Polyphenol-Rich Aronia melanocarpa Juice Consumption Affects LINE-1 DNA Methylation in Peripheral Blood Leukocytes in Dyslipidemic Women

ORIGINAL RESEARCH

published: 17 June 2021

doi: 10.3389/fnut.2021.689055

Frontiers in Nutrition | www.frontiersin.org 1 June 2021 | Volume 8 | Article 689055

Edited by:

Rosita Gabbianelli,

University of Camerino, Italy

Reviewed by:

Fabio Caradonna,

University of Palermo, Italy

Nenad Naumovski,

University of Canberra, Australia

*Correspondence:

Ljiljana Stojkovi

[email protected]

Specialty section:

This article was submitted to

Nutrigenomics,

a section of the journal

Frontiers in Nutrition

Received: 31 March 2021

Accepted: 26 May 2021

Published: 17 June 2021

Citation:

Stojkovi

c L, Zec M, Zivkovic M,

Bundalo M, Boškovi

c M, Glibeti

c M

and Stankovic A (2021)

Polyphenol-Rich Aronia melanocarpa

Juice Consumption Affects LINE-1

DNA Methylation in Peripheral Blood

Leukocytes in Dyslipidemic Women.

Front. Nutr. 8:689055.

doi: 10.3389/fnut.2021.689055

Polyphenol-Rich Aronia melanocarpa

Juice Consumption Affects LINE-1

DNA Methylation in Peripheral Blood

Leukocytes in Dyslipidemic Women

Ljiljana Stojkovi

, Manja Zec

2,3

, Maja Zivkovic

, Maja Bundalo

1,4

, Maja Boškovi

Marija Glibeti

and Aleksandra Stankovic

Laboratory for Radiobiology and Molecular Genetics, Department of Health and Environmental Research, “Vin

ca” Institute of

Nuclear Sciences—National Institute of the Republic of Serbia, University of Belgrade, Belgrade, Serbia,

Centre of Research

Excellence in Nutrition and Metabolism, Institute for Medical Research—National Institute of the Republic of Serbia, University

of Belgrade, Belgrade, Serbia,

Department of Nutritional Sciences, University of Arizona, Tucson, AZ, United States,

Institute of Experimental Biomedicine, University Hospital Würzburg, Würzburg, Germany

Cardiovascular disease (CVD) is associated with alterations in DNA methylation and

polyunsaturated fatty acid (PUFA) proﬁle, both modulated by dietary polyphenols. The

present parallel, placebo-controlled study (part of the original clinical study registered

as NCT02800967 at www.clinicaltrials.gov) aimed to determine the impact of 4-week

daily consumption of polyphenol-rich Aronia melanocarpa juice (AMJ) treatment on Long

Interspersed Nucleotide Element-1 (LINE-1) methylation in peripheral blood leukocytes

and on plasma PUFAs, in subjects (n = 54, age range of 40.2 ± 6.7 years) at

moderate CVD risk, including an increased body mass index, central obesity, high

normal blood pressure, and/or dyslipidemia. The goal was also to examine whether

factors known to affect DNA methylation (folate intake levels, MTHFR C677T gene

variant, anthropometric and metabolic parameters) modulated the LINE-1 methylation

levels upon the consumption of polyphenol-rich aronia juice. Experimental analysis of

LINE-1 methylation was done by MethyLight method. MTHFR C677T genotypes were

determined by the polymerase c hain reaction–restriction fragment length polymorphism

method, and folate intake was assessed by processing the data from the food frequency

questionnaire. PUFAs were measured by gas–liquid chromatography, and serum lipid

proﬁle wa s determined by using Roche Diagnostics kits. The statistical analyses were

performed using Statistica software package. In the comparison after vs. before the

treatment period, in dyslipidemic women (n = 22), we observed signiﬁcant decreases in

LINE-1 methylation levels (97.54 ± 1.50 vs. 98.39 ± 0.86%, respectively; P = 0.01) and

arachidonic acid/eicosapentae noic acid ratio [29.17 ± 15.21 vs. 38.42 (25.96–89.58),

respectively; P = 0.02]. The change (after vs. before treatment) in LINE-1 methylation

directly correlated with the presence of MTHFR 677T allele, average daily folate intake,

and the change in serum low-density lipoprotein cholesterol but inversely correlated with

the change in serum triacylglycerols (R = 0.72, R

= 0.52, adjusted R

= 0.36, P = 0.03).

Stojkovi

c et al. Aronia Polyphenols Affect LINE-1 Methylation

The current results imply potential cardioprotective effects of habitual polyphenol-rich

aronia juice consumption achieved through the modiﬁcations of DNA me thyla tion patt ern

and PUFAs in subjects at CVD risk, which should be further conﬁrmed. Hence, the

precision nutrition-driven modulations of both DNA methylation and PUFA proﬁle may

become targets for new approaches in the prevention of CVD.

Keywords: Aronia melanocarpa, polyphenols, polyunsaturated fatty acids, LINE-1, methylation, peripheral blood

leukocytes, cardiovascular risk

INTRODUCTION

Pathogenesis of human chronic diseases, such as cancer and

cardiovascular disease (CVD), is related to aberrant global and

locus-speciﬁc DNA methylation patterns (

1, 2). Methylation of

DNA, catalyzed by DNA methyltransferases (DNMTs), is one

of the main epigenetic processes, which most commonly occurs

at cytosine-guanine (CpG) dinucleotide clusters and results in

downregulation of gene expression (

3).

Various exogenous and endogenous factors modulate DNA

methylation. Folate (vitamin B9) is found in a variety of plant

foods and participates in one-carbon metabolism, resulting in

the formation of S-adenosyl-methionine (SAM) that acts as a

methyl group donor (4). Methylenetetrahydrofolate reductase

(MTHFR) activation catalyzes the conversion of homocysteine to

methionine, which is a direct precursor of SAM. The presence

of T allele at a common C677T (Ala2 22Val) MTHFR gene

polymorphic site is associated with a decreased activity of the

enzyme, thus reducing the methyl group bioavailability and

subsequently inhibiting the methylation of DNA (

5).

Methylation of Long Interspersed Nucleotide Element-1

(LINE-1), the largest member of the LINE retrotransposon

family of DNA repeat elements (comprising about 17% of

the human genome) (6), is considered a surrogate marker of

global DNA methylation (7, 8). Methylation status of LINE-1

in peripheral blood leukocytes is associated with metabolic

parameters, such as blood glucose and lipid proﬁles (9, 10),

and global DNA methylation in these cells has been found to

depend on demographic and lifestyle factors: age, gender, blood

pressure, body mass index (BMI), and dietary habits, including

polyunsaturated fatty acid (PUFA) supplementation (9, 11–14).

In addition, the studies have reported that the inhibition of DNA

methylation may prevent the progression of cancer and CVD

(

1, 2, 15). Hence, the methylation stat us of LINE-1 in peripheral

blood leukocytes represents a potential CVD biomarker, and

certain lifestyle factors, like diet, may modulate cardiovascular

risk by inﬂuencing alterations in DNA methylation patterns, thus

Abbreviations: AA, arachidonic acid; BMI, body mass index; COMT, catechol-

O-methyltransferase; CVD, cardiovascular disease; DBP, diastolic blood pressure;

DNMTs, DNA methyltransferases; EDTA, ethylenediaminetetraacetic acid;

EPA, eicosapentaenoic acid; Glu, glucose; HDL-C, high-density lipoprotein

cholesterol; LDL-C, low-density lipoprotein cholesterol; LINE-1, Long

Interspersed Nucleotide Element-1; MTHFR, methylenetetrahydrofolate reductase;

MUFA, monounsaturated fatty acid; PCR, polymerase chain reaction; PUFA,

polyunsaturated fatty acid; SAH, S-adenosyl-L-homocysteine; SAM, S-adenosyl-

methionine; SBP, systolic blood pressure; SFA, saturated fatty acid; TAG,

triacylglycerols; TC, total cholesterol; WC, waist circumference.

emphasizing t he importance of precision nutrition strategies in

the prevention of CVD.

Regular consumption of Aronia melanocarpa juice is

associated with CVD beneﬁcial eﬀects in human studies (

16–18)

and animal models (19). Aronia is rich in bioactive polyphenols

(

20) and, compared with ot h er berry fruits, contains higher levels

of polyphenolic compounds (21). Of note, dietary polyphenols

are reported to favorably modulate DNA methylation status by

inhibiting DNMT activity (22). In addition, aronia polyphenols

aﬀected the composition of plasma PUFAs in individuals at

CVD risk (23). To the best of our knowledge, no study has

examined the relation between polyphenols contained in aronia

berry and DNA methylation status. Therefore, the present

parallel, placebo-controlled, 4-week study aimed to investigate

the interconnection between daily consumption of polyphenol-

rich a ronia juice and LINE-1 methylation in peripheral blood

leukocytes and plasma fatty acids, in subjects at moderate CVD

risk. We also examined whether folate intake and MTHFR

C677T gene variant, as well as the anthropometric and metabolic

parameters, modulated the LINE-1 methylation levels upon the

consumption of polyphenol-rich aronia juice.

MATERIA LS AND METHODS

Study Design, Study Subjects, and

Intervention Treatments

The current research represents a parallel, placebo-controlled,

4-week nutritional intervention. The research is designed as a

substudy, forming part of t h e original clinical study that lasted 6

months, and is registered at ClinicalTrials.gov as NCT02800967.

The original study included nonsmoking adults at moderate

CVD risk, deﬁned as the presence of at least one of the

following: increased BMI (25–30 kg/m

), central obesity (waist

circumference ≥ 80 cm for women and ≥ 94 cm for men), and

high normal blood pressure [systolic/diastolic blood pressure

(SBP/DBP) > 120/80, ≤ 139/89 mm Hg]. E xclusion criteria

were the presence of chronic disease, self-reported allergy to

polyphenols, pregnancy, lactation, blood donation 16 weeks

before the start of the study, and parallel participation in another

clinical trial. During the course of the study, the participants were

asked to follow their habitual diet, including study treatments as

part of it, and to do their usual physical activity. They were also

asked to strictly refrain from berries and berry products and to

avoid excess amounts of polyphenol-rich food, inclusive of olive

oil, green tea, and nuts.

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Stojkovi

c et al. Aronia Polyphenols Affect LINE-1 Methylation

For purposes of the current substudy, additional a posterior i

inclusion criterion was the presence of dyslipidemia deﬁned as

either elevated serum total cholesterol (≥5.2 mmol/l), elevated

low-density lipoprotein cholesterol (LDL-C) (≥3.4 mmol/l),

or elevated serum triacylglycerols (≥1.7 mmol/l). In order

to address the objectives of the substudy, 54 subjects were

included, whose LINE-1 methylation status in peripheral blood

leukocytes and MTHFR C677T gene variant were additionally

analyzed. They received either original polyphenol-rich A.

melanocarpa juice (assigned as AMJ treatment, N = 34 subjects)

or polyphenol-free placebo beverage (assigned as PLB treatment,

N = 20 subjects). The research ﬂow diagram is shown in Figure 1.

The original polyphenol-rich aronia juice used in the study

was registered at the Serbian Ministry of Health as a dietary

supplement and was donated from “Nutrika” LTD (Belgrade,

Serbia). The placebo drink was made to match the appearance,

taste, and nutritional composition of the original aronia juice, but

without bioactive polyphenols. It was previously reported that

the daily amount of 100 ml of the placebo was safe for human

consumption (

24). Total polyphenols in the original aronia juice

were determined using a modiﬁed Folin-Ciocalteu method (25),

and it was found that the consumed daily amount of 100 ml

of the juice contained 1,177.11 mg of gallic acid equivalents of

polyphenols. Proanthocyanidins were hig hly represented among

the contained polyphenols, as demonstrated in the analysis of

the composition of herein used aronia juice (24). The study

compliance was assessed according to returned empty bottles of

intervention drinks and self-reports.

The study protocol adhered to the regulations of the 1975

Declaration of Helsinki and was approved by Clinical Hospital

Centre Zemun, Belgrade, Serbia, Ethics Committee Approval,

No: 2125, 2013. The written informed consent was given by all

participants before the commencement of the study.

Sample Collection

Study participants were instructed for overnight fasting,

and venous blood was collected the next morning between

8 and 9 AM into t h e sample tubes for serum and

ethylenediaminetetraacetic acid (EDTA)-evacuated tubes.

The sample collection was done at two time points, before and

after the corresponding 4-week treatments (AMJ and PLB).

Assessment of Study Variables

For the assessment of baseline dietary intake, trained staﬀ

conducted structured int erviews with study subjects and

collected data by using the food frequency questionnaire and

repeated 24-h dietary recalls. The subjects were assisted with

125-item photo-booklet containing simple foods and composite

dishes (

26). Data from dietary recalls were analyzed using the

nutritional platform for comprehensive diet evaluation (26, 27).

Bio-impedance analyzer TANITA UM072 balance (TANITA

Health Equipment H.K. Ltd, Hong Kong, China) was used for

the determination of body weight. Tot a l cholesterol, high-density

lipoprotein cholesterol (HDL-C), LDL-C, triacylglycerols, and

glucose from serum were determined by Roche Diagnostics Kits,

using t h e chemistry analyzer (Cobas c111, Roche Diagnostics,

Basel, Switzerland).

The procedure of determination of plasma phospholipid

fatty acids composition was previously described in detail by

Pokimica et al. (

23). Brieﬂy, plasma lipids were extracted

using a 2:1 chloroform–methanol mixture with 2,6-di-tert-

butyl-4-methylphenol (10 mg/100 ml) added as an antioxidant.

Phospholipids were separated from other lipid subclasses on

a thin-layer chromatography silica plate using a mixture of

petroleum ether, diethyl ether, and acetic acid (87:12:1). The

methyl esters of fatty acids were obtained by transmethylation

with 2M sodium hydroxide in methanol, at 85

◦

C for 1 h,

and with 1M sulfuric acid in methanol, at 85

◦

C for 2 h. The

mixture was cooled down to room temperature and centrifuged

at 1,860 × g for 15 min, and the upper phase was dried

up using a stream of nitrogen. The fatty acid methyl esters

were recovered in hexane and separated by using RTX 2330

capillary column (60 m × 0.25 mm × 0.2 µm; Restek, Bellefonte,

PA, United States), on Shimadzu GC-2014 gas chromatograph

(Kyoto, Japan) with ﬂame ionization detector. The ﬂow of

air, hydrogen, a nd helium (carrier gas) was 320, 30, and 5

ml/min, respectively. The temperature of the detector was

260

◦

C and of the injection port 220

◦

C. The initial column

temperature of 140

◦

C was maintained for 5 min and then

increased to 220

◦

C at a rate of 3

◦

C/min, and 220

◦

C was

kept for 20 min. Fatty acids were identiﬁed by comparing peak

retention times with calibration mixtures (PUFA-2, Supelco,

Bellefonte, PA, United States, and 37 FAMEs mix, Sigma

Chemical Co., St. Louis, MO, United States). The amounts of

individual f at ty acids in plasma phospholipids were presented

as the relative area percentage of the total pool of detected

fatty acids.

Analysis of LINE-1 Methylation

The fasting peripheral blood samples of each part icipant ,

collected with EDTA before and after the corresponding

treatments, were used for the total leukocyte genomic

DNA isolation, by a phenol–chloroform extraction-based

method (

28). The quantity of DNA was estimated wit h

BioSpecnano spectrophotometer (Shimadzu Biotech, Kyoto,

Japan).

Five hundred (500) ng of each sample genomic DNA was

used for sodium bisulﬁte conversion with EpiTect



Bisulﬁte kit

(Qiagen, Hilden, Germany). Bisulﬁte-converted DNA was then

used for the quantiﬁcation of LINE-1 methylation by MethyLight

real-time PCR. To normalize DNA input, an Alu sequence-based

real-time PCR control reaction was performed in parallel with

each LINE-1 reaction. The method was previously developed

and validated, its precision and reproducibility were conﬁrmed

(

8, 29), and the currently used protocol was described in detail by

Božovi

c et al. (29).

MTHFR Genotyping

The genotypes of MTHFR C677T variant were determined from

the total leukocyte genomic DNA samples, by PCR–restriction

fragment length polymorphism method reported in Coppedè

et al. (

30), in all subjects included in t he methylati on analysis.

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Stojkovi

c et al. Aronia Polyphenols Affect LINE-1 Methylation

FIGURE 1 | Study ﬂow diagram. N, number of subjects; AMJ, polyphenol-rich Aronia melanocarpa juice treatment; PLB, polyphenol-free bev erage;

placebo, treatment.

Statistical Analyses

The comparisons of MTHFR C677T genotype frequencies

between groups and estimation of deviations from

Hardy–Weinberg equilibrium were done with Fisher’s exact

test and th e χ

test. Due to a small number of the TT genotype

carriers (N < 3) in at least one of the analyzed groups, we

applied the dominant genotype model, CT + TT vs. CC, instead

of the additive. Depending on the distribution of continuous

variables (age, average daily energy intake, average daily folate

intake, SBP, DBP, waist circumference, BMI, serum glucose,

triacylglycerols, total cholesterol, HDL-C, LDL-C, plasma fatty

acids, and LINE-1 methylation levels), tested by the Shapiro–

Wilks test, the appropriate parametric or non-parametric tests

were performed. Between-group comparisons were done using

a t-test or the Mann–Whitney U-test, while within-group

comparisons (after vs. before each treatment) were done by t-test

for dependent samples or the Wilcoxon matched-pairs test, for

normally and non-normally distributed data, respectively. The

treatment eﬀects were investigated in the complete sample and

in women and men, separately. The relationship between LINE-1

methylation and the anthropometric and metabolic parameters

(age, average daily folate intak e, average daily energy intake, SBP,

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c et al. Aronia Polyphenols Affect LINE-1 Methylation

TABLE 1 | Baseline characteristics of the study participants.

AMJ PLB P

No. of subjects 34 20

Age (years) 41.1 ± 6.6 38.5 ± 6.8 0.17

Average daily energy intake (kCal) 2075 ± 555 1745 (1168–3385)

0.29

Average daily folate intake (µg) 239.7 ± 75.2 203.3 (98.2–461.5)

0.54

SBP (mmHg) 118.3 ± 13.4 120.6 ± 16.2 0.57

DBP (mmHg) 73.2 ± 10.0 74.0 ± 13.7 0.82

Waist circumference (cm) 89.8 ± 10.9 92.2 ± 15.7 0.51

BMI (kg/m

) 27.4 ± 3.5 27.8 ± 6.2 0.78

Glucose (mmol/l) 4.8 (3.8–7.2)

5.1 ± 0.8 0.27

TAG (mmol/l) 0.9 (0.4–4.1)

0.9 (0.5–5.0)

0.72

TC (mmol/l) 5.5 ± 1.1 5.2 ± 1.0 0.27

HDL-C (mmol/l) 1.5 (0.8–2.9)

1.6 ± 0.4 0.54

LDL-C (mmol/l) 3.5 ± 0.9 3.3 ± 1.0 0.56

†

MTHFR C677T genotype, No. (%)

CC 15 (44.1) 7 (35.0)

CT + TT 19 (55.9) 13 (65.0) 0.58

Continuous variables with a normal distribution are presented as mean ± standard deviation;

continuous variables with a non-normal distribution are presented as median

(minimum–maximum); P-values related to the between-treatment difference in the variable distribution, signiﬁcant difference at P < 0.05.

†

The observed MTHFR C677T genotype frequencies are consistent with Hardy–Weinberg equilibrium, in each analyzed group (χ

test, P > 0.05).

AMJ, polyphenol-rich Aronia melanocarpa juice treatment; PLB, polyphenol-free beverage, placebo treatment; SBP, systolic blood pressure; DBP, diastolic blood pressure; BMI , body

mass index; TAG, triacylglycerols; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.

DBP, waist circumference, BMI, serum glucose, triacylglycerols,

total cholesterol, HDL-C, LDL-C, and plasma fatty acids) was

investigated by regression analyses. T-test/Mann–Whitney

U-test was used to examine the association between LINE-1

methylation and MTHFR genotypes, by a genotype model,

CT+T T vs. CC. In the statistical tests, P < 0.05 were considered

statistically signiﬁcant. The statistica l analyses were performed

using Statistica 8.0 software package (StatSoft, Inc. 1984-2007).

RESULTS

Baseline Characteristics of the Study

Subjects and Effects of Polyphenol-Rich

Aronia Juice Consumption on

Cardiometabolic Parameters

Baseline characteristics, determined before each of the two

treatments, with polyphenol-rich aronia juice (AMJ) and

polyphenol-free beverage (placebo, PLB), for the whole treated

study groups are shown in Table 1. For the treated groups

separated by gender, the baseline characteristics are shown in

Supplementary Table 1. There were no signiﬁcant diﬀerences

in the baseline parameters (AMJ vs. PLB) within any of the

groups (whole sample, women and men), except for serum total

cholesterol in men [AMJ vs. PLB = 5.9 ± 1.0 vs. 4.9 ± 1.2 mmol/l;

P (t-test) = 0.04] (Table 1 and Supplementary Table 1).

Within-group comparisons of treatment eﬀects (after vs.

before treatment) toward anthropometric and metabolic

parameters are presented in Supplementary Table 2.

Even though signiﬁcant diﬀerences were found for DBP,

waist circumference, glucose, and HDL-C [P (t-test for

dependent samples/Wilcoxon matched pairs test) < 0.05]

(Supplementary Table 2), only values for waist circumference

dropped out of the clinical ranges.

Effects of Polyphenol-Rich Aronia Juice

Consumption on LINE-1 Methylation

The comparisons of LINE-1 methylation levels after vs. before

polyphenol-rich AMJ treatment and polyphenol-free beverage

(placebo) treatment (PLB) are presented in Figure 2. Although

the AMJ treatment tended to decrease LINE-1 methylation

levels, there was no signiﬁcant diﬀerence in the whole study

group [after vs. before treatment = 97.93 (93.58–99.84)% vs.

98.16 (93.58–99.91)%; P (Wilcoxon matched pairs test) =

0.14] (Figure 2A). We found a signiﬁcant decrease in LINE-1

methylation after the AMJ treatment in women (after vs. before

treatment = 97.54 ± 1.50 vs. 98.39 ± 0.86%; P (t-test for

dependent samples) = 0.01) (Figure 2C).

In women, where we demonstrated that polyphenol-rich AMJ

treatment signiﬁcantly changed LINE-1 methylation, the multiple

regression model showed signiﬁcant eﬀects of average daily

folate intake, MTHFR C677T genotypes (model CT+TT vs. CC),

change (1, after vs. before treatment) in triacylglycerols, and

change (1) in LDL-C on the change (1) in LINE-1 methylation

levels, while controlling for age (R = 0.72, R

= 0.52, adjusted R

= 0.36, P = 0.03) (Table 2).

Impact of Polyphenol-Rich AMJ Treatment

on the Proﬁle of Plasma Fatty Acids

Composition of plasma phospholipids fatty acids was analyzed

in women who consumed polyphenol-rich aronia juice, as,

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c et al. Aronia Polyphenols Affect LINE-1 Methylation

FIGURE 2 | Comparison of LINE-1 methylation levels (%) before and after treatment in: (A) all subjects on AMJ treatment (N = 34; Wilcoxon matched pairs test,

P = 0.14), (B) all subjects on PLB treatment (N = 20; Wilcoxon matched pairs test, P = 0.10), (C) women on AMJ treatment (N = 22; t-test for dependent samples,

P = 0.01), (D) women on PLB treatment (N = 10; Wilcoxon matched pairs test, P = 0.11), (E) men on AMJ treatment (N = 12; Wilcoxon matched pairs test,

P = 0.53), (F) men on PLB treatment (N = 10; Wilcoxon matched pairs test, P = 0.65). AMJ, polyphenol-rich Aronia melanocarpa juice treatment; PLB,

polyphenol-free beverage, placebo treatment; mean represents normally distributed LINE-1 methylation levels (%); median represents non-normally distributed LINE-1

methylation levels (%); SE, standard error; statistical signiﬁcance at P < 0.05 (

); N, number of subjects.

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c et al. Aronia Polyphenols Affect LINE-1 Methylation

TABLE 2 | Multiple regression analysis for the change (1) in LINE-1 methylation levels (%) in women who consumed polyphenol-rich Aronia melanocarpa juice (AMJ

treatment, N = 22).

Predictor variable β Std. error of β P

Age (years) −0.40 0.21 0.08

Average daily folate intake (µg) 0.49 0.19 0.02

MTHFR C677T genotypes (model CT + TT vs. CC) 0.50 0.19 0.02

1 Triacylglycerols (mmol/l) −0.53 0.20 0.02

1 Low-density lipoprotein cholesterol (mmol/l) 0.46 0.19 0.03

Regression summary: R = 0.72, R

= 0.52, adjusted R

= 0.36, P = 0.03.

1: change, after treatment value—before treatment value; signiﬁcant difference at P < 0.05 (bolded text).

only in these subjects, aronia juice consumption was associated

with a signiﬁcant change in LINE-1 methylation levels. Proﬁled

fatty acids are shown for 15 women treated with aronia juice

(Supplementary Table 3), whose plasma samples were available

for the analysis, representing a part of previously published data

(

23). Overall baseline plasma phospholipid fatty acids comprised

∼48% SFAs, ∼11% MUFAs, and ∼41% PUFAs. Among the most

abundant fatty acids were palmitic acid (∼30.5%) > linoleic

acid (∼23%) > stearic acid (∼17.5%) > arachidonic acid (AA)

(∼11%), while least quantitatively represented fatty acids were

eicosapentaenoic acid (EPA) and adrenic acid (∼0.3–0.4%).

Within PUFAs, the proportion of omega-6 was ∼10-fold higher

than that of omega-3 (∼37.4 vs. ∼3.6%). Comparisons of fatty

acid levels after vs. before the AMJ treatment in female subjects

revealed a signiﬁcant decrease in AA/EPA ratio (29.17 ± 15.21

vs. 38.42 (25.96–89.58), respectively; Wilcoxon matched pairs

test, P = 0.0 2), as AA levels signiﬁcantly decreased (10.18

± 2.16 vs. 11.16 ± 2.61 %, respectively; t-test for dependent

samples, P = 0.01) and EPA levels signiﬁcantly increased (0.43

± 0.20 vs. 0.28 ± 0.12 %, respectively; t-test for dependent

samples, P = 0.04) (Supplementary Table 3). Along with AA,

dihomo-γ linolenic acid also decreased following the treatment

in women [2.34 (1.59–4.33)% vs. 2.89 ± 1.04%, respe ctive ly;

Wilcoxon matched pairs test, P = 0.02]. The same type of change

was observed for adrenic acid [0.34 ± 0.12% vs. 0.40 (0.23–

0.98)%, respectively; Wilcoxon matched pairs test, P = 0.003]

(Supplementary Table 3).

The changes (1, after vs. before t reatment) in levels of

individual fatty acids did not signiﬁcantly correlate with a change

(1) in LINE-1 methylation levels, with the exception of adrenic

acid (R = 0.60, R

= 0.36, adjusted R

= 0.31, P = 0.02). Multiple

regression models revealed no signiﬁcant relations between a

change (1) in LINE-1 methylation levels and changes (1) in

levels of each class of fatty acids: SFAs (R = 0.53, R

= 0.28,

adjusted R

= 0.17, P = 0.13), MUFAs (R = 0.43, R

= 0.18,

adjusted R

= −0.04, P = 0.51), and PUFAs (R = 0.86, R

0.74, adjusted R

= 0.49, P = 0.09).

DISCUSSION

The main ﬁnding of this study is that the 4-week da ily

consumption of A. melanocarpa juice decreased the LINE-1

methylation levels in peripheral blood leukocytes in women

with CVD risk f actors, including overweight and dyslipidemia.

Daily tre atment with Aronia melanocarpa juice, assigned as AMJ

treatment, contained 1.18 g of total polyphenols. This amount

corresponds to an estimated average daily intake of about 1 g of

total polyphenols from th e dietary sources (

31), provided that,

in the composition of aronia polyphenols, the most abundant

are anthocyanins, proanthocyanidins, h ydroxycinnamic acids,

and ﬂavonols (32), which is in compliance with the ﬁndings

of a previous study that characterized the composition of

aronia juice used in the current research (24). Conﬁrmation

that the currently demonstrated signiﬁc ant reduction in LINE-

1 methylation is due to the impact of polyphenols, rather than

of other bioactive components of aronia juice, lies in the fact

that placebo treatment exerted no signiﬁcant eﬀects on LINE-

1 methylation. In line with t h is study, global DNA methylation

levels in peripheral leukocytes were signiﬁcantly reduced after

2-week-long consumption of polyphenol-rich cocoa product, in

individuals at cardiovasc ular risk (

13).

Herein observed decreased DNA methylation levels after the

consumption of polyphenol-rich aronia juice may be attributed

to the role of polyphenols as natural DNMT inhibitors (22).

Namely, catechins and th eir metabolites, which belong to

one of the main aronia polyphenol classes—proanthocyanidins

(

32), can increase the production of S-adenosyl-L-homocysteine

(SAH), a potent inhibitor of DNMTs and a participant of the

folate-methionine cycle, through the inhibition of catechol-O-

methyltransferase (COMT)-mediated O-methylation of catechol

substrates (33, 34). Among the main endogenous substrates

for both liver and leukocyte, COMT is a c atechol estrogen

(33, 35), potentially explaining the ﬁnding of the present

methylation analysis exclusively in women. In addition, catechins

suppress MTHFR activity, hence blocking th e folate-methionine

cycle and reducing the methyl group bioavailability (36). The

antioxidant and anti-inﬂammatory activities of polyphenol-rich

aronia products, by which they contribute to cardiovascular

protection (

17, 37), might be attributed to the role of polyphenols

as DNMT inhibitors. By reducing the activity of DNMTs, the

prevalent aronia ﬂavonol, quercetin, decreases the promoter

methylation le vels and activates t h e expression of target genes,

such as a transcription regulator nuclear factor erythroid 2-

related factor 2 (38), which is involved in t he anti-inﬂammatory

and antioxidant mechanisms in vascular cells and macrophages

(38–40). Moreover, aronia polyphenols may be involved in the

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Stojkovi

c et al. Aronia Polyphenols Affect LINE-1 Methylation

inﬂammatory response by aﬀecting t h e activity/production of

DNMTs via cytokines, since it has been demonstrated that aronia

anthocyanins aﬀected the production of inﬂammatory mediators

(37), and under the inﬂuence of IL-1, there have been changes in

DNMT expression and genomic methylation in human cells in

vitro (

41).

With regard to our results, the proposed mechanisms of

polyphenol-induced inhibition of DNA methylation (33, 34, 36)

would depend on daily folate intake and MTHFR C677T gene

variant. We found that the change (after vs. before the AMJ

treatment) in LINE-1 methylation levels in women correlated

directly with average daily folate intake, and this is consistent

with previously deﬁned eﬀects of folate on the modulation

of DNA methylation (4). Concerning the eﬀects of MTHFR

C677T gene variant, our ﬁnding is in line with the study of

Nojima et al. (42), which showed t hat the global methylation

levels in peripheral blood leukocytes were signiﬁcantly increased

in carriers of the 677T allele. This was obtained when the

MTHFR variant was analyzed in interaction with another factor

that aﬀected the methylation of DNA—an inﬂammatory marker

blood concentration (

42). Similarly, except with folate intake,

our regression model denoted an interaction of the MTHFR

variant with circulating LDL-C and triacylglycerol levels, as

metabolic factors have been associated with LINE-1 methylation

(9, 10, 43). Namely, the current change in LINE-1 methylation

in women correlated positively with the change in LDL-C a nd

negatively with the change in t riacylglycerols, which should be

pointed out along with the fact that our female subjects are

dyslipidemic. So far, few studies have investigated the relationship

between LINE-1 methylation and lipid proﬁle and yielded

conﬂicting results (9, 10, 43), emphasizing the need for further

research. The present positive correlation of peripheral blood

leukocyte LINE-1 methylation with circulating LDL-C levels is

in line with two studies, which have investigated middle-aged

subjects with cardiovascular risk factors and no evidence of

CVD (10, 43). Since we have established this positive correlation

between changes in LINE-1 methylation and circulating LDL-

C levels in subjects whose LINE-1 methylation was signiﬁcantly

decreased, following the consumption of aronia juice, our ﬁnding

may support the currently proposed cardioprotective action

of polyphenol-rich a ronia products attributed to the role of

polyphenols as DNMT inhibitors. Accordingly, the same type

of correlation would be expected between changes in LINE-

1 methylation and serum triacylglycerols levels, but we found

them to correlate inversely. Still, ﬁndings regarding the link

of LINE-1 met hylat ion with serum triacylglycerols are rather

inconsistent, as one of the mentioned studies reported a positive

correlation (

10), while in the other there was no signiﬁca nt

relation (43). With respect to pathogenesis of CVD and the

proposed explanation, LDL-C was shown to cause vascular

endothelial dysfunction in part by changing the DNMT activity

and t h us altering the DNA methylation, given that, in endothelial

cells, LDL-C inhibited the transcription of a gene i mportant

in maintaining the endothelial function, KLF2, through the

activation of DNMT1 (

44).

Along with the investigated classical lipid parameters

(circulating cholesterol and triacylglycerols), we examined

plasma f atty acids proﬁle in women who consumed polyphenol-

rich aronia juice, since the treatment signiﬁcantly changed the

LINE-1 methylation levels only in these individuals, and this

change in LINE-1 methylation was found to correlate with the

changes in lipid parameters. We observed a signiﬁcant reduction

of t h e AA/EPA ratio due to a signiﬁcant decre ase in plasma AA

and a signiﬁcant increase in EPA levels, in ta rget dyslipidemic

female subjects who underwent AMJ treatment. Similarly, in a

larger group of study participants at cardiovascular risk, from

which our substudy group was sorted out, there had also been a

signiﬁcant AA/EPA reduction in aronia polyphenols consumers,

noting that this reduction had been due to a signiﬁcant

decrease in AA, while EPA had not increased signiﬁcantly (

23).

Furthermore, in a recent study that has investigated the link

between FADS2 gene variants, fatt y acid metabolism, and aronia

polyphenol inta ke in the overweight, there was a trend of

AA/EPA reduction in individuals who consumed aronia juice,

compared to placebo consumers (

45). There has been evidence

for an association between total plasma AA and ischemic

stroke (46), and the increase in circulating EPA levels has been

shown to have beneﬁcial eﬀects on cardiovascular health (47).

Both ﬁndings (46, 47) are expected because the ratio of AA

to EPA reﬂects regulatory mechanisms of the inﬂammatory

process, as these two PUFAs compete for the conversion to

two classes of bioactive eicosanoids: pro- and anti-inﬂammatory

(48, 49). Hence, in addition to the well-known markers, such

as circulating total cholesterol, LDL-C, and triacylglycerols, the

AA/EPA ratio has recently been identiﬁed as a sensitive marker

of cardiovascular risk, given that a lower AA/EPA ratio was

associated with a decreased risk of coronary artery disease,

acute coronary syndrome, myocardial infarction, stroke, chronic

heart fa ilure, and peripheral artery disease [reviewed in Davinelli

et al. (

50)]. Our female study subjects who consumed aronia

juice had hig h baseline AA/EPA levels, indicating a chronic

low-grade inﬂammation and an increased risk of cardiovascular

events. Thus, a signiﬁcant decrease in AA/EPA ratio following

the A MJ treatment is in line with proposed anti-inﬂammatory

and cardioprotective eﬀe ct s of aronia juice polyphenols, also

supporting a sug gested relation of these eﬀects with the currently

obser ved decrease in LINE-1 methylation levels in women.

The ﬁndings of the present study should be interpreted in

light of limiting factors, primarily a sample size. Nevertheless, the

study design has strength regarding the measurement of LINE-1

methylation levels both before and after each applied treatment,

together with the corresponding anthropometric and metabolic

parameters, allowing the accurate determination of treatment-

dependent changes in parameter values. Another strength of

the study is the use of a reliable food frequency questionnaire

for the baseline dietary intake assessment with the subsequent

analysis of data collected from 24-h dietary recalls, performed

by using a validated nutritional platform for comprehensive diet

evaluation. This allowed balancing of baseline dietary i ntake

of the study subjects across each of the two interventional

treatments, assuming that dietary intake did not change within

the 4-week treatment period. Still, the possible confounding

eﬀects of temporal changes in dietary habits may not be excluded

and represent a limiting factor of the study design. Another

Frontiers in Nutrition | www.frontiersin.org 8 June 2021 | Volume 8 | Article 689055

Stojkovi

c et al. Aronia Polyphenols Affect LINE-1 Methylation

limiting factor is a possible interindividual variability in response

to polyphenol treatment, related to polyphenol bioavailability

and ot her factors that fell beyond the s cope of this study.

In conclusion, the main novelty brought by the present study

is a change in LINE-1 methylation levels after the 4-week habitual

consumption of polyphenol-rich aronia juice, which indicates an

impact of aronia polyphenols on DNA methylation. The current

results suggest cardioprotective eﬀects of aronia polyphenols

in subjects at CVD risk, achieved through the modiﬁcations

of LINE-1 DNA methylation pattern and AA/EPA ratio. Our

ﬁndings merit further investigation, particularly in view of the

fact that the precision nutrition-driven modulations of both DNA

methylation and PUFA proﬁle may become pertinent targets for

new approaches in the prevention and treatment of CVD.

DATA AVAILABILITY STATEMENT

The original contributions presented in the study are included

in the article/Supplementary Material, further inquiries can be

directed to the corresponding author/s.

ETHICS STATEMENT

The studies involving human participants were reviewed and

approved by Ethics Committee of the Clinical Hospital Centre

Zemun, Belgrade, Serbia. The patients/participants provided

their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS

LS performed the epigenetic, genetic and statistical analyses,

interpreted the results, and wrote the manuscript. MZe was

involved in the assessments of dietary intake, anthropometric

and metabolic parameters, and the analysis of plasma fatty acids

composition. AS, MZi, MZe, and LS designed the study. MZe,

MZi, and AS revi sed the manuscript. MBu assisted with the

epigenetic analysis. MBo was involved in laboratory work and

sample collection. MG contributed to the conceptualization of

the study. All authors read and agreed to the ﬁnal version of

the manuscript.

FUNDING

This study was supported by funding from the EU Seventh

Framework Programme (FP7/2007–2013), under Agreement No

2312090 (BACCHUS), and the Ministry of Education, Science

and Technological Development of the Republic of Serbia, under

the contract numbers 451-03-9/2021-14/200017 and 451-03-

9/2021-14/200015.

ACKNOWLEDGMENTS

The authors are grateful to Nevena Vidovi

c and Aleksandra

Koni

c-Risti

c (Centre of Research Excellence in Nutrition and

Metabolism/CENM, Institute for Medical Research, Belgrade) for

assistance in study design, sample collection, and biochemical

assessments; Marina Nikoli

c (CENM, Institute for Medical

Research, Belgrade) for the assessment of t he dietary intake

data; Biljana Pokimica (CENM, Institute for Medical Research,

Belgrade) for participation in the analysis of fatty acids; Ana

Kolakovi

c and M. S. Ivana Koli

c (Laboratory for Radiobiology

and Molecular Genetics, Vin

ca Institute of Nuclear Sciences,

Belgrade) for assistance in processing DNA samples; and Filip

Stojanovi

c (CENM, Institute for Medical Research, Belgrade) for

technical assistance. The authors are also thankful to Nutrika

d.o.o., Belgrade, Serbia, for the provision of study treatments.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found

online at: https://www.frontiersin.org/articles/10.3389/fnut.2021.

689055/full#supplementary-material

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24871

Conﬂict of Interest: The authors declare that the research was conducted in the

absence of any commercial or ﬁnancial relationships that could be construed as a

potential conﬂict of interest.

c, Zec, Zivkovic, Bundalo, Boškovi

c, Glibeti

c and

Stankovic. This is an open-access article distributed under the terms of the Creative

Commons Attribution License (CC BY). The use, distribution or reproduction in

other forums is permitted, provided the or i ginal author(s) and the copyright owner(s)

are credited and that the orig i nal publication in this journal is cited, in accordance

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which does not comply with these terms.

Frontiers in Nutrition | www.frontiersin.org 11 June 2021 | Volume 8 | Article 689055