.261
Inflammation
IFNg Interferon, ␥1.048 ⫾0.464 ⫺0.109 ⫾0.330 0.049
IL10 Interleukin 10 0.915 ⫾0.360 0.609 ⫾0.270 0.506
CHUK Conserved helix-loop-helix ubiquitous kinase 0.325 ⫾0.192 0.036 ⫾0.140 0.236
ADAM17 ADAM metallopeptidase domain 17 (tumor necrosis
factor, ␣, converting enzyme) 0.290 ⫾0.153 0.008 ⫾0.112 0.148
ADAMTS1 ADAM metallopeptidase with thrombospondin type 1
motif, 1 0.166 ⫾0.208 ⫺0.120 ⫾0.150 0.277
IFNA1 Interferon, ␣1 0.726 ⫾0.356 0.001 ⫾0.258 0.117
TNFSF10 Tumor necrosis factor (ligand) superfamily, member 10 0.195 ⫾0.219 ⫺0.195 ⫾0.156 0.157
TNFSF12_13 Tumor necrosis factor (ligand) superfamily, member
12-member 13 ⫺0.021 ⫾0.102 0.133 ⫾0.075 0.235
IL6 Interleukin 6 ⫺0.017 ⫾0.588 0.356 ⫾0.401 0.612
IL7R Interleukin 7 receptor 0.580 ⫾0.182 0.095 ⫾0.132 0.037
USP48 Ubiquitin specific peptidase 48 0.380 ⫾0.179 0.203 ⫾0.131 0.431
MPO Myeloperoxidase ⫺0.159 ⫾0.121 ⫺0.013 ⫾0.090 0.343
RGS2 Regulator of G-protein signaling 2, 24 kDa 0.439 ⫾0.268 0.289 ⫾0.196 0.656
NFKB2 Nuclear factor of light polypeptide gene enhancer in
B-cells 2 ⫺0.098 ⫾0.082 0.008 ⫾0.063 0.315
Nuclear receptors
and fatty acids
receptors
NR1H2 Nuclear receptor subfamily 1, group H, member 2 ⫺0.081 ⫾0.070 ⫺0.003 ⫾0.050 0.369
NRIH3 Nuclear receptor subfamily 1, group H, member 3 0.166 ⫾0.108 0.034 ⫾0.077 0.331
PPARA Peroxisome proliferator-activated receptor ␣0.088 ⫾0.123 0.068 ⫾0.092 0.897
PPARBP PPAR binding protein 0.341 ⫾0.160 0.022 ⫾0.105 0.084
PPARG Peroxisome proliferator-activated receptor ␥0.002 ⫾0.242 0.235 ⫾0.175 0.463
PPARD Peroxisome proliferator-activated receptor ␦0.066 ⫾0.128 0.010 ⫾0.096 0.732
Oxidative stress
LIAS Lipoic acid synthetase 0.228 ⫾0.197 0.188 ⫾0.148 0.874
PTGS1 Prostaglandin-endoperoxide synthase 1 ⫺0.176 ⫾0.171 ⫺0.170 ⫾0.117 0.978
PTGS2 Prostaglandin-endoperoxide synthase 2 0.170 ⫾0.545 ⫺0.231 ⫾0.379 0.557
OLR1 Oxidized low-density lipoprotein (lectin-like) receptor 1 0.521 ⫾0.948 0.113 ⫾0.580 0.724
OSBP Oxysterol binding protein 0.219 ⫾0.130 0.035 ⫾0.093 0.260
ADRB2 Adrenergic, -2, receptor, surface 0.225 ⫾0.135 ⫺0.138 ⫾0.098 0.036
OGT O-linked N-acetylglucosamine (GlcNAc) transferase 0.373 ⫾0.235 0.014 ⫾0.162 0.218
ALDH1A1 Aldehyde dehydrogenase 1 family, member A1 ⫺0.101 ⫾0.187 ⫺0.116 ⫾0.135 0.949
DNA repair
CCNG1 Cyclin G1 0.396 ⫾0.192 0.004 ⫾0.139 0.106
POLK Polymerase (DNA directed) 0.595 ⫾0.275 ⫺0.115 ⫾0.204 0.045
TP53 Tumor protein p53 ⫺0.071 ⫾0.077 ⫺0.048 ⫾0.056 0.812
DCLRE1C DNA cross-link repair 1C 0.406 ⫾0.169 0.052 ⫾0.123 0.100
ERCC5 Excision repair cross-complementing rodent repair
deficiency, complementation group 5 0.401 ⫾0.227 0.049 ⫾0.169 0.221
XRCC5 X-ray repair complementing defective repair in Chinese
hamster cells 5 (double-strand-break rejoining; Ku
autoantigen, 80 kDa)
0.267 ⫾0.152 0.000 ⫾0.111 0.166
Gene expression changes, adjusted by age and sex, are presented as means ⫾se of the RQ log
2
ratio (posttreatment vs. basal values).
8 Vol. 24 July 2010 KONSTANTINIDOU ET AL.The FASEB Journal 䡠www.fasebj.org
The ADRB2 gene was also down-regulated after 3
mo of TDM intervention, particularly in the TDM⫹
VOO intervention group. A recent study has demon-
strated that the ADRB2 blockade reduces macro-
phage cytokine production and improves survival
after traumatic injury (56). ADRB2 agonists can affect
glucose homeostasis through the modulation of in-
sulin and glucagon secretion, hepatic glucose pro-
duction, and glucose uptake into muscle (57). In this
sense, we have previously reported up-regulation of
the ADRB2 expression in human PBMNCs at the
postprandial state after ingestion of 50 ml of VOO
(22). This olive oil ingestion promoted a postpran-
dial peak of insulin, lipid oxidative damage, and
triglycerides, and the ADRB2 expression at 6 h post-
prandial was inversely correlated with plasma oxLDL
and triglyceride concentrations (22). Oxidation of
the lipids and apoproteins present in LDL leads to a
change in the lipoprotein conformation by which
LDL is better able to enter the monocyte/macro-
phage system of the arterial wall and promote the
atherosclerotic process (58). In functional studies
the ADRB2 receptor appears to be protective against
oxidative stress (22, 59). In our present study, after 3
mo of TMD⫹VOO intervention, an improvement in
the oxidative status of the volunteers was observed.
These data are in agreement with those obtained in
the EUROLIVE study, in which a dose-dependent
decrease of the lipid oxidative damage was observed
with the phenol content of the administered olive oil
(13). One of our trial’s strengths is that the study
design is able to provide first-level scientific evidence
(60), reflecting eating habits of community-dwelling
individuals. Compliance of the volunteers was good
as reflected in the changes of olive oil consumption
patterns and urinary tyrosol and hydroxytyrosol. The
lack of significance in the increase in urinary olive oil
phenolics in the TMD⫹VOO group vs. the control
group could be due to the fact that the control group
participants followed their habitual diet, which in
Mediterranean countries includes VOO, or the high
interindividual variation in urinary phenolic values,
particularly in the case of hydroxytyrosol (28). We
worked with whole dietary patterns at real-life doses
of food. Administration of isolated antioxidants (i.e.,
hydroxytyrosol) at high doses has been shown to
promote the atherosclerosis lesion, as well as an
increase in oxidative damage, in apolipoprotein E-
Figure 3. Gene expression changes in adrenergic 
2
-receptor (ADRB2; A), Rho GTPase activating protein 15 (ARHGAP15; B),
INF-␥(IFN␥;C), and IL-7 receptor (IL7R; D) genes after the 3-mo interventions. P⬍0.05 for linear trend in all cases; *P⬍0.05
vs. control group.
9NUTRIGENOMIC EFFECTS OF OLIVE OIL POLYPHENOLS
deficient mice (61). This finding points out the
importance of the matrix and the dose of antioxi-
dants. Changes in gene expression were modest, as
was expected in real-life intervention conditions. The
lack of a washout period at the beginning of the study
could also be one factor responsible for the relative
low gene response observed. We worked against our
own hypothesis, by using the current Spanish dietary
pattern in our control group, to maintain real-life
conditions in all groups. A study limitation was the
inability to assess potential interactions between the
olive oil and other diet components that might affect
the generalization of the results. However, the effects
of food components are subtle and must be consid-
ered in the context of chronic exposure. Whether
additional or different effects would have been ob-
served over longer periods is unknown. A longer
study, however, could have impaired the compliance
of the participants.
In summary, a down-regulation in the expression
of atherosclerosis-related genes occurs in human
PBMNCs after 3 mo of TMD. Our results point out a
significant role of olive oil polyphenols in the down-
regulation of proatherogenic genes in the frame of
the Mediterranean diet. Changes in gene expression
were concomitant with decreases in lipid oxidative
damage and systemic inflammation markers. Our
results support the idea that the benefits associated
TABLE 5. Functional annotation clustering (biological processes level 5)
Functional
group Enrichment Gene Ontology Gene symbol
Adjusted
Pvalue
1 3.86 GO:0008203; cholesterol metabolic
process ABCG1, ABCA1, CETP, PPARD, SCARB1 ⬍0.001
GO:0016125; sterol metabolic
process ABCG1, ABCA1, CETP, PPARD, SCARB1 ⬍0.001
GO:0008202; steroid metabolic
process OSBP, ABCG1, ABCA1, CETP, PPARD,
SCARB1
⬍0.001
2 2.67 GO:0006915; apoptosis IL6, IFNG, MPO, ERCC5, TP53, IL10,
TNFSF10, ARHGEF6, PPARD, ANXA1,
SCARB1, ADRB2
⬍0.001
GO:0042981; regulation of
apoptosis IL6, MPO, ERCC5, TP53, IL10, TNFSF10,
ANXA1, ADRB2
0.001
GO:0043066; negative regulation
of apoptosis IL6, MPO, ERCC5, IL10, ANXA1 0.005
GO:0043065; positive regulation of
apoptosis TP53, TNFSF10, ADRB2 0.181
3 2.19 GO:0031325; positive regulation of
cellular metabolic process IL6, IFNG, MED1, TP53, IL10, PPARG,
ABCA1, PPARA, ADRB2
⬍0.001
GO:0045935; ositive regulation of
nucleobase, nucleoside,
nucleotide, and nucleic acid
metabolic process
IL6, IFNG, MED1, TP53, PPARG, ABCA1,
PPARA, ADRB2
⬍0.001
GO:0045893; positive regulation of
transcription, DNA-dependent IL6, IFNG, MED1, TP53, PPARG, PPARA,
ADRB2
⬍0.001
GO:0031324; negative regulation
of cellular metabolic process IL6, TP53, IL10, PPARG, NR1H2, PPARD 0.006
GO:0006357; regulation of
transcription from RNA
polymerase II promoter
MED1, TP53, PPARG,PPARA, PPARD,
ADRB2
0.014
GO:0019219; regulation of
nucleobase, nucleoside,
nucleotide, and nucleic acid
metabolic process
IL6, MED1, TP53, PPARA, XRCC5,
NR1H3, IL7R, IFNG, PPARG,NFkB2,
ABCA1, PPARD, ADRB2
0.047
4 1.64 GO:0006631; fatty acid metabolic
process CD36, PTGS1, PTGS2, PPARA, PPARD 0.002
GO:0032787; monocarboxylic acid
metabolic process CD36, PTGS1, PTGS2, PPARA, PPARD 0.006
GO:0008544; epidermis
development PTGS2, PPARA, PPARD 0.075
GO:0009888; tissue development PTGS2, PPARA, PPARD, ADRB2 0.084
5 1.58 GO:0048534; hemopoietic or
lymphoid organ development IL6, IL7R, CHUK, IL10, NFkB2 0.003
GO:0002521; leukocyte
differentiation IL6, CHUK, IL10 0.045
GO:0030097; hemopoiesis IL6, CHUK, IL10 0.116
10 Vol. 24 July 2010 KONSTANTINIDOU ET AL.The FASEB Journal 䡠www.fasebj.org
with a Mediterranean-type diet and olive oil polyphe-
nol consumption on CHD risk can be mediated
through changes in the expression of atherosclerosis-
related genes. Data from this study provide further
evidence to recommend the TMD and rich-polyphe-
nol olive oils, such as VOO, as a useful tool for the
prevention of atherosclerosis.
The CIBER de Fisiopatología de la Obesidad y Nutricio´ n is an
initiative of the Instituto de Salud Carlos III, Madrid, Spain. This
work was supported by Fóndo de Investigación Sanitaria–Fóndo
Europeo de Desarrollo Regional (FIS-FEDER; PI041308), by
Sistema National de Salud (SNS) contract Miguel Servet (CP06/
00100) Instituto de Salud Carlos III, and by the Greek State
Scholarship Foundation (Athens, Greece), and partially sup-
ported by the Generalitat of Catalunya (2005 SGR 00577). The
authors declare no conflicting financial interests.
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Received for publication October 28, 2009.
Accepted for publication January 21, 2010.
12 Vol. 24 July 2010 KONSTANTINIDOU ET AL.The FASEB Journal 䡠www.fasebj.org
