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