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Maternal micronutrients and brain global


methylation patterns in the offspring

Article in Nutritional Neuroscience · November 2013


DOI: 10.1179/1476830513Y.0000000097 · Source: PubMed

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Original research paper
Maternal micronutrients and brain global
methylation patterns in the offspring
Pratiksha Sable, Karuna Randhir, Anvita Kale, Preeti Chavan-Gautam,
Sadhana Joshi
Department of Nutritional Medicine, Interactive Research School for Health Affairs, Bharati Vidyapeeth
University, Pune, India

Objectives: Studies have established the association of maternal nutrition and increased risk for non-
communicable diseases. It has been suggested that this involves epigenetic modifications in the genome.
However, the role of maternal micronutrients in the one-carbon cycle in influencing brain development of
the offspring through methylation is unexplored. It is also unclear whether epigenomic marks established
during early development can be reversed by a postnatal diet. The present study reports the effect of
maternal micronutrients and omega-3 fatty acids on global DNA methylation patterns in the brain of the
Wistar rat offspring at three timepoints (at birth, postnatal day 21, and 3 months of age).
Method: Pregnant rats were divided into control (n = 8) and five treatment groups (n = 16 dams in each
group) at two levels of folic acid (normal and excess folate) in the presence and absence of vitamin B12
(NFBD, EFB, and EFBD). Omega-3 fatty acid supplementation was given to vitamin B12 deficient groups
(NFBDO and EFBDO). Following delivery, eight dams from each group were shifted to control diet and
remaining continued on the same treatment diet.
Results: Our results demonstrate that maternal micronutrient imbalance results in global hypomethylation in
the offspring brain at birth. At adult age the cortex of the offspring displayed hypermethylation as compared
with control, in spite of a postnatal control diet. In contrast, prenatal omega-3 fatty acid supplementation was
able to normalize methylation at 3 months of age.
Discussion: Our findings provide clues for the role of omega-3 fatty acids in reversing methylation patterns
thereby highlighting its contribution in neuroprotection and cognition.
Keywords: Cortex, Docosahexaenoic acid, Folic acid, Global methylation, Hippocampus, Omega-3 fatty acids, Vitamin B12

Introduction reported altered placental global methylation patterns


Studies have well established the association between in pregnancy complications like preeclampsia6 and in
maternal nutrition and health of the offspring as an preterm7 pregnancies wherein these women had
adult and are mainly considered to be a consequence altered levels of maternal micronutrients like folic
of epigenetic modifications in the genome.1 Reports acid, vitamin B12, and omega-3 fatty acids.8 We have
have suggested that the constituents of the ‘one extensively discussed the association between folic
carbon cycle’, especially folic acid and vitamin B12, acid, vitamin B12, and docosahexaenoic acid (DHA)
can mediate foetal programming possibly through epi- in the one-carbon cycle both in the human9 and
genetic modifications.2 Among the epigenetic mechan- animal10 studies. We have also reported reduced pla-
isms, DNA methylation and histone modifications are cental global DNA methylation levels in Wistar rats
the most widely studied.3 Most of the studies assessing as a consequence of altered maternal micronutrients
the risk of disease in the offspring as a consequence of like folic acid and vitamin B12.10 Recent reports
altered maternal nutrition have mainly been in connec- suggest that the epigenetic patterns displayed by the
tion with placental–foetal development4 and con- placenta could be suggestive of the patterns in the
ditions like diabetes and obesity.5 We have earlier pup brain.11
It is reported that maintaining a balanced supply of
Correspondence to: Sadhana Joshi, Department of Nutritional Medicine, methyl groups to the brain is necessary for sustaining
Interactive Research School for Health Affairs, Bharati Vidyapeeth normal functioning.12 Nutrition is known to play a
University, Pune Satara Road, Pune 411043, India.
Email: srjoshi62@gmail.com key role in regulating brain development and

© W. S. Maney & Son Ltd 2013


DOI 10.1179/1476830513Y.0000000097 Nutritional Neuroscience 2013 VOL. 0 NO. 0 1
Sable et al. Maternal micronutrients and brain global methylation patterns

functioning13,14 and any alteration may cause brain randomly allocated to the six dietary groups. Diet
disorders through epigenetic modifications like DNA composition was as described by us earlier.21,24
methylation.15 In spite of this, limited studies have Immediately after confirmation of pregnancy (day 0)
examined the association of maternal micronutrients dams were randomly assigned to either control or any
and methylation changes in the brain of the offspring of the treatment groups. The effect of dietary vitamin
and their role remains unclear. Some studies have B12 deficiency during pregnancy was analysed at two
demonstrated that there is no major association levels of folic acid (i.e. 2 and 8 mg folic acid/kg diet).
between maternal methyl donor deficiency and Both the vitamin B12 deficient groups were sup-
expression or methylation of genes like glucocorticoid plemented with omega-3 fatty acids (DHA + EPA).
receptors in the brain of the offspring.16 In contrast, Maxepa tablets (fish oil, Merck, Darmstadt,
maternal choline deficiency has been shown to affect Germany) were used to provide the omega-3 fatty
methylation patterns and possibly impair brain devel- acid supplementation, which is a combination of
opment in the mouse offspring.17 DHA (120 g) and eicosapentaenoic acid (EPA) (180 g).
Furthermore, it is unclear whether the epigenomic The groups were as follows: control: normal folic
marks established early in life can be reversed during acid, normal vitamin B12; NFBD: normal folic acid,
the postnatal period. Most of the studies examining vitamin B12 deficient; NFBDO: normal folic acid,
the possibility of reversal, using nutrients like leptin, vitamin B12 deficient, omega-3 fatty acid sup-
are related to the markers of the metabolic syn- plemented; EFB: excess folic acid, normal vitamin
drome.18 Postnatal supplementation of nutrients like B12; EFBD: excess folic acid, vitamin B12 deficient;
choline has been shown to normalize methylation EFBDO: excess folic acid, vitamin B12 deficient,
changes in the rat offspring.19 In contrast, studies in omega-3 fatty acid supplemented. There were 8 dams
the humans demonstrate that folic acid supplemen- in the control group and 16 dams in each of the treat-
tation for a period of 3 years had no effect on blood ment groups. After delivery, eight dams from each
leucocyte global DNA methylation patterns.20 Our group were shifted back to control diet and the remain-
earlier studies indicate that maternal micronutrient ing eight continued on the same treatment diet. In case
imbalance leads to a reduction in protein and of control, all the animals that delivered continued on
mRNA levels of neurotrophins like brain-derived neu- a control diet until 3 months of age (Table 1).
rotrophic factor (BDNF) at birth and we discussed the All experimental procedures were in accordance
possibility of BDNF levels being altered as a conse- with guidelines of Institutional Animal Ethics
quence of altered methylation.21,22 However, prenatal Committee. The institute is recognized to undertake
omega-3 fatty acid supplementation, but not a post- experiments on animals as per the committee for the
natal control diet, normalized the levels at day 21
(d21) suggesting the possibility of BDNF being an
Table 1 Dietary treatment groups
imprinted gene.22 In the present study, we hypothesize
that an imbalance in maternal micronutrients will alter Diet during Diet during lactation up
brain global methylation levels which will be normal- Groups pregnancy to 3 months of age

ized only by a prenatal omega-3 fatty acid Control Control Control


supplementation. NFBD-C NFBD Control
NFBD–NFBD NFBD NFBD
Brain development is known to be dependent upon EFB-C EFB Control
critical windows and is suited to target intervention EFB–EFB EFB EFB
studies aimed at re-programming.23 It therefore EFBD-C EFBD Control
EFBD–EFBD EFBD EFBD
makes it necessary to examine changes in global NFBDO-C NFBDO Control
methylation levels in the brain in a time-dependent NFBDO–NFBDO NFBDO NFBDO
fashion. The present study for the first time reports EFBDO-C EFBDO Control
EFBDO–EFBDO EFBDO EFBDO
the effect of a maternal micronutrient imbalanced
diet and the effects of omega-3 fatty acid supplemen- NFBD: normal folic acid, vitamin B12 deficient; NFBDO: normal
folic acid, vitamin B12 deficient, omega-3 fatty acid
tation (both short term and long term) on global supplemented; EFB: excess folic acid, normal vitamin B12;
DNA methylation patterns in the brain of the Wistar EFBD: excess folic acid, vitamin B12 deficient; EFBDO: excess
rat offspring at three timepoints, i.e. at birth, postnatal folic acid, vitamin B12 deficient, omega-3 fatty acid
supplemented.
d21, and 3 months of age. NFBD–NFBD: NFBD continuing on NFBD after delivery; NFBD-
C: NFBD shifting to control after delivery; NFBDO-C: NFBDO
shifting to control after delivery; NFBDO–NFBDO: NFBDO
Materials and methods continuing on NFBDO after delivery; EFB–EFB: EFB continuing
Animals on EFB after delivery; EFB-C: EFB shifting to control after
The protocol for the study has been described by us in delivery; EFBD–EFBD: EFBD continuing on EFBD after delivery;
EFBD-C: EFBD shifting to control after delivery;
detail earlier.21,24,25 The dietary groups designed were EFBDO–EFBDO: EFBDO continuing on EFBDO after delivery;
based on AIN-93 guidelines and pregnant dams were EFBDO-C: EFBDO shifting to control after delivery.

2 Nutritional Neuroscience 2013 VOL. 0 NO. 0


Sable et al. Maternal micronutrients and brain global methylation patterns

purpose of control and supervision of experimental


animals, Government of India (No. 258/CPCSEA).

Global DNA methylation


Genomic DNA extraction from brain tissues was
carried out using the Qiagen Blood and Tissue kit.
Global DNA methylation was measured in duplicate
using the Methylamp™ Global DNA Methylation
Quantification Kit as per the manufacturer’s instruc-
1 tions (Epigentek Group Inc., NY, USA) and the
method has been reported by us earlier.6,7 This kit
measures methylcytosine content as a percentage of
total cytosine content. DNA extracted from brain Figure 1 Global DNA methylation in the rat brain at birth.
*P < 0.05, **P < 0.01 as compared with control; @@P < 0.01
samples was immobilized on a strip well. The strip
as compared with NFBD. NFBD: normal folic acid, vitamin B12
well was specifically treated to have a high affinity deficient; NFBDO: normal folic acid, vitamin B12 deficient,
for DNA. DNA methylation was then quantified by omega-3 fatty acid supplemented; EFB: excess folic acid,
an enzyme-linked immunosorbent assay-like reaction normal vitamin B12; EFBD: excess folic acid, vitamin B12
using 5-methylcytosine antibody. The amount of deficient; EFBDO: excess folic acid, vitamin B12 deficient,
methylated DNA is proportional to the optical omega-3 fatty acid supplemented.

density and the degree of DNA methylation can be


calculated using the following formula: DNA methylation in the brain as compared with
   control (Fig. 1).
Per cent methylation = OD sample − blank /
 
2OD positive control − blank × 100 Global DNA methylation in offspring brain at d21
The per cent global methylation in the brain in all
where OD is the optical density, blank is buffer treatment groups was comparable with that of
without DNA, positive control is methylated control control at d21 (Fig. 2). As compared with the results
DNA. The amount of methylated DNA was expressed at birth, neither a sustained vitamin B12 deficiency,
in terms of per cent methylation. at both levels of folic acid, nor postnatal shift to a
control diet had an effect on the methylation patterns
Statistical analysis at d21. Similarly, an omega-3 supplementation during
Per cent methylation values were reported as mean ± pregnancy or up to d21 did not affect the methylation
SD. The data were analysed using SPSS/PC+ patterns.
package (Version 20, Chicago, IL, USA). The treat-
ment groups were compared with the relevant Global DNA methylation in offspring
control group by analysis of variance (ANOVA) and hippocampus at 3 months of age
the post hoc least significant difference test (P < 0.05 In the hippocampus, a sustained vitamin B12
was considered significant). One-way ANOVA was deficiency at normal levels of folic acid (NFBD–
used to examine changes in the control group from NFBD) displayed higher (1.8 fold, P < 0.05) levels
birth to d21. compared with when the group was shifted back to
control (NFBD-C). An omega-3 supplementation at
Results normal levels of folic acid (NFBDO–NFBDO) had
Global DNA methylation in offspring brain at higher levels (1.7 fold) as compared with control
birth (P < 0.01) and as compared with NFBDO-C (1.5
A maternal vitamin B12 deficient diet at normal level fold, P < 0.05). However, there was no effect on per
of folic acid (NFBD) did not affect the global DNA cent methylation at excess folic acid supplementation
methylation in the brain, while excess folic acid sup- both in the presence and absence of vitamin B12
plementation to a vitamin B12 deficient (EFBD) diet (Fig. 3).
reduced (0.7 fold) the per cent DNA methylation as
compared with control (P < 0.05) and NFBD (P < Global DNA methylation in offspring cortex at 3
0.01). Furthermore, maternal omega-3 fatty acid sup- months of age
plementation to these vitamin B12 deficient diets at In the cortex, vitamin B12 deficiency at normal folic
normal folic acid levels (NFBDO) reduced (0.6 fold) acid (NFBD–NFBD) had no effect on the methylation
the per cent DNA methylation in the brain as com- patterns. Shifting this group back to control diet
pared with control and NFBD (P < 0.01 for both). (NFBD-C) led to an increase (1.9 fold, P < 0.01) in
Omega-3 fatty acid supplementation at excess folic methylation levels as compared with control. Excess
acid (EFBDO) levels reduced (P < 0.01) the per cent folic acid in the presence of vitamin B12 (EFB-EFB)

Nutritional Neuroscience 2013 VOL. 0 NO. 0 3


Sable et al. Maternal micronutrients and brain global methylation patterns

Figure 2 Global DNA methylation in the rat brain at the end of lactation. NFBD–NFBD: NFBD continuing on NFBD after delivery;
NFBD-C: NFBD shifting to control after delivery; NFBDO–NFBDO: NFBDO continuing on NFBDO after delivery; EFB–EFB: EFB
continuing on EFB after delivery; EFB-C: EFB shifting to control after delivery; EFBD–EFBD: EFBD continuing on EFBD after
delivery; EFBD-C: EFBD shifting to control after delivery; EFBDO–EFBDO: EFBDO continuing on EFBDO after delivery; EFBDO-C:
EFBDO shifting to control after delivery.

Figure 3 Global DNA methylation in the rat hippocampus at 3 months of age. **P < 0.01 as compared with control; #P < 0.05 as
compared with NFBD-C; &P < 0.05 as compared with NFBDO-C. NFBD–NFBD: NFBD continuing on NFBD after delivery; NFBD-C:
NFBD shifting to control after delivery; NFBDO–NFBDO: NFBDO continuing on NFBDO after delivery; EFB–EFB: EFB continuing
on EFB after delivery; EFB-C: EFB shifting to control after delivery; EFBD–EFBD: EFBD continuing on EFBD after delivery; EFBD-
C: EFBD shifting to control after delivery; EFBDO–EFBDO: EFBDO continuing on EFBDO after delivery; EFBDO-C: EFBDO shifting
to control after delivery.

Figure 4 Global DNA methylation in the rat cortex at 3 months of age. *P < 0.05, **P < 0.01 as compared with control; ##P < 0.01
as compared with NFBD-C;ˆP < 0.05 as compared with EFB-C; ++P < 0.01 as compared with EFBD-C; ΩP < 0.05 as compared with
EFBDO-C. NFBD–NFBD: NFBD continuing on NFBD after delivery; NFBD-C: NFBD shifting to control after delivery;
NFBDO–NFBDO: NFBDO continuing on NFBDO after delivery; EFB–EFB: EFB continuing on EFB after delivery; EFB-C: EFB
shifting to control after delivery; EFBD–EFBD: EFBD continuing on EFBD after delivery; EFBD-C: EFBD shifting to control after
delivery; EFBDO–EFBDO: EFBDO continuing on EFBDO after delivery; EFBDO-C: EFBDO shifting to control after delivery.

caused an increase (1.8 fold, P < 0.01) in methylation control diet (EFB-C). Excess folic acid in the
levels, which remained high (P < 0.05) as compared absence of vitamin B12 (EFBD-EFBD) caused an
with control even if the group was shifted to a increase (2.2 fold, P < 0.01) in methylation levels,

4 Nutritional Neuroscience 2013 VOL. 0 NO. 0


Sable et al. Maternal micronutrients and brain global methylation patterns

which remained high (P < 0.01) as compared with development. Vitamin B12 deficiency during preg-
control even if the group was shifted to a control diet nancy at normal folic acid levels shows a trend
(EFBD-C). Long-term (EFBDO-EFBDO) maternal towards a higher methylation at birth and decrease
omega-3 fatty acid supplementation at excess folic at d21. However, the methylation changes were more
acid increased per cent methylation (1.7 fold) com- dramatic in the imbalanced group (i.e. excess folic
pared with control and EFBDO-C (P < 0.05 for acid and vitamin B12 deficiency). This result is
both). A prenatal omega-3 fatty acid supplementation similar to the levels of global methylation observed
(EFBDO-C) was able to normalize the hypermethyla- in the placenta and have been reported by us
tion displayed by the micronutrient imbalanced group earlier.10 We have recently demonstrated that
during pregnancy (EFBD-C) (Fig. 4). maximum adverse effects were observed when there
is an imbalance in maternal micronutrients (folic
Global DNA methylation in control from birth to acid and vitamin B12) on brain fatty acids, oxidative
d21 stress and neurotrophin levels, and expression from
The per cent methylation values from birth to d21 birth till adult age.21,22,24,31 Similarly, in case of the
(0.938 ± 0.239 at birth; 2.092 ± 0.71 at d21) reveal a excess folic acid supplementation although methyl-
significant time-dependent increase. ation levels showed a trend towards reduction at
birth, there was a significant increase at 3 months.
Discussion This was exacerbated in the absence of vitamin B12,
This study for the first time reports changes in global i.e. imbalanced diet possibly as a result of the methyl
DNA methylation patterns in the offspring brain at trap. Studies by others also suggest that high folate
birth, d21, and at 3 months of age as a consequence in addition to the low vitamin B12 creates an imbal-
of a maternal micronutrient imbalance. It also exam- ance in the one-carbon cycle, which may lead to
ines the effect of omega-3 fatty acid supplementation altered programming of the offspring during preg-
to these maternal micronutrient imbalanced diets on nancy.5 A combination of high folate and low
DNA methylation levels in the offspring brain. The vitamin B12 is also suggested to have a negative
key findings of this study are: (1) maternal micronutri- impact upon the nervous system.32 However, in vitro
ent imbalance led to reduction in brain global methyl- cultures and in vivo studies are yet to demonstrate a
ation levels at birth. (2) At d21, both a maternal clear link between vitamin B12 deficiency, increased
micronutrient imbalanced diet and a postnatal genomic instability, and DNA methylation.33
control diet did not alter methylation patterns in the In contrast to the results observed at birth, global
brain. (3) At 3 months of age, in the hippocampus methylation levels at d21 were similar among all
there was no change in the methylation levels in the groups. Possible explanation for the observed effects
imbalanced groups (EFBD-EFBD), while in the in our study could be the increased amount of DHA
cortex the micronutrient imbalanced group, in spite in the breast milk, which may have played a role in
of a postnatal control diet (EFBD-C), showed higher the tight regulation of nutrient availability to the off-
levels of methylation as compared with control. (4) spring which we have reported earlier.22,25 Our find-
Prenatal omega-3 fatty acid supplementation ings support the concept that nutrient alterations
(EFBDO-C) was able to normalize the levels of result in differential effects on methylation patterns.34
methylation, while a long-term omega-3 fatty acid Similarly, other studies have demonstrated that a
supplementation (EFBDO-EFBDO) increased the dietary folate supplementation in rats initiated at
levels of methylation as compared with control in the weaning or later in life failed to modulate DNA
cortex at 3 months of age. methylation.35,36
Global DNA methylation is considered to be repre- In the current study, methylation patterns in the
sentative of the status of repetitive DNA elements, brain of the adult offspring at 3 months of age were
transposons, and intergenic sequences.26 Loss of examined in a region wise manner (cortex and hippo-
DNA methylation is known to have multiple conse- campus), since it is known that methylation patterns
quences including genomic instability27 and loss of display region specificity.19 Furthermore, these
function in the central nervous system.28 However, regions are also known to play a differential role in
little is known about the effects of altered global cognitive functions.37 In contrast to our results
DNA hypomethylation on the central nervous obtained at d21, at 3 months of age in the adult off-
system.29 The brain, during early development, is spring, the micronutrient imbalanced group showed
suggested to be highly sensitive towards external influ- hypermethylation in the cortex but no change in the
ences and the nature of insult is also subject to the time hippocampus. The cognitive abilities of these adult
at which it occurs.30 Therefore, an adequate maternal animals using the Morris water maze test showed
nutritional status during early phases of brain growth that the animals from the imbalanced group displayed
could play an important role in determining brain longer escape latencies.31 The cortex is considered to

Nutritional Neuroscience 2013 VOL. 0 NO. 0 5


Sable et al. Maternal micronutrients and brain global methylation patterns

play a vital role in decision-making and cognitive be- from foetal life to early childhood.44 Our study
haviour38 and our results may have implications for suggests that perturbation of epigenetic mechanisms
altered brain development. Moreover, global hyper- may play a role in long-term cognitive impairment
methylation of DNA has been reported in brain dis- possibly through specific epigenetic modifications in
orders like Alzheimer’s disease and bipolar disorder.39 vital genes involved in neurodevelopment.
The hypermethylation in the cortex observed in the To summarize, the in utero effects of maternal
current study could have serious implications for adult micronutrient imbalance showed differential change
health as the hypermethylated state could adversely in the offspring at birth, d21, and 3 months of age.
affect the expression of key genes involved in learning The limitations of this study are: (1) the analysis
and memory. We have earlier reported that maternal carried out at the first two timepoints (at birth and
micronutrient imbalanced group in the cortex dis- end of lactation) was on whole brain samples, while
played lower levels of BDNF, a key gene involved in the analysis at 3 months of age was region-specific;
neurodevelopment, at birth and at d21 of age,21,22- (2) global methylation analysis is mainly used as an
which are important molecules involved in learning indicator of genomic stability27,45 and cannot be
and memory formation. used to predict changes taking place at the gene-level.
In the current study, a long-term omega-3 fatty acid Nevertheless, our findings for the first time demon-
supplementation to the imbalanced group also showed strate changes in global methylation patterns in the
hypermethylation as compared with control while, a brain as a consequence of altered maternal micronutri-
short-term prenatal omega-3 fatty acid supplemen- ents. We also demonstrate the important role of prena-
tation was able to normalize the hypermethylation tal omega-3 fatty acids in normalizing methylation
changes to that of control in the cortex at 3 months patterns.
of age. Dietary omega-3 fatty acids are known to
have a wide range of physiological effects that influ- Acknowledgements
ence gene expression and are also capable of influen- We thank the Department of Biotechnology, New
cing epigenetic processes.40 Delhi, India for partially funding this project. We
Micronutrients are a vital part of the one-carbon thank Mr Vinayak Dhawale for his assistance at the
cycle, which generates methyl groups which are animal house.
accepted by DNA, RNA, hormones, neurotransmit-
ters, and membrane phospholipids.41 We have earlier
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