Research Paper
Dietary omega-3 fatty acid intake impacts peripheral blood DNA methylation -anti-inflammatory effects and individual variability in a pilot study

https://doi.org/10.1016/j.jnutbio.2021.108839Get rights and content

Abstract

Omega-3 or n-3 polyunsaturated fatty acids (PUFAs) are widely studied for health benefits that may relate to anti-inflammatory activity. However, mechanisms mediating an anti-inflammatory response to n-3 PUFA intake are not fully understood. Of interest is the emerging role of fatty acids to impact DNA methylation (DNAm) and thereby modulate mediating inflammatory processes. In this pilot study, we investigated the impact of n-3 PUFA intake on DNAm in inflammation-related signaling pathways in peripheral blood mononuclear cells (PBMCs) of women at high risk of breast cancer.

PBMCs of women at high risk of breast cancer (n=10) were obtained at baseline and after 6 months of n-3 PUFA (5 g/d EPA+DHA dose arm) intake in a previously reported dose finding trial. DNA methylation of PBMCs was assayed by reduced representation bisulfite sequencing (RRBS) to obtain genome-wide methylation profiles at the single nucleotide level. We examined the impact of n-3 PUFA on genome-wide DNAm and focused upon a set of candidate genes associated with inflammation signaling pathways and breast cancer.

We identified 24,842 differentially methylated CpGs (DMCs) in gene promoters of 5507 genes showing significant enrichment for hypermethylation in both the candidate gene and genome-wide analyses. Pathway analysis identified significantly hypermethylated signaling networks after n-3 PUFA treatment, such as the Toll-like Receptor inflammatory pathway. The DNAm pattern in individuals and the response to n-3 PUFA intake are heterogeneous. PBMC DNAm profiling suggests a mechanism whereby n-3 PUFAs may impact inflammatory cascades associated with disease processes including carcinogenesis.

Introduction

Dietary n-3 polyunsaturated fatty acids (PUFAs) are associated with health benefits for a wide range of diseases in both preclinical and clinical studies, particularly for conditions with underlying chronic inflammation such as cardiovascular disease, rheumatoid arthritis, dementia, and cancer [1,2]. In humans, blood and tissue concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are largely dependent on consumption of foods or supplements enriched in these fatty acids such as fatty fish or fish oils, given the inefficient conversion of plant-derived alpha linolenic acid to long chain n-3 PUFAs [3]. Intervention studies of dietary n-3 PUFAs/fish oil have shown increased EPA, DHA, and n-3:n-6 PUFA ratio in plasma, adipose tissue, and circulating cells such as erythrocytes and peripheral blood mononuclear cells (PBMCs) [4], [5], [6].

Clinical investigations with dietary EPA and DHA have demonstrated anti-inflammatory effects on PBMC responses. Fish oil supplementation reduces the production of inflammatory cytokines including interleukin 2 (IL-2), interleukin 1 (IL-1), and tumor necrosis factor alpha (TNFα) by stimulated mononuclear cells [7], [8], [9]. Healthy subjects taking fish oil (775 mg/d EPA) and borage oil (831 mg/d gamma linolenic acid) supplements for four weeks demonstrate decreased PBMC gene expression of PI3Kα and PI3Kγ, key mediators of pro-inflammatory signal transduction, and cytokines (IL-1, IL-10, IL-23) [10]. In older adults, treatment with 1.8 g/d EPA+DHA for 6 months also showed a decrease in PBMC pro-inflammatory gene expression involving the interleukin 6, MAP kinase, NF-κB, and Toll-like receptor signaling pathways [11]. In an Alzheimer's disease cohort, supplementation with DHA-rich n-3 PUFA (1.7 g/d DHA, 0.6 g/d EPA) vs. corn oil for 6 months led to decreased secretion of IL-6, IL-1β, and granulocyte colony stimulating factor by PBMCs stimulated ex vivo by lipopolysaccharide (LPS), without impacting TNFα, IL-10 [12]. In parallel, PBMC gene expression showed changes consistent with anti-inflammatory effects of omega-3 fatty acid intake [13].

Epigenetic regulation of gene expression may mediate, at least in part, the anti-inflammatory effects of dietary n-3 PUFAs. DNAm is one mechanism by which pro-inflammatory genes are silenced, as shown in prior reports of epigenetic regulation of inflammation in a wide range of cell types including PBMCs [14,15], CD4+ lymphocytes [16], and cancer cell lines [11,[17], [18], [19]]. Regarding the role of dietary n-3 PUFAs in modulating the epigenome, a cross-sectional study of DNAm in PBMCs of Yup'ik Alaskan Native Americans at the lowest and highest range of long chain marine n-3 PUFA intake showed potential impact of DNAm on anti-inflammatory pathway genes [20]. Modulating effects of n-3 PUFAs on DNAm that impact inflammatory and PUFA metabolism pathways are also evident in short term intervention trials of n-3 PUFAs [21], [22], [23]. However, to date, very few n-3 PUFA intervention studies conducted in humans investigate DNAm on a genome-wide scale [23].

Towards our goal of investigating n-3 PUFA in breast cancer prevention, we conducted this pilot study to determine whether DNAm changes could be detected in PBMCs. We utilized reduced representation bisulfite sequencing (RRBS) to analyze DNAm on a genome-wide level with single base-pair resolution as well as coverage of many more sites than array-based methods. Our work shows that DNAm changes after dietary n-3 PUFA treatment in women at high risk of breast cancer are detectable in PBMCs. Additionally, our findings implicate two inflammatory pathways and uncover variability in DNAm that may indicate variable treatment effects.

Section snippets

PBMCs

PBMCs were isolated via ficoll-hypaque separation from peripheral blood collected in acid citrate dextrose (ACD) blood tubes as part of a study of n-3 PUFA supplementation in women at high risk of breast cancer; the main results of the trial were previously reported [4]. In brief, 48 women at high risk of breast cancer were randomly assigned to one of four daily doses of n-3 PUFAs (0.84, 2.52, 5.04, or 7.56 g/d of EPA+DHA) for 6 months of treatment. The study was conducted with the approval of

n-3 PUFA treatment does not alter average genome-wide DNA methylation patterns

We previously reported the effects of four different doses of n-3 PUFAs on fatty acid profiles of women at high risk of breast cancer following 6 months of treatment [4]. This PBMC DNAm substudy focused on 10 participants from the 6 capsule/d arm with highest increases in DHA and EPA at 6 months. Analysis of serum and breast adipose fatty acid profiles of these samples yielded similar results to those previously reported [4]. The 10 women had an average age of 51±6.8 years, weight of 74.4±11.3

Discussion

With this study, we demonstrate that n-3 PUFA supplementation in women at high risk of breast cancer elicits locus-specific DNAm changes in PBMCs. While average genome-wide CpG methylation is essentially unchanged in PBMCs after six months of n-3 PUFA supplementation, analysis of DNAm changes at CpGs (DMCs) revealed marked enrichment of hypermethylation in the promoter regions of candidate genes selected for potential for epigenetic regulation of inflammatory and breast carcinogenesis signaling

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Funding sources

This work was supported by the Cancer Metabolism Training Program [T32 CA221709]; the Pelotonia Graduate Research Fellowship; Integrated Training in Biomedical Systems [T32 GM068412]; the National Cancer Institute [R01 CA164019]; The Ohio State University Comprehensive Cancer Center Support Grant [P30CA016058]; Ohio Supercomputer Center.

References (67)

  • JK Kiecolt-Glaser et al.

    Omega-3 supplementation lowers inflammation and anxiety in medical students: a randomized controlled trial

    Brain Behav Immun

    (2011)
  • S Huang et al.

    Saturated fatty acids activate TLR-mediated proinflammatory signaling pathways

    J Lipid Res

    (2012)
  • SM Eraky et al.

    Modulating effects of omega-3 fatty acids and pioglitazone combination on insulin resistance through toll-like receptor 4 in type 2 diabetes mellitus

    Prostaglandins Leukot Essent Fatty Acids

    (2018)
  • GC Burdge et al.

    Conversion of α -linolenic acid to longer-chain polyunsaturated fatty acids in human adults

    Reprod Nutr Dev

    (2005)
  • S Straka et al.

    Incorporation of eicosapentaenioic and docosahexaenoic acids into breast adipose tissue of women at high risk of breast cancer: a randomized clinical trial of dietary fish and n-3 fatty acid capsules

    Mol Nutr Food Res

    (2015)
  • CG Walker et al.

    Age and sex differences in the incorporation of EPA and DHA into plasma fractions, cells and adipose tissue in humans

    Br J Nutr

    (2014)
  • S Endres et al.

    Dietary supplementation with n-3 fatty acids suppresses interleukin-2 production and mononuclear cell proliferation

    J Leukoc Biol

    (1993)
  • S Endres et al.

    The effect of dietary supplementation with n—3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells

    N Engl J Med

    (1989)
  • M Fisher et al.

    Effects of dietary fish oil supplementation on polymorphonuclear leukocyte inflammatory potential

    Inflammation

    (1986)
  • I Vedin et al.

    Effects of DHA- Rich n-3 fatty acid supplementation on gene expression in blood mononuclear leukocytes: the OmegAD study

    PLoS One

    (2012)
  • M Bam et al.

    Evidence for epigenetic regulation of pro-inflammatory cytokines, interleukin-12 and interferon gamma, in peripheral blood mononuclear cells from PTSD patients

    J Neuroimmune Pharmacol

    (2016)
  • PJ Robson-Ansley et al.

    Dynamic changes in dna methylation status in peripheral blood Mononuclear cells following an acute bout of exercise: potential impact of exercise-induced elevations in interleukin-6 concentration

    J Biol Regul Homeost Agents

    (2021)
  • N-H Kwon et al.

    DNA Methylation and the expression of IL-4 and IFN-γ promoter genes in patients with bronchial asthma

    J Clin Immunol

    (2008)
  • R Hammamieh et al.

    Differential effects of omega-3 and omega-6 fatty acids on gene expression in breast cancer cells

    Breast Cancer Res Treat

    (2007)
  • JE Pérez-Mojica et al.

    Docosahexaenoic acid and oleic acid induce altered DNA methylation of individual CpG loci in Jurkat T cells

    Prostaglandins Leukot Essent Fat Acids

    (2020)
  • V Ceccarelli et al.

    Eicosapentaenoic acid activates RAS/ERK/C/EBPβ pathway through H-Ras intron 1 CpG island demethylation in U937 leukemia cells

    PLoS One

    (2014)
  • SP Hoile et al.

    Supplementation with N-3 long-chain polyunsaturated fatty acids or olive oil in men and women with renal disease induces differential changes in the DNA methylation of FADS2 and ELOVL5 in peripheral blood mononuclear cells

    PLoS One

    (2014)
  • CL do Amaral et al.

    DNA methylation pattern in overweight women under an energy-restricted diet supplemented with fish oil

    Biomed Res Int

    (2014)
  • BL Tremblay et al.

    Epigenetic changes in blood leukocytes following an omega-3 fatty acid supplementation

    Clin Epigenetics

    (2017)
  • MA Belury et al.

    Conjugated linoleic acid modulates hepatic lipid composition in mice

    Lipids

    (1997)
  • Y Benjamini et al.

    Controlling the false discovery rate: a practical and powerful approach to multiple testing

    J R Stat Soc Ser B

    (1995)
  • H Gu et al.

    Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution

    Nat Methods

    (2010)
  • M. Martin

    Cutadapt removes adapter sequences from high-throughput sequencing reads

    EMBnetJournal

    (2011)
  • Cited by (4)

    1

    Departments of Diabetes Complications & Metabolism and Population Sciences, Beckman Research Institute, City of Hope, 1500 E. Duarte Rd, Duarte CA 91010, USA

    2

    Department of Pathology, Duke University, DUMC 3712 Durham NC 27710, USA

    3

    Department of Surgery, City of Hope, 1500 E. Duarte Rd, Duarte CA 91010, USA

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