Epigenetic regulation of human buccal mucosa mitochondrial superoxide
dismutase gene expression by diet
Roman Thaler1, Heidrun Karlic2,3, Petra Rust1and Alexander G. Haslberger1*
1Department of Nutritional Sciences, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
2Ludwig Boltzmann Institute for Leukaemia Research, Hanusch Krankenhaus, Vienna, Austria
3Hanuschkrankenhaus der Wiener Gebietskrankenkasse, Heinrich Collinstraße 30, A-1140 Vienna, Austria
(Received 30 October 2007 – Revised 24 June 2008 – Accepted 26 June 2008 – First published online 7 August 2008)
The impact of nutrition on the epigenetic machinery has increasingly attracted interest. The aim of the present study was to demonstrate the effects
of various diets on methylation and gene expression. The antioxidative enzyme mitochondrial superoxide dismutase (MnSOD) was chosen as the
model system because epigenetic regulation has been previously shown in cell lines for this gene. Promoter methylation and gene expression of
MnSOD in buccal swabs from three sample groups were analysed. The three groups included: (1) forty vegetarians (aged 20–30 years); (2) age-
matched omnivores; (3) elderly omnivores (aged . 85 years). A 3-fold increase in the expression of the MnSOD gene was associated with
decreased CpG methylation of the analysed promoter region in the vegetarian group compared with the age-matched omnivores group. Expression
and promoter methylation of the MnSOD gene in elderly omnivores showed no significant differences compared with younger omnivores.
In accordance with previous findings in various tissues, DNA global methylation was found to be significantly higher (30%) in buccal swabs
of younger subjects (independent of the diet), than in those of elderly omnivores. In the control experiment which was designed to verify the find-
ings of the human buccal swab studies, the Caco-2 cell line was treated with zebularine. Results of the control study showed a 6-fold increase of
MnSOD expression, an approximately 40% decreased methylation of specified CpG in the MnSOD promoter and a 50% reduction of global DNA
methylation. These results indicate that diet affects the epigenetic regulation of human MnSOD.
Mitochondrial superoxide dismutase: Epigenetic regulation: Buccal mucosa: Vegetarian diet
The impact of nutrition on the epigenetic machinery(1–3)has
increasingly attracted interest. There is increasing evidence of
the determinant role of the environment, including nutrition,
on the expression of many genes related to health status(4–8).
Many environmental factors, for example, irradiation or toxins,
are known to induce signalling pathways that provoke oxi-
dative stress in tissues. These same pathways are associated
Hypermethylation of CpG islands located in promoter
regions of genes has been shown to alter gene expression(11,12).
DNA methylation is mediated by DNA methyltransferases
(DNMT). Inhibitors such as zebularine restore expression of
methylated genes by inhibition of the methyltransferases(13).
Additional mechanisms at the histone(14)and small interfering
RNA(15)levels also contribute to the epigenetic regulatory
Epidemiological data suggest that epigenetic mechanisms are
responsible for a correlation between parental nutrition and the
risk of progeny developing certain diseases(16,17). Experiments
utilising the agouti mouse model show a transgenerational
alteration of the mouse phenotype by hypermethylation of the
agouti gene due to methyl donor-rich diets(18).
Several dietary compounds are implicated in the regulation
of the DNA-methylation pathway. Vitamin B12is a cofactor in
the folate-mediated remethylation of homocysteine to methio-
nine, which is further activated to S-adenosylmethionine
(SAM), the methyl donor for DNA methylation. SAM con-
verts to S-adenosylhomocysteine (SAH) after DNA methyl-
ation. Reversiblehydrolysis of SAH to homocysteine
completes the cycle. Under conditions of elevated homocys-
teine concentrations, this reaction is reversed resulting in an
increased concentration of the potent SAM-inhibitor SAH.
Deficiency in vitamin B12leads to an accumulation of serum
homocysteine(19). The vegetarian diet is low in vitamin B12,
thereby reducing the remethylation of homocysteine and
resulting in low methionine content(19), which may reduce
DNA-methylation machinery in vegetarians. Studies con-
ducted at our Nutrition Department at the University of
Vienna have previously demonstrated that vegetarians dis-
played decreased vitamin B12 and enhanced homocysteine
plasma concentrations compared with omnivores(20).
*Corresponding author: Dr Alexander G. Haslberger, fax þ43 1 8795896, email email@example.com
Abbreviations: DNMT, DNAmethyltransferase;G6PD,glucose-6-phosphatedehydrogenase;MnSOD,mitochondrialsuperoxidedismutase;SAH,S-adenosylhomocysteine;
SAM, S-adenosylmethionine; TA, telomerase activity.
British Journal of Nutrition (2009), 101, 743–749
q The Authors 2008
British Journal of Nutrition
DNA methylation of CpG of specific genes needs to
be differentiated from global DNA methylation because
during ageing the DNA total methylation level declines,
whereas some gene promoters tend to be hypermethy-
lated(21–23). Genes that change methylation status with
age are tissue specific(21). This process is also described in
the pathology of many cancers(22)and may be attributed
to a decreased expression of the global methylation-related
DNMT1 and an increased expression of the specific methyl-
ation-related DNMT3b during ageing(9).
Mitochondrial superoxide dismutase (MnSOD) is an antioxi-
dant enzyme and converts superoxide radicals (Oz2), which
are highly reactive and damaging to cellular structures, to
many pathological mechanisms and ageing, but also have fun-
damental beneficial roles, for example, in the innate immune
response(25). The cancer-promoting effects of superoxide radi-
cals have been linked to experimental data demonstrating
changes in the tumour-suppressor activity of MnSOD(26).
Close regulation of such proteins is essential. Hereditary influ-
ences(24,27)but also epigenetic regulation(28)are proposed for
the expression of the MnSOD gene.
The present paper focuses on the analysis of epigenetic
regulation of the MnSOD gene. It describes the effect of
different diets and age on expression of the MnSOD gene
and methylation of its promoter as well as the affects on
global methylation in comparison with a cell model. In
addition, we analysed the expression of telomerase because
its expression is associated with ageing in several tissues(29).
The findings demonstrate that diet may impact on epigenetic
regulation of MnSOD, the consequences of which should be
(24). Reactive oxygen species are known to contribute to
Groups and sample collection
Buccal swabs from three subject groups (sex matched)
were collected and stored at 2208C in PBS. The subject
groups consisted of: (1) ovo-lacto vegetarians including
those who consumed fish (aged 20–30 years); (2) young
omnivores (aged 20–30 years); (3) elderly omnivores
(aged . 85 years). The reliability and reproducibility of
buccal mucosa for the investigation of gene expression and
methylation has previously been established(2,30). The volun-
teers were selected after the evaluation of a questionnaire
characterising nutritional habits, food preferences and food
intake, daily liquid intake, alcohol intake, vitamin supplemen-
tation, smoking, drug intake, and health status. Only subjects
who had consumed an ovo-lacto vegetarian diet for at least
5 years were admitted to the study. Volunteers who reported
vitamin supplementation, a high alcohol intake, or smoking
were excluded from the study. Written formal consent was
obtained from all subjects. The study protocol is covered by
an agreement of the ethical committee of the city of Vienna.
Cell line and treatment with methyltransferase inhibitor
The human endodermal carcinoma cell line Caco-2 was grown
in Roswell Park Memorial Institute (RPMI) 1640 media sup-
plemented with 4 mM-glutamine, 10% heat-inactivated fetal
bovine serum, and 100 units each of streptomycin and penicil-
lin. For inhibition of DNMT1, cells were treated with zebular-
ine at a concentration of 250mmol/l for 48h. Cells were
washed twice with cooled PBS (48C) before nucleotide
Genomic DNA isolation and bisulfite modification
Hilden, Germany) and bisulfite conversion of unmethylated
cytosines was performed using the Epitect Bisulfite Kit
(Qiagen). Both kits were used according to the manufacturer’s
Polymerase chain reaction of bisulfite-treated DNA
Two sequential PCR were used to amplify the modified DNA
fragments of interest. The primers for the first PCR were:
sense primer 50-GTA TTT TTA GGG G[C/T]G GAT
[C/T]GG AGG TAG GGT TT-30and antisense primer
50-CCA AAC CC[A/G] ATA C[A/G]A CCA CTA TC[A/G]
CCA TTA C-30. The primers for the second PCR were:
sense primer 50-GGG T[C/T]G TAT TAA TTT TA[C/T]
GGG GGT AGG GGT-30and antisense primer 50-AAC CCC
TTA CCC CTT AAA AC[A/G] TAA CC[A/G] AAT
CCC-30(reference sequence: GenBank L34157). Conditions
for the first PCR cycling were: 958C for 3min followed by
thirty-five cycles at 948C for 30s, annealing at 558C for
1min and extension at 728C for 2min; and finally 7min at
728C. The second PCR was performed under the same con-
ditions as the first run with the exception of the annealing tem-
perature which was modified to 578C. The PCR reaction of
25ml contained 12·5ml of 2 £ PCR master mix (Biotools,
Madrid, Spain), 1ml of each primer at a concentration of
30pmol/ml and 2ml template. The amplified DNA fragments
of intended size were purified by agarose-gel extraction with
the QIAquick Gel Extraction Kit (Qiagen) and subsequently
directly sequenced by an ABI sequencing system (Applied
Biosystems, Foster City, CA, USA). Gene expression-related
AP-2 and SP-1 binding sites(28,31,32)were selected for detailed
methylation quantification. No differences were seen between
sequencing of cloned samples compared with directly
Analysis of mitochondrial superoxide dismutase gene
Total mRNA was extracted from cells using the mRNA Iso-
lation Kit (Roche, Mannheim, Germany) and reverse tran-
scribed using the single-strand cDNA Synthesis Kit (Roche,
Mannheim, Germany). Gene expression was performed by
real-time TaqMan PCR using the Corbett Rotor-Gene 3000.
The target mRNA expression was normalised to the glucose-
6-phosphate dehydrogenase (G6PD) expression. The follow-
ing thermocycling conditions were selected for all genes:
958C for 3min and forty-five cycles of 958C for 30s and
678C for 45s. Real-time PCR were set up in 10ml final
volumes. Optimum reaction conditions were obtained for the
MnSOD gene with 5ml of 2 £ PCR master mix (Quantimix
Easy Probes Kit; Biotools, Madrid, Spain), 1ml (8pmol/ml)
of each primer and 1ml (2pmol/ml) probe and for the G6PD
R. Thaler et al.744
British Journal of Nutrition
gene with 5ml of 2 £ PCR master mix, 1ml (10pmol/ml) of
each primer and 1ml (2·4pmol/ml) probe. Finally, 2ml tem-
plate cDNA was added to the reaction mixture. The primers
and probes were as follows: MnSOD primer sense 50-AAG
GGA GAT GTT ACA GCC CAG ATA-30, MnSOD primer
antisense 50-TCC AGA AAA TGC TAT GAT TGA TAT
GAC-30, MnSOD probe 50-CCA CCA TTG AAC TTC AGT
GCA GGC TG-30, G6PD primer sense 50-ATC GAC CAC
TAC CTG GGC AA-30, G6PD primer antisense 50-TTC
TGC ATC ACG TCC CGG A-30and G6PD probe 50-AAG
ATC CTG TTG GCA AAT CTC AGC ACC A-30. All
probes were marked with flavin–adenine mononucleotide as
the fluorophor and black hole quencher (BHQ) 1 as the
DNA total methylation analysis
DNA global methylation was analysed by the Methylampe
Global DNA Methylation Quantification Kit (Epigentek,
New York, USA) according to the manufacturer’s instructions.
DNA methylation status was compared with an artificially
fully methylated DNA standard.
Relative telomerase activity
The relative telomerase activity (TA) was determined by the
real-time quantitative telomeric repeat amplification pro-
tocol (RTQ-TRAP) assay(33). For Sybr Green real-time PCR
the following primers at a concentration of 10 pmol/ml
were used: sense primer 50-AAT CCG TCG AAG AGT T-30
and antisense primer 50-GCG CGG CTT ACT AAC C-30.
The reactions were set up with 5ml Sybr Green Master Mix
(Biotools, Madrid, Spain), 1ml of each primer and 1–3ml of
sample, depending on the calculated protein concentration.
Cycling conditions were: 3min at 958C followed by fifty
cycles of 20s at 958C, 30s at 508C and 90s at 728C.
Role of diet on methylation and expression of mitochondrial
When gene expression and MnSOD promoter methylation
status of human buccal swabs of subjects consuming two
different diet forms and zebularine-treated or -untreated
Caco-2 endodermal cells were analysed, the results suggested
that MnSOD expression is epigenetically regulated by diet or
factors associated with diet choice.
CpG methylation.Fig. 1 (b) illustrates the reduced meth-
ylation patterns in the MnSOD promoter region in cells of
buccal swabs sampled from the vegetarian group. Analysis
of bisulfite-treated DNA using genomic sequencing of the pro-
moter region by the ABI Prism system indicated an approxi-
mately 30–40% methylation of the relevant CpG (Fig. 1 (a)).
No significant difference in methylation of the MnSOD region
was observed when buccal swabs of groups of elderly (.85
years) and young (20–30 years) subjects consuming the
same diet were compared.
11 12 13 14 15 16 17 18 19 20
Relative CpG methylation
Fig. 1. (a) Mitochondrial superoxide dismutase promoter region showing CpG, activating protein (AP)-2 and stimulatory protein (SP)-1 binding sites.
(b) Percentage of methylation of twenty CpG (^10%; shown in bold and numbered in (a)) in vegetarian subjects ( ), and young ( ) and elderly ( ) omnivores.
Values are means, with standard deviations represented by vertical bars. Relative CpG methylation refers to the proportion of cytosine–thymine signals at these
sites in the electropherograms.
Regulation of superoxide dismutase by diet 745
British Journal of Nutrition
be significantly higher in the group of forty vegetarians
compared with the group of forty age-matched omnivores.
Compared with the expression of G6PD, vegetarians showed
a 3·2 (SD 0·7)-fold (P,0·05) increase in MnSOD mRNA
expression. No significant difference in MnSOD gene exp-
ression was observed between the group of forty elderly
subjects (.85 years) and the group of forty subjects aged
20–30 years, both of whom adhered to a conventional
middle European diet.
Expression of MnSOD was found to
Effect of age on DNA global methylation
DNA global methylation.
1·4% was detected in the buccal mucosa samples of young
omnivores. No significant difference in DNA global methyl-
ation levels could be seen in the vegetarian subject group.
However, reduced genome-wide methylation (by about 4%;
P,0·05) was found in the elderly subject group (Fig. 2 (c)).
Telomerase activity. The relative TA was determined by
the real-time quantitative telomeric repeat amplification proto-
col (RTQ-TRAP) assay. Significant (P,0·05) differences
were observed between young and elderly subjects, indepen-
dent of diet. Vegetarians showed a TA of 0·79 (SD 0·97),
young omnivores showed a TA of 0·80 (SD 0·02) and elderly
omnivores showed a TA of 0·15 (SD 0·01).
DNA global methylation of about
Effect of zebularine on epigenetic regulation of the
mitochondrial superoxide dismutase gene
Methyltransferase inhibition results in gene-specific and
global hypomethylation in Caco-2 cells.
ment caused demethylation of CpG in the analysed MnSOD
promoter region of Caco-2 cells. Analysis of bisulfite-treated
DNA using genomic sequencing of the promoter region
by ABI sequencing indicated an approximately 60–70%
methylation of the relevant CpG which was reduced to
about 30–40% methylation due to zebularine treatment
(data not shown).
When the DNA global methylation was measured by
ELISA, the level in zebularine-treated Caco-2 cells was
decreased by approximately 30% compared with an artifi-
cially fully methylated DNA standard.
Methyltransferase inhibition induces gene expression in
Caco-2 cells.Gene expression (Fig. 2 (a)) and methylation
status of the MnSOD promoter region was analysed in
Caco-2 cells after incubation with the methyltransferase
inhibitor zebularine for 48h. Several concentrations and incu-
bation times were tested (data not shown). A concentration of
zebularine at 250mmol/l for 48h showed the greatest DNMT1
inhibition. The expression of MnSOD in zebularine-treated
cells increased 6·3 (SD 0·3)-fold (P,0·05), referenced to
G6PD mRNA expression levels. The results obtained for
MnSOD promoter methylation and gene expression in the
Caco-2 cell-line experiments agree with previously reported
observations from a multiple myeloma cell line(28). Analysis
of methylation of CpG focused on those CpG that are situated
in the AP-2 and SP-1 transcription factor-binding sites(28,31,32).
The influence of environmental factors and the role of nutri-
tion on the epigenetic regulation of gene expression are
important topics in the understanding of gene expression and
Relative DNA global methylation
Relative MnSOD expression
Relative MnSOD expression
Fig. 2. (a) Increase of mitochondrial superoxide dismutase (MnSOD) gene
expression after a 48h zebularine treatment in the endodermal Caco-2
cell line. *MnSOD expression increased in zebularine-treated cells by 6·3
(SD 0·3)-fold compared with zebularine-untreated cells (P,0·05). (b) Relative
MnSOD gene expression in elderly (1·81 (SD 0·7)) and young (2·02 (SD 0·8))
omnivores and in vegetarians (6·46 (SD 0·7)). MnSOD gene expression is
referenced to glucose-6-phosphate dehydrogenase expression levels. *Veg-
etarians showed a 3·2-fold (P,0·05) increase in MnSOD expression com-
pared with young subjects and a 3·6-fold (P,0·05) increase compared with
elderly omnivores. (c) DNA global methylation in the omnivorous and veg-
etarian (1·44 (SD 0·64)) diet groups, groups of elderly (.85 years; 1·02 (SD
0·05)) and young omnivores (20–30 years; 1·45 (SD 0·53)), in percentage of
totally methylated control DNA. *DNA global methylation was significantly
higher (30%) in the younger subjects (vegetarian and omnivorous) than in
the elderly omnivores (P,0·05). Values are means, with standard deviations
represented by vertical bars.
R. Thaler et al. 746
British Journal of Nutrition
prevention of diseases. Based on results of in vitro studies, we
investigated the methylation-dependent epigenetic regulation
of the MnSOD gene as well the DNA total methylation in
mucosal swabs of subjects consuming different diets and of
different ages. Results of the present study demonstrated a sig-
nificantly higher expression of MnSOD mRNA in young vege-
tarian subjects (20–30 years) compared with omnivores of the
same age. Higher MnSOD expression correlated with a CpG
demethylation in the promoter region of the gene in the vege-
tarian group. Furthermore, our findings show significant
differences in the DNA global methylation and TA between
young and elderly subjects.
Previous studies analysing the B-vitamin status of compa-
rable groups of vegans, vegetarians and omnivores, conducted
by our department in Austria, showed a higher dietary supply
of folate and a lower dietary supply of cobalamin for the vege-
tarian and vegan group. A correlation between plasma folate
and vitamin B12values was seen and study findings showed
that the vegetarian and vegan group had the highest plasma
homocysteine concentrations(20). DNA hypomethylation due
to high homocysteine levels has been reported in vitro and
in vivo(34,35). Several other studies support these observations
These characteristics of the vegetarian diet may explain the
decreased activity of the DNA-methylation pathway resulting
in a decreased methylation of the MnSOD gene.
Geisel et al.(19)also compared vegetarians and omnivores
and observed an inverse correlation between SAH concen-
trations and DNA global methylation levels in blood. But,
Geisel et al. were unable to correlate the degree of CpG meth-
ylation of the promoter of the p66Shc gene (involved in oxi-
dative stress) and homocysteine, SAM or SAH levels. These
different methylation results seen for the MnSOD and
p66Shc promoter region might be explained by the activity
of different DNMT for both genes. Also, no correlation
between homocysteine concentrations and the degree of
DNA global methylation was found by Geisel et al.(19).
They suggested that the generation of SAM may be critical
in vegetarians because formation of the precursor methionine
is decreased by the disturbed remethylation of homocysteine
due to the lower vitamin B12supply.
However, vegetarians display a tendency for higher
intakes and serum concentrations of the methyl donor
folate(20), and human, animal and in vitro studies suggest
that folate-dependent DNA methylation is highly complex,
gene and site specific(37–40). In addition, the vegetarian diet
is characterised by a higher intake in secondary dietary com-
pounds such as diallyl sulfide(9), an organosulfur compound
found in garlic, genistein, the main flavonoid in soya(4), and
vitamin D3 or all-trans-retinoic-acid(41)which have been
shown to influence DNA methylation by altering histones
and chromatin structure. Analysis of the impact of nutrition
on gene regulation therefore needs to consider complex and
A higher MnSOD gene expression in vegetarians was
seen in the present study. Thus, a better defence against
superoxide radicals might be expected as a consequence of
a vegetarian diet. Because of this activity and its role
in the regulation of apoptosis(42), MnSOD was suggested as
a tumour-suppressor gene(26). Epidemiological data suggest
that vegetarians show lower rates of several types of cancer
and chronic cardiovascular diseases compared with omni-
vores(43–45). So far, these findings were explained by a
higher intake of antioxidant vitamins, Cu and secondary diet-
ary compounds(46,47). With the results of the present study, we
suggest that the higher protection against chronic diseases
in vegetarians may be explained by both epigenetic and
Findings of the present study showed a significantly lower
DNA global methylation status as well as TA in elderly
omnivores. No differences in DNA global methylation were
seen between vegetarian subjects and age-matched omnivores.
Several alterations, such as global hypomethylation, CpG
island hypermethylation or telomere shortening develop pro-
gressively as a result of ageing(23). Methylation of CpG islands
in non-malignant tissues increases but the total number of
methylated cytosine residues decreases with age(48,49).
Age-dependent chromosomal instability and DNA double-
strand breaks are mechanisms that have been demonstrated
in laboratory rats fed hypomethylated diets throughout their
lifespan as well as in postmenopausal women with methyl
donor-deficient diets(50). This chromosomal instability pro-
motes the progressive methylation of individual genes
during ageing(21). Highly differentiated methylation of the
same gene has been observed in various tumour tissues(11).
The present study has demonstrated that nutritional stimuli
contribute to epigenetic regulation of MnSOD in buccal
mucosa. These results could provide a foundation for targeted
dietary approaches(50)designed to alleviate or minimise the
consequences of environmental and toxic influences on
DNA methylation or other epigenetic mechanisms. In order
to better understand the implications of diet, the influence of
nutrients on DNA methylation enzymes and histone modifi-
cation needs to be further analysed.
The present study was supported by the Federal Ministry of
Health, Family and Youth. A. G. H. and R. T. designed the
study. R. T. also performed the data analysis. H. K. and
P. R. contributed to the drafting of the paper. The authors
declare that they have no conflicts of interest.
We acknowledge critical reading by Dr Franz Varga, Eliza-
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