Epigenetic changes with dietary soy in cynomolgus monkeys.
ABSTRACT Nutritional interventions are important alternatives for reducing the prevalence of many chronic diseases. Soy is a good source of protein that contains isoflavones, including genistein and daidzein, and may alter the risk of obesity, Type 2 diabetes, osteoporosis, cardiovascular disease, and reproductive cancers. We have shown previously in nonhuman primates that soy protein containing isoflavones leads to improved body weight, insulin sensitivity, lipid profiles, and atherosclerosis compared to protein without soy isoflavones (casein), and does not increase the risk of cancer. Since genistein has been shown to alter DNA methylation, we compared the methylation profiles of cynomolgus monkeys, from multiple tissues, eating two high-fat, typical American diets (TAD) with similar macronutrient contents, with or without soy protein. DNA methylation status was successfully determined for 80.6% of the probes in at least one tissue using Illumina's HumanMethylation27 BeadChip. Overall methylation increased in liver and muscle tissue when monkeys switched from the TAD-soy to the TAD-casein diets. Genes involved in epigenetic processes, specifically homeobox genes (HOXA5, HOXA11, and HOXB1), and ABCG5 were among those that changed between diets. These data support the use of the HumanMethylation27 BeadChip in cynomolgus monkeys and identify epigenetic changes associated with dietary interventions with soy protein that may potentially affect the etiology of complex diseases.
- SourceAvailable from: Mary S Anthony[show abstract] [hide abstract]
ABSTRACT: We sought to determine if arterial LDL metabolism contributes to the decreased atherosclerosis seen with soy and if isolated isoflavones would have similar effects. Ovariectomized monkeys were fed an atherogenic diet for 20 weeks with a protein source of (1) casein/lactalbumin (CAS, n=20), (2) soy protein isolate (SOY, n=20), or (3) casein/lactalbumin with isolated soy isoflavones (ISO, n=17). Plasma lipoprotein concentrations were improved with SOY but not ISO. Arterial LDL metabolism was characterized with one subset (n=12/group) injected with dual-labeled tyramine-cellobiose (TC)-LDL (125I-TC-131I-LDL) 24 hours before necropsy to determine LDL degradation and accumulation, while another subset (n=8/group) was injected with 125I-TC-LDL 1 hour before necropsy to determine LDL permeability and delivery. Coronary artery LDL degradation was reduced by 50% (P=0.02) with SOY but not with ISO compared with CAS. Neither treatment altered arterial permeability. Reduced LDL degradation with SOY was due to decreased arterial LDL delivery (P=0.02). Carotid artery cholesterol ester was also decreased with SOY, but not with ISO. Plasma isoprostanes or plasma markers of inflammation did not differ among treatment groups. Thus, the decreased arterial LDL delivery and subsequent LDL degradation may explain, in part, the atheroprotective effects of soy.Arteriosclerosis Thrombosis and Vascular Biology 01/2004; 23(12):2241-6. · 6.34 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The human estrogen receptor (hER) exists as two subtypes, hER alpha and hER beta, that differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activities of soy isoflavones after digestion with enteric bacteria in competition binding assays with hER alpha or hER beta protein, and in a gene expression assay using a yeast system. The estrogenic activities of these isoflavones were also investigated by the growth of MCF-7 breast cancer cells. Isoflavone glycoside binds weakly to both receptors and estrogen receptor-dependent transcriptional expression is poor. The aglycones bind more strongly to hER beta than to hER alpha. The binding affinities of genistein, dihydrogenistein and equol are comparable to the binding affinity of 17 beta-estradiol. Equol induces transcription most strongly with hER alpha and hER beta. The concentration required for maximal gene expression is much higher than expected from the binding affinities of the compounds, and the maximal activity induced by these compounds is about half the activity of 17 beta-estradiol. Although genistin binds more weakly to the receptors and induces transcription less than does genistein, it stimulates the growth of MCF-7 cells more strongly than does genistein.Biological & Pharmaceutical Bulletin 05/2001; 24(4):351-6. · 1.85 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The major soy isoflavones are daidzin and genistin, the glycoside conjugates of daidzein (DZ) and genistein (GTN). After ingestion, they are metabolized into diverse compounds in the gut. The marked inter-individual variation has been suggested in their metabolism. The clinical effects may be modulated by the metabolic ability to produce a more potent metabolite than the precursor. Our study was, therefore, designed to analyze and compare in vitro biologic activities of their metabolites: DZ, GTN, dihydrogenistein (DGTN), dihydrodaidzein (DDZ), tetrahydrodaidzein (TDZ), O-desmethylangolensin (ODMA), and equol (EQL). Furthermore, we investigated their modulatory effects in the presence of estrogen using several in vitro systems. The intermediate metabolites, such as DGTN, DDZ, and TDZ, bind much weakly to both ERs and induce less potently in transcriptional activity, gene expression, and mammary cell proliferation than their precursors. EQL has the strongest binding affinities and estrogenic activities especially for ERbeta among the daidzin metabolites and shows the ability to suppress osteoclast formation at high doses. The test isoflavonoids act like estrogen antagonists with the premenopausal dose of E2 and thus inhibit estrogenic actions by E2, whereas they exert estrogen agonist activity with the lower dose of estrogen close to the serum levels of postmenopausal women. Our results suggest that phytoestrogens such as isoflavones may exert their effects as estrogen antagonists in a high estrogen environment, or they may act as estrogen agonists in a low estrogen environment.The Journal of Steroid Biochemistry and Molecular Biology 12/2006; 101(4-5):246-53. · 3.98 Impact Factor
Epigenetic Changes with Dietary Soy in Cynomolgus
Timothy D. Howard1*, Shuk-Mei Ho3,4,5, Li Zhang2, Jing Chen3,4, Wei Cui1, Rebecca Slager1, Stanton
Gray2, Gregory A. Hawkins1, Mario Medvedovic3,4, Janice D. Wagner2
1Center for Genomics and Personalized Medicine Research, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America, 2Department of
Pathology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America, 3Department of Environmental Health, University of Cincinnati,
Cincinnati, Ohio, United States of America, 4Center for Environmental Genetics, University of Cincinnati, Cincinnati, Ohio, United States of America, 5Cincinnati Veteran
Affairs Medical Center, Cincinnati, Ohio, United States of America
Nutritional interventions are important alternatives for reducing the prevalence of many chronic diseases. Soy is a good
source of protein that contains isoflavones, including genistein and daidzein, and may alter the risk of obesity, Type 2
diabetes, osteoporosis, cardiovascular disease, and reproductive cancers. We have shown previously in nonhuman primates
that soy protein containing isoflavones leads to improved body weight, insulin sensitivity, lipid profiles, and atherosclerosis
compared to protein without soy isoflavones (casein), and does not increase the risk of cancer. Since genistein has been
shown to alter DNA methylation, we compared the methylation profiles of cynomolgus monkeys, from multiple tissues,
eating two high-fat, typical American diets (TAD) with similar macronutrient contents, with or without soy protein. DNA
methylation status was successfully determined for 80.6% of the probes in at least one tissue using Illumina’s
HumanMethylation27 BeadChip. Overall methylation increased in liver and muscle tissue when monkeys switched from the
TAD-soy to the TAD-casein diets. Genes involved in epigenetic processes, specifically homeobox genes (HOXA5, HOXA11,
and HOXB1), and ABCG5 were among those that changed between diets. These data support the use of the
HumanMethylation27 BeadChip in cynomolgus monkeys and identify epigenetic changes associated with dietary
interventions with soy protein that may potentially affect the etiology of complex diseases.
Citation: Howard TD, Ho S-M, Zhang L, Chen J, Cui W, et al. (2011) Epigenetic Changes with Dietary Soy in Cynomolgus Monkeys. PLoS ONE 6(10): e26791.
Editor: Brian P. Chadwick, Florida State University, United States of America
Received June 30, 2011; Accepted October 3, 2011; Published October 25, 2011
Copyright: ? 2011 Howard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported, in part, by the National Institutes of Health awards P40RR021380 from the National Center for Research Resources (JDW),
ES015584 (MM, SMH), ES018758 (MM, SMH), ES018789 (MM, SMH), ES006096 (MM, SMH), ES019480 (SMH), and an award from the Department of Veterans Affairs
(SMH). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Dietary soy has been proposed to be a ‘‘heart healthy’’ food
supplement. The FDA approved a health claim for soy protein and
soy-based food products, based largely on the evidence that soy
consumption improves plasma lipid and lipoprotein concentra-
tions and might reduce the risk of cardiovascular disease (CVD),
yet does not appear to increase cancer risk . Isoflavones, in
particular genistein and daidzein, are abundant in soy protein and,
as they are structurally similar to estradiol, can bind to both
estrogen receptor (ER) a and b, but with greater affinity for ER b
. Depending on their concentration, the concentration of
endogenous estrogen, and ER number and type (e.g., ER a or b),
these isoflavones can act with estrogen-like activity (i.e., agonists)
or act as estrogen antagonists . Thus, isoflavones have the
potential to be endocrine disrupters, especially if given during key
periods of development or reproductive stage [4,5]. Soy is
commonly used in infant formulas, and the safety of these
formulas in developing infants has been controversial [6,7]. The
mechanisms involved in soy’s beneficial and potentially adverse
effects are complex and poorly understood.
The ‘‘fetal basis of adult disease’’ hypothesis  postulates
‘‘that nutrition and other environmental factors during prenatal
and early development influence cellular plasticity, thereby
altering susceptibility to adult CVD, type 2 diabetes (T2D),
obesity, and other chronic diseases’’ . An abundance of
literature in animal models suggests that prenatal or early
exposure to either estrogenic compounds or genistein specifically
affects later risk of obesity , congestive heart failure ,
immune function , reproductive effects in males  and
females , and cancers of the reproductive system, such as
breast and prostate [15–19].
There is a significant body of evidence demonstrating that
environmental exposures, including diet, lead to epigenetic
changes. These changes offer one mechanism for ‘‘fetal program-
ming’’ that can affect the onset of diseases later in life. For
example, in utero exposure to dietary genistein modified coat color
in inbred mice by methylation of a CpG island that regulates
expression of the Agouti coat color gene [9,20]. This in utero gene
regulation also modified the risk of adult obesity in the offspring
and persisted into adulthood. These studies indicate the potential
for dietary genistein to affect chronic diseases in mice through an
epigenetic pathway, namely DNA methylation. While these rodent
studies have been helpful in establishing the relationship between
diet and DNA methylation, we propose using a more relevant
primate model, cynomolgus monkeys, where sex hormone levels
PLoS ONE | www.plosone.org1October 2011 | Volume 6 | Issue 10 | e26791
and risk of disease follow the same disease risk profile as in humans
We hypothesized that changes in DNA methylation contribute
to the effects we and others have observed in humans and animals
consuming soy-based diets . The diets for this study had
similar macronutrient content with the exception of protein
source, which was either soy- or casein/whey-based. To better
understand this mechanism, we have performed DNA methylation
analysis with multiple tissues from cynomolgus monkeys eating one
of two diets based on that of a typical North American. DNA
microarrays designed for human DNA methylation studies (the
Illumina HumanMethylation27 Beadchip) were used to examine
DNA from tissues relevant to common, complex diseases to
determine if these two diets contributed to epigenetic changes that
may be involved in complex disease susceptibility.
Materials and Methods
Approval for all procedures in this study was obtained from the
Wake Forest University (WFU) Animal Care and Use Committee
(A08-219). In addition, an appropriate environmental enrichment
SOP for this project was approved by the WFU NHP
Environmental Enrichment committee. This included, but was
not limited to: monkeys pair-housed the majority of the time, but
single-housed temporarily for feeding and other experimental
requirements; monkeys located to have auditory, visual, and
olfactory stimulation from other monkeys in the room, and;
monkeys received species-appropriate toys and manipulata rotated
every two weeks at minimum. Animals in this project were fully
under the care of veterinarians at the WFU School of Medicine in
accordance with the standards incorporated in The Guide to the
Care and Use of Laboratory Animals (1996). All experimental
procedures were performed only with sedated animals and
appropriate analgesia was administered. Animal welfare and steps
were taken to ameliorate suffering in accordance with the
recommendations of the Weatherall report (2006).
Most rodent and nonhuman primate studies are done with
animals fed commercial chows that contain low fat and cholesterol
content, with the major portion of the protein from soy. This diet
results in quite high serum concentrations of isoflavones [22–24]
and is unlike most Western diets. We designed two diets that
mimic the macronutrient content of the typical American diet
(TAD), with 35% of calories from fat, but with differing protein
sources . One diet (TAD-casein, LabDietH 5L0P) has casein
and whey as the primary protein source and is nearly devoid of soy
protein, more typical of the diet consumed by North Americans.
The other diet (TAD-soy, LabDietH 5L0R) contains soy as the
protein source to mimic a high soy supplementation diet, similar to
a diet in the human population consuming soy and tofu products
or monkeys consuming standard chow. It provides the equivalent
on a caloric basis to 180 mg/person/day of soy isoflavones,
resembling the isoflavone content of chow consumed by almost all
monkeys used in research [22,23,25]. The high soy protein chows
likely do not model disease patterns of people in Western
countries, who eat very limited amounts of soy protein, and thus
have relatively low isoflavone concentrations.
This study utilized monkeys that had been consuming two
different diets at baseline, and then two diet changes were
performed for each group of monkeys (Figure 1). Monkeys (N=4
in each diet arm) were previously eating either standard monkey
chow (with soy as the protein source) or a low cholesterol, high
fructose diet with casein as the protein source . Monkeys
eating the standard chow diet (Figure 1, top) were switched to the
TAD-soy diet due to their similar exposure to soy from chow (Diet
Arm 1). These animals continued to eat the TAD-soy diet for eight
weeks, at which time they were switched to the TAD-casein diet.
The four monkeys eating the fructose-casein diet (Figure 1,
bottom) were switched to the TAD-casein diet for 8 weeks, due to
the similar exposure to casein, and then switched to the TAD-soy
diet for an additional 8 weeks (Diet Arm 2). After four weeks of
consuming each diet, blood samples were taken to determine
plasma lipids and carbohydrate measures, and intravenous glucose
tolerance tests (GTT) were done. Blood samples and fat, muscle,
and liver biopsies were collected from all eight monkeys at baseline
and at the end of each diet phase. As there was evidence for
differences between monkeys in the two diet arms at baseline,
physiologic measurements for TAD soy and TAD casein diets
were evaluated within each arm using linear mixed models,
adjusting for age of the animals and baseline measures. For all
statistical comparisons, a two-sided p value ,0.05 was considered
significant. All procedures were approved by the Institutional
Animal Care and Use Committee.
Monkeys were sedated with ketamine HCl (15–20 mg/kg)
intramuscularly. Aseptic surgical technique was used to collect
subcutaneous adipose tissue, muscle, and liver. For subcutaneous
adipose and muscle tissue, a 2 cm skin incision was made on the
upper thigh and adjacent to the umbilicus, respectively. Subcu-
taneous adipose tissue over the abdomen was bluntly dissected,
and quadriceps muscle was taken with a 6 mm punch biopsy, for
approximately 0.25–0.50 g of tissue each. Liver samples were
obtained by ultrasound-guided needle biopsies. A small, 2 mm
skin incision was made caudal to the last rib on the right side of the
ventral abdominal region. A 14-gauge liver biopsy needle was
inserted through the skin incision to a depth of 1.5–3.0 cm.
Ketoprofen (5 mg/kg, IM) and buprenorphine (0.01 mg/kg, IM)
was given post-operatively for analgesia.
DNA methylation analysis
DNA was isolated from blood, muscle, fat and liver after eight
weeks on each diet. DNA was bisulfite-converted with the EZ
DNA MethylationTMKit (Zymo Research) and analyzed with
Illumina Human Methylation27 BeadChips, which assay 27,578
human CpG sites from 14,000 highly annotated genes. To
calculate the total DNA methylation proportions, only probes with
a detection p-value less than 0.05, according to the program
GenomeStudio (Illumina, Inc.), for all tissues was used. For all
other analyses, probe-level data was normalized using quantile
normalization and a gene-level methylation ratio was determined.
Differential methylation between diets was determined using a
linear model with a block effect.
Principal Component Analysis (PCA) was performed using data
from all CpG loci with a detection p-value less than 0.05 in all
monkeys (N=8,002) and JMP Genomics software (SAS Institute,
Cary, NC). Both loci (rows) and samples (columns) were centered
to a mean zero prior to PCA determination.
Sequencing and Pyrosequencing of Cynomolgus DNA
To obtain the DNA sequence of our differentially methylated
genes in cynomolgus macaques, PCR and sequencing primers
were designed by using a human/rhesus macaque consensus
sequence. Sequencing was attempted around the seven CpG sites
Epigenetic Changes with Soy in Cynomolgus Monkeys
PLoS ONE | www.plosone.org2October 2011 | Volume 6 | Issue 10 | e26791
with significant changes between the two TAD diets, and four of
these sites generated high-quality, unambiguous sequence. Pyro-
sequencing assays were designed for these four sites using
PyroMark Assay Design software (Qiagen, Inc.). Pyrosequencing
was performed on a Biotage PSQ96 instrument and data was
analyzed with Pyro Q-CpG (Biotage, Inc.).
Monkeys eating two distinct protein diets were studied to
delineate the effects of soy protein in animals initially fed standard
monkey chow (which contains soy) or a casein-based diet (Figure 1).
These monkeys had been consuming their initial diets for a
minimum of two years before the current study, and as such, some
phenotypic differences were observed between the two groups at
baseline (Table 1). For this reason, the two diet arms were
analyzed separately. Insulin resistance, as determined by HOMA-
IR, was significantly higher in animals eating the initial high-
fructose diet compared to animals eating chow (4.33 versus 3.00
HOMA units; p=0.005). In addition, proinsulin levels were
higher in the high fructose group (131.13 pmol/L versus
42.5 pmol/L, p=0.025) and total plasma cholesterol was lower
(91.75 mg/dL versus 102.25 mg/dL, p=0.031). Over the course
of the study, there were no significant changes in the clinical
characteristics of monkeys in Diet Arm 1 (Table 1). In Diet Arm 2,
fasting insulin levels were lower after changing from the TAD-
casein to the TAD-soy diet (35.75 mU/ml versus17 mU/ml,
p=0.01). Related to the insulin levels, lower insulin resistance
was also observed with the TAD-soy diet (2.67 versus 6.03 HOMA
units, p=0.02). No other clinical or physiologic parameters
changed significantly between animals eating the different diets.
We performed DNA methylation analysis using the Human-
Methylation27 BeadChip (Illumina, Inc.). Using the ‘‘detection p-
value’’ metric in GenomeStudio (which evaluates the probe
intensity for each CpG site compared to a negative control),
80.6% of the assays generated a sufficient signal in at least one
sample from cynomolgus monkeys. Using methylation data from
all probes with a detection p-value less than 0.05, PCA analysis
was performed with all monkeys, on each diet. Four clusters were
observed, predominately defined by tissue type (Figure 2). Samples
from blood provided the most compact cluster, indicating that the
DNA methylation pattern from blood was highly similar among all
samples. Six liver samples were outside of the liver cluster (three of
these were from the same monkey), but all other samples formed
easily observable clusters based on the tissue type (i.e., blood, liver,
fat, or muscle). Using all animals and each diet condition, a total of
95 samples (one sample was unacceptable for analysis) were
DNA methylation levels varied by diet and tissue type, and
overall DNA methylation levels increased in liver and muscle after
transitioning from the TAD-soy to the TAD-casein diet (Figure 3,
top). The levels in blood remained the same while levels in fat
decreased slightly. In liver, the proportion of overall DNA
methylation increased from 0.175 to 0.209 (a 19% increase) and
in muscle the increase was from 0.165 to 0.186 (a 13% increase)
with the TAD-soy to TAD-casein change. Methylation also
increased in liver and muscle after switching from the high-
fructose casein diet to the high-fat TAD-casein diet (Figure 3,
bottom). The proportion in liver increased from 0.181 to 0.212 (a
17% increase) and muscle increased from 0.162 to 0.197 (a 22%
Gene-specific analyses were performed with a focus on the
transition between the two TAD-based diets. Separate tissue-
specific analyses were performed to identify changes at the p,0.01
and p,0.001 levels. Genes with p,0.01 were used to identify
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathways that were over-represented with each
transition (Table 2). Significant pathways were identified from
blood only with the TAD-soy to TAD-casein transition with Diet
Arm 1; all tissues except muscle revealed significant pathways with
the TAD-casein to TAD-soy change with Diet Arm 2.
DNA methylation levels for seven individual genes changed
significantly (False Discovery Rate, FDR, p,0.20) after switching
diets – four with the TAD-soy to TAD-casein transition and three
with the TAD-casein to TAD-soy diet change (Table 3). These
genes included homeobox genes (HOXA5, HOXA11, HOXB1) and
ATP-binding cassette, sub-family, member 5 (ABCG5). The sites
for HOXA5 and HOXA11 are separated by ,43 kb, suggesting
that this entire genomic region may be epigenetically marked. No
genomic sequence for cynomolgus macaques is currently available,
so to further investigate these findings the region around ten of the
50bp probes from these seven genes was sequenced to determine
Figure 1. Study Design. Four cynomolgus monkeys were included in each diet arm. Measurements for clinical variables were performed after four
weeks on each diet. DNA was isolated from blood, fat, liver, and muscle at the end of each diet period.
Epigenetic Changes with Soy in Cynomolgus Monkeys
PLoS ONE | www.plosone.org3 October 2011 | Volume 6 | Issue 10 | e26791
the identity between the human and cynomolgus sequences.
Identities between human and cynomolgus DNA sequences
ranged from 0.94 to 1.0, suggesting that these specific probes
were assaying the correct CpG loci of the respective human sites.
To validate the HumanMethylation27 BeadChip data, we
performed pyrosequencing of four of the top seven CpG sites with
high-quality sequence data from cynomolgus monkeys. Data from
three of these sites (ABCG5, TBX5, and HOXB1) were highly
correlated (Figure 4). For sites in ABCG and TBX5, the data were
virtually identical between the two assays (r2=0.97 for both),
which is consistent with the 100% identity between the human and
cynomolgus sequences that encompass the 50bp Illumina probes.
The correlation was lower for HOXB1 (r2=0.89), where a single
base difference exists between the human probe and cynomolgus
monkey sequences. Since the base difference is a ‘‘C’’ in
cynomolgus monkeys and a ‘‘T’’ in humans, this difference has
no effect after bisulfite conversion, where both bases would
become a ‘‘T’’. A second sequence difference 2bp 39 of the probe
creates an adjacent CpG in the cynomolgus monkeys that is absent
in humans. This change would alter the fluorescent nucleotide
extended in the assay and therefore affect the signal. Importantly,
this change alters the apparent magnitude of the methylation but
does not affect the difference between samples. A fourth site
examined was located in HOXA11, where a significant difference
was detected in muscle tissue (Table 3). Unfotunately, we did not
have sufficient DNA from muscle, so DNA from fat tissue was used
for the validation assay. This site had the lowest correlation of the
four tested (r2=0.74), most likely due to a single base difference
between the cynomolgus monkey and human sequences. This
change (a ‘‘C’’ in humans is a ‘‘G’’ in cynomolgus monkeys), which
is located at a CpG site within the Illumina probe, would still differ
after bisulfite conversion, decreasing the affinity of the probe for
the genomic DNA.
To elucidate the genetic and molecular mechanisms of complex
diseases and disentangle the age- and environment-related factors,
relevant animal models are needed. One limitation of animal
models, specifically nonhuman primates, is that the current
genomic tools have been developed primarily for humans, where
well-annotated sequence data is easily available. This pilot study
was done to determine the usefulness of the HumanMethylation27
BeadChip (Illumina, Inc.) in cynomolgus monkeys. In addition, we
characterized tissue-specific changes in DNA methylation that
occurred after altering diets that differed in protein content. DNA
methylation data was easily distinguished by tissue type using
PCA, supporting the ability of the HumanMethylation27 Bead-
Chip to properly assay the cynomolgus genome. Overall DNA
methylation levels increased in liver and muscle tissues when
Table 1. Characteristics of monkeys on each diet at baseline and after diet changes.
Diet Arm 1 (mean, SD)
Baseline descriptivesChow TAD SoyTAD CaseinP-value*
Body weight, kg6.07 (0.68) 5.86 (0.92)5.77 (0.63) 0.61
Fasting glucose, mg/dL55.46 (5.73)56.25 (2.22)61.25 (3.95)0.12
Fasting insulin, mU/mL 21.24 (13.6)13.25 (6.34)19 (10.1) 0.22
Insulin resistance index (HOMA units)3 (2.08) 1.81 (0.98) 2.93 (1.72)0.21
C-peptide, pg/mL 463.38 (226.52)502.75 (296.91)573 (235.42)0.29
Proinsulin, pmol/L42.5 (27.65)38.25 (9.91)45.25 (32.43)0.57
Fructosamine, mmol/L177.95 (12.22)166.18 (12.35)169.8 (8.06)0.29
TPC, mg/dL102.25 (12.28)131.25 (31.63) 124.75 (45.51)0.40
TG, mg/dL85.75 (70.18)67.75 (50.36)61 (40.01)0.71
HDL, mg/dL46.75 (4.11) 63 (17.49)62 (19.41)0.62
CRP, ng/mL657.32 (406.62) 637.98 (447.70)933.2 (1121.92)0.80
Diet Arm 2 (mean, SD)
Baseline descriptivesHigh Fructose TAD Casein TAD SoyP-value*
Body weight, kg8.4 (1.79) 7.36 (1.76)7.49 (1.67)0.59
Fasting glucose, mg/dL66.69 (9.32)68 (8.29)62.75 (8.42)0.27
Fasting insulin, mU/mL26.38 (11.76)35.75 (7.23)17 (2.58)0.01
Insulin resistance index (HOMA units) 4.33 (1.98) 6.03 (1.7)2.67 (0.74)0.02
C-peptide, pg/mL 296.25 (60.14)352 (82.92)303.75 (69.14)0.34
Proinsulin, pmol/L 131.13 (35.86)118.75 (41.19)106.5 (31.94) 0.53
Fructosamine, mmol/L170.7 (15.41) 155.25 (14.6)168.9 (17.4) 0.10
TPC, mg/dL91.75 (10.78) 121 (24.47)128.75 (28.48)0.73
TG, mg/dL107.5 (24.56) 81 (26.55)62 (16.87)0.34
HDL, mg/dL45 (6.98)66.25 (19.31)71.75 (9.54) 0.59
CRP, ng/mL900.63 (186.94)766.03 (279.24)868.9 (271.96)0.39
*adjusted for age and baseline values.
Epigenetic Changes with Soy in Cynomolgus Monkeys
PLoS ONE | www.plosone.org4 October 2011 | Volume 6 | Issue 10 | e26791
animals were switched to the TAD-casein diet. Gene specific
changes in specific pathways, such as those involved in epigenetic
regulation, were also identified. Significant DNA methylation
changes in seven individual genes from muscle and fat samples
were also observed after diet changes, and four of these were
validated using an alternative assay.
DNA methylation changes were detected after the relatively
short dietary study periods, most notably in liver and muscle. It is
unclear why liver and muscle demonstrated the largest changes in
DNA methylation, but it may be due to the effect of soy
components (e.g., isoflavones) on these tissues specifically. Based
on our previous studies comparing the effects of dietary soy versus
casein in cynomolgus monkeys, changes in triglyceride levels and
glycemic control were noted . These changes are consistent
with variable methylation in genes in liver and muscle, as observed
in this study. Also consistent is that measures of insulin and insulin
sensitivity, which are affected by the ability of peripheral muscles
to respond to insulin, were the only phenotypic traits significantly
different in this study with the transition from the TAD-casein to
the TAD-soy diet (Table 1).
Genes with changes in DNA methylation were more likely to be
found in specific GO and KEGG pathways (Table 2). Some of
these pathways, such as histone methyltransferase activity and
protein methyltransferase activity, are directly related to epigenetic
modifications. Other processes appear to be more tissue-specific.
Gene-specific analysis identified seven individual genes that
changed significantly after the diet changes – four after the
transition from TAD-soy to TAD-casein and three after
transitioning from TAD-casein to TAD-soy. All four of the genes
from Diet Arm 1 were from muscle tissue and included two
homeobox genes, HOXA5 and HOXA11, which are located
approximately 37 kb apart on chromosome 7. For both of these
genes there was a decrease in methylation when the monkeys
changed from the TAD-soy to the TAD-casein diet, consistent
with an overall decrease of DNA methylation of this entire
genomic region. Homeobox (Hox) genes encode a unique set of
transcription factors that play key roles in development. More
recently, epigenetic modification of Hox genes, in general, has
been shown to be important in a variety of cancers. The other
gene of interest with a significant change was ABCG5, which
functions to limit intestinal absorption and promote biliary
excretion of sterols. In fact, mutations in this gene cause the
autosomal recessive disease sitosterolemia, which is characterized
by markedly increased absorption of sterols by the intestine and a
limited ability to secrete sterols into bile . Individuals with this
condition are at an increased risk of developing atherosclerosis and
coronary artery disease. Notably, dietary soy consumption has
been shown to lower plasma lipids and decrease the progression of
There is a significant body of evidence demonstrating that
environmental exposures, including diet, alter important DNA
methylation patterns, resulting in ‘‘fetal programming’’ that may
affect adult disease. For example, maternal nutrition prior and
during gestation, and during lactation, plays an essential role in the
establishment of epigenetic patterns in the offspring. Individuals
who were prenatally exposed to famine during the Dutch Hunger
Figure 2. PCA analysis of DNA methylation levels from all monkeys eating each diet. Four clusters can be observed, distinguished by the
tissue type. The axes are based on the eigenvalues of the three principal components determined. Tissues are differentiated by color: liver, gold;
muscle, blue; fat, green; and blood, red.
Epigenetic Changes with Soy in Cynomolgus Monkeys
PLoS ONE | www.plosone.org5 October 2011 | Volume 6 | Issue 10 | e26791
Winter in 1944-45 had, six decades later, less DNA methylation of
the paternally imprinted IGF2 gene compared with their
unexposed, same-sex siblings . The effects of genistein has
been examined in rodents, where gene- and tissue-specific DNA
methylation changes have been observed [20,28,29]. Here, we
show changes in primate gene methylation associated with soy
diets containing genistein, consistent with these rodent studies.
There was a significant decrease in fasting insulin and HOMA
index values in animals that transitioned from the TAD-casein to
the TAD-soy diet, suggesting improved insulin sensitivity with the
TAD-soy diet. There was a complementary increase (although not
statistically significant) with these same measures in animals that
transitioned from the TAD-soy to TAD-casein. No significant
change in body weight was observed in monkeys in either diet
arm, likely due to the short study periods and the small number of
animals studied. In studies with longer diet interventions, monkeys
consuming TAD-soy diets had lower body weight in addition to
improved lipids and measures of glycemic control .
We and others have previously reported significant variation in
complex disease phenotypes that appears to be mediated by
dietary soy (e.g., [22,30–32]). To study this variability systemat-
ically, we have leveraged the available molecular tools designed for
Figure 3. Overall proportion of DNA methylation in each tissue, by diet. Values are the means (with standard errors) of probes with a
detection p-value ,0.05 for all samples (N=8,002) in each tissue. Four males were included in each diet group.
Epigenetic Changes with Soy in Cynomolgus Monkeys
PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26791
the human genome with nonhuman primates, which have a high
degree of DNA identity with humans, but can be studied in a
controlled environmental setting (including diet). We identified
significant, tissue-specific changes in DNA methylation due to diet
changes. These data provide evidence that dietary soy affects
global DNA methylation, which may contribute to soy’s beneficial
Table 2. Pathways detected after TAD diet changes.
Diet Change Tissue
P, ,0.001Over-represented GO and KEGG pathways*
TAD-soy to TAD-caseinBlood 1373 preassembly of GPI anchor in ER membrane 0.030
GPI anchor metabolic process0.067
GPI anchor biosynthetic process0.067
protein amino acid lipidation 0.076
histone methyltransferase activity0.076
lipoprotein biosynthetic process0.082
P-P-bond-hydrolysis-driven protein transmembrane transp. act.0.082
macromolecule transmembrane transporter activity0.082
protein methyltransferase activity 0.089
Fat212 18No associated pathways NA
Liver 35925No associated pathwaysNA
Muscle 67782No associated pathwaysNA
TAD-casein to TAD-soyBlood 1265 autophagic vacuole0.059
Fat264 22embryonic skeletal system development 0.0068
cranial nerve development0.011
skeletal system morphogenesis 0.028
proton-transp. two-sector ATPase complex proton-transp. domain0.039
forelimb morphogenesis 0.044
striated muscle cell proliferation 0.054
embryonic skeletal system morphogenesis0.054
cardiac muscle cell proliferation0.054
proton-transporting ATP synthase complex coupling factor F(o)0.099
Liver 693 anion:cation symporter activity 0.00012
hydro-lyase activity 0.0011
carbon-oxygen lyase activity 0.019
protein kinase A binding0.053
carbonate dehydratase activity0.068
Muscle 18917 No associated pathwaysNA
*All genes with p,0.01 were used to identify GO and KEGG pathways.
Table 3. Genes with significant DNA methylation changes (FDR P,0.20) after the diet switch.
Diet TissueGene Symbol
TAD-soy to TAD-casein muscleHOXA5cg02248486
TAD-casein to TAD-soy fatABCG5 cg25781162
Epigenetic Changes with Soy in Cynomolgus Monkeys
PLoS ONE | www.plosone.org7 October 2011 | Volume 6 | Issue 10 | e26791
effects on multiple complex phenotypes. Additional studies in
nonhuman primates and humans are necessary to determine the
long-term physiologic and potentially pathologic consequences of
soy and other dietary supplements on epigenetic regulation and
Conceived and designed the experiments: JDW TDH. Performed the
experiments: TDH LZ WC SG GAH JDW. Analyzed the data: TDH
SMH JC RS SG MM. Wrote the paper: TDH JDW.
1. Wagner JD, Schwenke DC, Greaves KA, Zhang L, Anthony MS, et al. (2003)
Soy protein with isoflavones, but not an isoflavone-rich supplement, improves
arterial low-density lipoprotein metabolism and atherogenesis. Arterioscler
Thromb Vasc Biol 23: 2241–2246.
2. Morito K, Aomori T, Hirose T, Kinjo J, Hasegawa J, et al. (2002) Interaction of
phytoestrogens with estrogen receptors alpha and beta (II). Biol Pharm Bull 25:
3. Hwang CS, Kwak HS, Lim HJ, Lee SH, Kang YS, et al. (2006) Isoflavone
metabolites and their in vitro dual functions: they can act as an estrogenic
agonist or antagonist depending on the estrogen concentration. J Steroid
Biochem Mol Biol 101: 246–253.
4. Cline JM, Wood CE (2009) Estrogen/isoflavone interactions in cynomolgus
macaques (Macaca fascicularis). Am J Primatol 71: 722–731.
5. Zhang X, Ho SM (2011) Epigenetics meets endocrinology. J Mol Endocrinol 46:
6. Badger TM, Ronis MJ, Hakkak R, Rowlands JC, Korourian S (2002) The
health consequences of early soy consumption. J Nutr 132: 559S–565S.
7. Bhatia J, Greer F (2008) Use of soy protein-based formulas in infant feeding.
Pediatrics 121: 1062–1068.
8. Barker DJ, Winter PD, Osmond C, Margetts B, Simmonds SJ (1989) Weight in
infancy and death from ischaemic heart disease. Lancet 2: 577–580.
9. Dolinoy DC, Jirtle RL (2008) Environmental epigenomics in human health and
disease. Environ Mol Mutagen 49: 4–8.
10. Newbold RR, Padilla-Banks E, Snyder RJ, Jefferson WN (2005) Developmental
exposure to estrogenic compounds and obesity. Birth Defects Research Part A-
Clinical and Molecular Teratology 73: 478–480.
11. Souzeau E, Belanger S, Picard S, Deschepper CF (2005) Dietary isoflavones
during pregnancy and lactation provide cardioprotection to offspring rats in
adulthood. Am J Physiol Heart Circ Physiol 289: H715–H721.
12. Klein SL, Wisniewski AB, Marson AL, Glass GE, Gearhart JP (2002) Early
exposure to genistein exerts long-lasting effects on the endocrine and immune
systems in rats. Mol Med 8: 742–749.
13. Wisniewski AB, Cernetich A, Gearhart JP, Klein SL (2005) Perinatal exposure to
genistein alters reproductive development and aggressive behavior in male mice.
Physiol Behav 84: 327–334.
14. Jefferson WN, Padilla-Banks E, Goulding EH, Lao SP, Newbold RR, et al.
(2009) Neonatal exposure to genistein disrupts ability of female mouse
reproductive tract to support preimplantation embryo development and
implantation. Biol Reprod 80: 425–431.
15. Lamartiniere CA, Cotroneo MS, Fritz WA, Wang J, Mentor-Marcel R, et al.
(2002) Genistein chemoprevention: timing and mechanisms of action in murine
mammary and prostate. J Nutr 132: 552S–558S.
16. Ho SM, Tang WY, Belmonte dF, Prins GS (2006) Developmental exposure to
estradiol and bisphenol A increases susceptibility to prostate carcinogenesis and
epigenetically regulates phosphodiesterase type 4 variant 4. Cancer Res 66:
17. Prins GS, Birch L, Tang WY, Ho SM (2007) Developmental estrogen
exposures predispose to prostate carcinogenesis with aging. Reprod Toxicol 23:
18. Prins GS, Tang WY, Belmonte J, Ho SM (2008) Developmental exposure to
bisphenol A increases prostate cancer susceptibility in adult rats: epigenetic
mode of action is implicated. Fertil Steril 89: e41.
19. Prins GS, Tang WY, Belmonte J, Ho SM (2008) Perinatal exposure to oestradiol
and bisphenol A alters the prostate epigenome and increases susceptibility to
carcinogenesis. Basic Clin Pharmacol Toxicol 102: 134–138.
20. Dolinoy DC, Weidman JR, Waterland RA, Jirtle RL (2006) Maternal genistein
alters coat color and protects Avy mouse offspring from obesity by modifying the
fetal epigenome. Environ Health Perspect 114: 567–572.
Figure 4. Comparison of HumanMethylation27 BeadChip with pyrosequencing. The gene associated with each CpG site is shown to the
left of each graph. The correlation (r2) between pyrosequencing (black bars) and the HumanMethylation27 BeadChip (white bars) is shown under
each gene symbol. The four pairs of bars on the left of each graph represent the percent DNA methylation from monkeys eating the TAD Casein diet,
and the four pairs on the right half represent the percent DNA methylation from monkeys eating the TAD Soy diet.
Epigenetic Changes with Soy in Cynomolgus Monkeys
PLoS ONE | www.plosone.org8 October 2011 | Volume 6 | Issue 10 | e26791
21. Wagner JD, Kavanagh K, ward d, Kaplan J (2006) Old world primate models of
type 2 diabetes mellitus. Institute for Laboratory Animal Research 47: 259–271.
22. Wagner JD, Jorgensen MJ, Cline JM, Lees CJ, Franke AA, et al. (2009) Effects of
soy vs. casein protein on body weight and glycemic control in female monkeys
and their offspring. Am J Primatol 71: 802–811.
23. Thigpen JE, Setchell KD, Ahlmark KB, Locklear J, Spahr T, et al. (1999)
Phytoestrogen content of purified, open- and closed-formula laboratory animal
diets. Lab Anim Sci 49: 530–536.
24. Jensen MN, Ritskes-Hoitinga M (2007) How isoflavone levels in common rodent
diets can interfere with the value of animal models and with experimental results.
Lab Anim 41: 1–18.
25. Stroud FC, Appt SE, Wilson ME, Franke AA, Adams MR, et al. (2006)
Concentrations of isoflavones in macaques consuming standard laboratory
monkey diet. J Am Assoc Lab Anim Sci 45: 20–23.
26. Berge KE, Tian H, Graf GA, Yu L, Grishin NV, et al. (2000) Accumulation of
dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC
transporters. Science 290: 1771–1775.
27. Heijmans BT, Tobi EW, Stein AD, Putter H, Blauw GJ, et al. (2008) Persistent
epigenetic differences associated with prenatal exposure to famine in humans.
Proc Natl Acad Sci U S A 105: 17046–17049.
28. Day JK, Bauer AM, DesBordes C, Zhuang Y, Kim BE, et al. (2002) Genistein
alters methylation patterns in mice. J Nutr 132: 2419S–2423S.
29. Dolinoy DC, Huang D, Jirtle RL (2007) Maternal nutrient supplementation
counteracts bisphenol A-induced DNA hypomethylation in early development.
Proc Natl Acad Sci U S A 104: 13056–13061.
30. Wagner JD, Zhang L, Shadoan MK, Kavanagh K, Chen H, et al. (2008) Effects
of soy protein and isoflavones on insulin resistance and adiponectin in male
monkeys. Metabolism 57: S24–S31.
31. Droke EA, Hager KA, Lerner MR, Lightfoot SA, Stoecker BJ, et al. (2007) Soy
isoflavones avert chronic inflammation-induced bone loss and vascular disease.
J Inflamm (Lond) 4: 17.
32. Adams MR, Golden DL, Williams JK, Franke AA, Register TC, et al. (2005)
Soy protein containing isoflavones reduces the size of atherosclerotic plaques
without affecting coronary artery reactivity in adult male monkeys. J Nutr 135:
Epigenetic Changes with Soy in Cynomolgus Monkeys
PLoS ONE | www.plosone.org9 October 2011 | Volume 6 | Issue 10 | e26791