Mechanisms of primary cancer prevention by butyrate and other products formed
during gut flora-mediated fermentation of dietary fibre
Daniel Scharlau*, Anke Borowicki, Nina Habermann, Thomas Hofmann, Stefanie Klenow,
Claudia Miene, Umang Munjal, Katrin Stein, Michael Glei
Institute for Nutrition, Friedrich Schiller University Jena, Dornburger Strasse 24, 07743 Jena, Germany
1.Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.The association between gut health, colorectal cancer and diet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.Induction of GSTs by SCFA as a possible mechanism of chemoprevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experimental approaches to study effects of butyrate in human colon cells in vitro. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Human colon cells in different neoplastic stages were used for the studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Differential effects of butyrate on different cell types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.Analysis of GST expression and enzyme activity in different colon cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mutation Research 682 (2009) 39–53
A R T I C L EI N F O
Received 5 November 2008
Received in revised form 8 April 2009
Accepted 14 April 2009
Available online 19 April 2009
In respectful memory of
Prof. Dr. Beatrice L. Pool-Zobel.
Drug metabolising enzymes
A B S T R A C T
Dietary fibres are indigestible food ingredients that reach the colon and are then fermented by colonic
bacteria, resulting mainly in the formation of short-chain fatty acids (SCFA) such as acetate, propionate,
and butyrate. Those SCFA, especially butyrate, are recognised for their potential to act on secondary
chemoprevention by slowing growth and activating apoptosis in colon cancer cells. Additionally, SCFA
can also act on primary prevention byactivation ofdifferent drug metabolising enzymes.This can reduce
the burden of carcinogens and, therefore, decrease the number of mutations, reducing cancer risk.
Activation of GSTs by butyrate has been studied on mRNA, protein, and enzyme activity level by real-
time RT-PCR, cDNA microarrays, Western blotting, or photometrical approaches, respectively. Butyrate
had differential effects incoloncells of differentstages of cancer development. In HT29 tumour cells, e.g.,
mRNA GSTA4, GSTP1, GSTM2, and GSTT2 were induced. In LT97 adenoma cells, GSTM3, GSTT2, and
MGST3 were induced, whereas GSTA2, GSTT2, and catalase (CAT) were elevated in primary colon cells.
Colon cells of different stages of carcinogenesis differed in post-transcriptional regulatory mechanisms
because butyrate increased protein levels of different GST isoforms and total GST enzyme activity in
HT29 cells, whereas in LT97 cells, GST protein levels and activity were slightly reduced.Because butyrate
increased histone acetylation and phosphorylation of ERK in HT29 cells, inhibition of histone
deacetylases and the influence on MAPK signalling are possible mechanisms of GST activation by
butyrate. Functional consequences of this activation include a reduction of DNA damage caused by
carcinogens like hydrogen peroxide or 4-hydroxynonenal (HNE) in butyrate-treated colon cells.
Treatment of colon cells with the supernatant from an in vitro fermentation of inulin increased GST
activity and decreased HNE-induced DNA damage in HT29 cells. Additional animal and human studies
are needed to define the exact role of dietary fibre and butyrate in inducing GST activity and reducing the
risk of colon cancer.
? 2009 Elsevier B.V. All rights reserved.
Abbreviations: ACF, aberrant crypt foci; AeAx, alkali extractable arabinoxylans; ARE/ERE, antioxidant/electrophile-responsive element; BSO, L-buthionine-(S,R)-sulfoximine;
CAT, catalase; CDNB, 1-chloro-2,4-dinitrobenzene; COX-2, cyclooxygenase-2; ERK, extracellular signal-regulated protein kinase; GST, glutathione-S-transferase; HDAC,
histone deacetylases; HDACi, histone deacetylase inhibitors; HNE, 4-hydroxynonenal; Keap1, kelch-like ECH associated protein 1; MAPK, mitogen-activated protein kinase
AP-1, activator protein-1; Nrf2, nuclear factor E2-related factor 2; SCFA, short-chain fatty acids; SOD2, superoxid-dismutase 2; UDP, uridine 50-diphosphate; WeAx, water
* Corresponding author at: Friedrich Schiller University Jena, Department of Nutritional Toxicology, Dornburger Str. 24, 07743 Jena, Germany. Tel.: +49 3641 949674;
fax: +49 3641 949672.
E-mail address: email@example.com (D. Scharlau).
Contents lists available at ScienceDirect
Mutation Research/Reviews in Mutation Research
journal homepage: www.elsevier.com/locate/reviewsmr
Community address: www.elsevier.com/locate/mutres
1383-5742/$ – see front matter ? 2009 Elsevier B.V. All rights reserved.
3.Activation of GSTs by butyrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.GST-gene activation by butyrate may be mediated by different mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.Butyrate treatment enhanced mRNA gene expression, protein expression and enzyme activity of GSTs and other stress response genes
The modulation of gene and protein expression by butyrate may protect cells from genotoxic carcinogens. . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.DNA damage caused by H2O2and HNE was reduced by butyrate pre-treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.Is it a double edged sword to enhance detoxification in both normal cells and in tumour cells? . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of complete gut fermentation samples better reflect in vivo exposure conditions than butyrate alone . . . . . . . . . . . . . . . . . . . . . . . .
5.1.Experimental approaches to obtain samples simulating gut lumen contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.Some key results on complex fermentation samples and their effects on GSTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Summary and conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. The association between gut health, colorectal cancer and diet
of the World Cancer Research Fund on ‘‘Food, Nutrition and the
Prevention of Cancer: A Global Perspective’’ . With this report a
new dimension of the field came into view because the
accumulated evidence was showing that cancer and diet were
strongly interrelated. Moreover, on account of the possibility to
adjust diet, it was deemed also possible in the long run to prevent
diet-related cancers. Because the amount of available literature on
this topic has significantly increased within 10 years, just recently
the second edition of this expert report was published . While
many of the described aspects have been refined in the update due
to the large amount of new literature, the general picture that
adjustment of diet could prevent some cancers is maintained. Of
of which a significant proportion is diet or exposure related.
Avoidable risk factors are considered to be diets rich in meat,
especially in conjunction with genetic predisposing genotypes [3–
6]. Opposed to this, healthy behaviours such as the consumption of
high quantities of vegetables, fruits, or dietary fibres, coupled to
physical exercise have been shown to be associated with reduced
risk [7,8]. The epidemiological evidence in humans is supported by
animal studies which have shown e.g. that dietary haem increased
faecal cation content, cytolytic activity of faecal water and colonic
epithelial proliferation in rats , a property that can be inhibited
by dietary calcium . Furthermore, Pierre et al. reported that
diets with meat promoted the formation of aberrant crypt foci
(ACF) in azoxymethane treated rats on low calcium diets and that
this was associated with high concentrations of lipoperoxides in
the faeces and faecal water cytotoxicity . In F344 female rats
fed with N-nitroso-N-methylurea it was shown that the simulta-
neous feeding of a fat diet and haem-iron caused a significant
increase in the incidence of colon cancer compared to a diet
without haemoglobin . On the other hand, Xu and Dashwood
summarised results from ACF and tumour bioassays which
demonstrated that constituents of tea, green vegetables, crucifer-
ous vegetables and dairy products all have a potential to protect
carcinogens [13,14]. Additionally,the results of Vogelet al. showed
that greenvegetablesinthe dietof ratssignificantlyreducetherisk
of meat-inducedcolorectal cancer . Reviews on the potential of
are available by Bruce and Corpet, both pioneers in the
development of appropriate experimental models [16,17].
Increasingly, however, doubt is cast on the strength of reported
inverse relationships, since recent human studies are not showing,
for instance, that fruit and vegetable intakes, the major sources of
dietary fibre, are associated with a reduced cancer incidence in
general and in particular with reduced colorectal cancer risk
amines which are colon
[18,19]. Moreover, intervention with a diet low in fat and high in
polyp recurrence . Based on the knowledge of mechanisms of
actionof fibre fromexperimental and animal studies, it istherefore
becoming apparent that more detailed studies are needed on how
different types of foods and dietary fibres contribute to gut health
and how they may act on a molecular basis. In particular, it is of
interest to better understand overall metabolism, the roles of the
products formed during the process of gut fermentation and how
these products interact with each other in a chemopreventive
modeof actionwith thecolon mucosa.Consequently,it islogical to
postulate that beneficial effects are possibly not only mediated by
‘‘vegetables and fruits’’ or ‘‘dietary fibres from plant foods’’ in
general, but also by specific types of dietary fibres that are
beneficially fermented by the gut flora to yield products that
contribute to chemoprotection. In other words, it is now
recognised that the traditional chemoprotective role of dietary
fibre, which formerly consisted of faecal bulking , rapid transit
 and enhanced defaecation, may have added benefits. These
purported health promoting properties could include the so called
prebiotic activity , putatively encompassing cell protective
effects of particular antioxidants that can be liberated in the colon
after fermentation by the gut flora [24,25]. A prebiotic was first
defined as ‘‘a nondigestible food ingredient that beneficially affects
the host by selectively stimulating the growth and/or activity of
one ora limitednumberof bacteria inthe colon,and thusimproves
host health’’ . After the first definition of prebiotics, a lot of
research on these carbohydrates has been done leading to clear
criteria that characterise prebiotics, namely (1) resistance to
gastric acidity, to hydrolysis by mammalian enzymes, and to
gastrointestinal absorption; (2) fermentation by intestinal micro-
flora; and (3) selective stimulation of the growth and/or activity of
those intestinal bacteria that contribute to health and well-being
. Hence, prebiotics can have an impact on gut health in general,
and are believed to play an important role in the prevention of
colorectal cancer, as has been highlighted by a recent review .
In this context,results of a recent study by Schatzkinet al. are of
interest . The authors aimed to investigate the relation
between dietary fibre and whole-grain food intakes and the
incidence of invasive colorectal cancer in the prospective National
Institutes of Health—AARP Diet and Health Study. Key results were
that total dietary fibre intake was not associated with colorectal
cancer, whereas whole-grain intake was inversely associated with
colorectal cancer risk. This indicates a particular role of whole
grains as source of dietary fibres (and of other gut health
promoting ingredients) for which it will be of interest to better
understand molecular mechanisms of activities . Examples of
the outstanding prebiotic types of fibres in this context are inulin,
oligofructoses and related fructans—in short, inulin-type fructans.
It is however important to note that it depends on the definition
whether these inulin-type fructans can be classified as dietary
fibres. Originally dietary fibres were defined as the remnants of the
D. Scharlau et al./Mutation Research 682 (2009) 39–53
plant cell wall that are not hydrolysed by the alimentary enzymes
of man, which included mainly non-starch polysaccharides and
does not include inulin. Since this definition is still accepted in
some parts of the world, inulin is not classified as dietary fibre
worldwide. Anyway, since newer definitions in many parts of the
world are based on physiological properties and thus include more
or less all carbohydrates (typically with a degree of polymerisation
?3) that are resistant to digestion, inulin-type fructans are
classified as dietary fibres in many countries. Inulin-type fructans
(b(2-1)fructans) are extracted from chicory roots (Cichorium
intybus) and prepared to be added to various types of food. They
are also present in a number of foods, such as garlic, onion,
artichoke and asparagus as natural ingredients. Their average
consumption in the normal human diet has been evaluated to
amount to several grams per day . They are defined to be
prebiotic food ingredients since they selectively increase growth of
bifidobacteria, which have anticancer potential , or enhance
formation of short-chain fatty acids (SCFA), such as acetate,
butyrate, and propionate. Of these, butyrate, and propionate also
have beneficial properties [32,33]. In non-transformed cells,
butyrate is utilised as an energy source  whereas in tumour
cells butyrate reduces survival by inducing apoptosis and
inhibiting proliferation . Thus butyrate most likely acts on
secondary chemoprevention by reducing the number of cells in
cancerous lesions and thereby slowing or inhibiting formation of
Cancer chemoprevention is characterised by the use of natural,
synthetic, or biologic (from a living source) substances to reverse,
suppress, or prevent the development of cancer . The under-
lying principles of chemoprevention are summarised in Fig. 1.
Basically three different phases of prevention can be distinguished,
namely primary prevention, secondary prevention and therapy.
Primary prevention describes the inhibition of initiation, the first
step of tumourigenesis by reduction of toxification or induction of
detoxification. This can be accomplished e.g. by preventing the
formation of ultimate carcinogens or reactive oxygen species as
well as by antioxidative effects and is also called blocking activity.
The promotion of initiated cells to preneoplastic cells is inhibited
by secondary prevention, e.g. by reduction of cell growth or
enhancement of differentiation and apoptosis in initiated cells.
Agents that affect secondary prevention are suppressing agents.
Blockage of progression of preneoplastic cells to neoplastic cells,
which occurs comparably late during carcinogenesis, is termed
tertiary chemoprevention and includes therapeutic approaches.
The role of butyrate in secondary chemoprevention is subject of
numerous studies and has been extensively reviewed [37,38].
Another mechanism of chemoprotection by fermentation pro-
ducts, especially by butyrate, has been hypothesised to be the
induction of glutathione-S-transferases (GSTs) and other stress
response genes . This action of complex fermentation products
and butyrate on primary chemoprevention is the subject of the
1.2. Induction of GSTs by SCFA as a possible mechanism of
GSTs [EC Nr 18.104.22.168] are enzymes of biotransformation that
detoxify many carcinogens . The increased cellular levels of
such enzyme systems has been shown to protect against food-
derived genotoxic compounds such as 4-hydroxynonenal (HNE) in
tumour-derived cell lines . Similar mechanisms occurring in
non-transformed cells may very well reduce cancer initiation and
thus be considered an effective means of primary cancer
chemoprevention , since GSTs are capable of detoxifying
endogenous and exogenous (food- or smoking-derived) carcino-
gens like HNE or benzo(a)pyrene .
There have already been a number of reviews on SCFA and their
role in gut health. Cummings has reported of SCFA occurrence in
the colonic lumen and in hepatic tissues decades ago [32,44].
Csordas wrote a comprehensive compilation on the toxicology of
these compounds , while other examples of reviews on SCFA
and gut health were by Mortensen and Claussen , Topping and
Clifton , as well as by Augenlicht et al. . Some of the
findings have led to a number of critical editorials highlighting
controversies that pertain to opposing effects of butyrate in vitro
and in vivo or to findings on its different types of activities in non-
transformed versus transformed colon cells [49–51]. The main
purpose of the following review is to compile own laboratory
experiments on the effects of butyrate, and if available, on gut
fermentation products from dietary fibres (especially from inulin),
in different colonocyte cultures in vitro. Hereby the focus is on
recent findings of modulated gene or protein expression, in
particular of genes and proteins related to drug metabolism and to
stress response. Newer publications on effects of butyrate by other
Fig. 1. Different phases of chemoprevention. Primary prevention inhibits the initiation of cells by reducing toxification or inducing detoxification. Secondary prevention
describes the reduced promotion of initiated cells to preneoplastic cells by preventing proliferation and/or inducing apoptosis or differentiation. Therapy focusses on limiting
progression of cells from preneoplastic to neoplastic status (ROS, reactive oxygen species).
D. Scharlau et al./Mutation Research 682 (2009) 39–53
authors will also be reviewed in the context of these objectives.
Thus, this review will gather data on the butyrate-mediated
induction of GSTs and other drug metabolising enzymes. Excellent
reviews on GSTs have been published by Hayes [40,43,52] and
others [53–56]. More specific articles on the roles of GSTs in
nutrition and their expression levels in human cells or their
particular relation to factors of colon cancer are also available
[39,57–65], as are reviews on the anticancer properties of inulin-
type fructans [66,67], which is another focus of the present report.
2. Experimental approaches to study effects of butyrate in
human colon cells in vitro
It has been of interest to explore whether butyrate could
contribute to chemoprotection in colon cells not only by reducing
growth of tumour cells , committing them to more rapidly go
into apoptosis , serving as survival factor for normal non-
transformed colon cells [34,50] or enhancing mucin synthesis ,
but also via the mechanism of favourably altering patterns of drug
metabolism . For this, several of our studies have first dealt
human colon cells related to different stages of cell transformation
[71–73]. Then, the potential of butyrate to modulate expression of
genes related to stress response and to drug metabolism were
studied [70,73]. Finally, functional consequences arising from
potential to prevent genotoxicity caused by cancer-relevant risk
2.1. Human colon cells in different neoplastic stages were used for the
Primary colon cells were used as an example of non-
transformed healthy cells. They were isolated from patients who
had given their informed consent after being admitted to the
hospital for surgery of colorectal tumours, diverticulitis and colon
polyps [74,75]. The human colon epithelium was separated from
the tissue by a perfusion-supported mechanical disaggregation
and epithelial stripes were either conserved for RNA isolation or
they were further incubated in vitro and treated with butyrate.
The human colon adenoma cell line LT97 was used as an
example of an intermediary stage of the colorectal carcinogenesis
process, and represents adenomatous colorectal cells. The cell line
was a kind gift from Professor Brigitte Marian (Institute for Cancer
Research, University of Vienna, Austria) who established the cell
line from colon microadenomas of a patient with familial
adenomatous polyposis . The epithelial origin of the LT97 cell
line was confirmed,as was the stabilityof the karyotype . As an
example for highly transformed neoplastic colorectal cells, HT29
cells were used. They were isolated from a colon adenocarcinoma
of a female Caucasian and were obtained from the American Tissue
Culture Collection (ATCC; Rockville, MD, USA). Both LT97 cells and
HT29 cells were characterisedfor GST genotypes (Table 1). Primary
which some of the data has been published .
Genotyping of cellular DNA from LT97 and HT29 revealed the
presence of the GSTT1*1 gene, whereas no GSTM1 gene was
detectable in LT97 cells. Since the GSTT1 null genotype is found in
only 20% of Caucasians , presence of the wild-type allele in
both cell lines is not surprising.
Furthermore, both the *A wild-type allele and the three base
pair deletion containing *B allele of GSTM3 were detected in LT97,
the wild-type allele GSTP1*A as well as the heterozygous sequence
polymorphism at nucleotide +313 (Alw26I restriction site) present
in the GSTP1*1B and GSTP1*1C alleles were detected in both cell
lines. Whether polymorphisms in the GSTP gene increase the risk
for colorectal cancer, and therefore would be expected to occur in
coloncancercells asobserved,cannotbe assured,because onlyfew
studies were performed and the results are inconsistent .
Genetic polymorphisms were also determined for catalase
(CAT). However, a common polymorphism in the promoter region
of the CAT gene which consists of a C ! T substitution at position
?262 in the 50region , thought to result in reduced activity,
was not found in our cell lines HT29 and LT97 (unpublished
2.2. Differential effects of butyrate on different cell types
Effects of butyrate on the growth properties of HT29 and LT97
cells and on the survival of primary colon cells were assessed in
detail. Based on these studies, each of the cell types was incubated
and treated for a defined period of time with the maximum
butyrate concentration (<EC50) that had no significant effects on
the growth rates in LT97 and HT29 cells [71,72,82] or that were
well tolerated by primary colon cells . Therefore, the cell-
specific, sub-toxic and optimal conditions varied in terms of time
between plating and treatment, duration of treatment and
concentration of butyrate. Major results from butyrate uptake
studies were that LT97 adenoma cells retained 2-fold more
butyrate than HT29 cells treated with 2 mM butyrate resulting in
estimated intracellular concentrations of 0.8, 1.6 and 1.8 mM after
24, 48 and 72 h treatment, respectively. LT97 cells were also more
sensitive to growth inhibition than HT29 cells (EC50of 1.9 mM and
4.0 mM, respectively) . Primary colon cells retained 1.5 and
0.5 mM butyrate after 4 and 12 h treatment with 10 mM butyrate,
2 h treatment (0–50 mM), whereas cell yields decreased after 1 h.
Metabolic activity of remaining primary cells was either increased
(4 h, 50 mM) or retained at 97% (8 h, 50 mM).
The findings indicated that butyrate, at concentrations that are
well below the exposure levels in the gut lumen (estimated to
range from approximately 5–15 mM, depending on type and
availability of dietary fibre) had suppressing-activities in human
colon cell lines by inhibiting cell growth, with LT97 more sensitive
than HT29 cells. In primary and non-transformed cells, in contrast,
concentrations of butyrate higher than physiological concentra-
tions (up to 50 mM) were well tolerated. These have however only
low capacity to survive ex vivo, which makes it difficult to compare
their sensitivity to cell lines after long term exposure. The overall
conclusion was that colonocyte-exposure to butyrate in the gut
lumen of humans could be protective by reducing survival of
transformedcoloncells, whileat the same timepromotingsurvival
of non-transformed colonocytes.
2.3. Analysis of GST expression and enzyme activity in different colon
Expression levels of GSTs in different colon cells on the mRNA
level were measured by cDNA macroarrays and/or real-time RT-
PCR. Cellular protein concentrations of GSTA1, GSTM1, GSTM2,
Patterns of genetic polymorphisms of GSTs forLT97 adenoma cells in comparison to
gene patterns of HT29 cells. The polymorphisms for GSTM1 and GSTT1 were
determined simultaneously by multiplex PCR, and the sequence polymorphisms of
GSTM3 and GSTP1 were assessed by RFLP-PCR technique as described previously
D. Scharlau et al./Mutation Research 682 (2009) 39–53
GSTP1 and GSTT1 were quantified by Western blotting using
appropriate protein standards as described previously . Total
GST activity was determined spectrophotometrically at 340 nm
and 30 8C using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate
. Catalase activity was assayed spectrophotometrically at 25 8C
by following the extinction of H2O2at 240 nm .
A major finding for GSTs was that GSTP1 and GSTT2 were the
most expressed of all 12 GSTs spotted on the cDNA macroarray
in all three cell types. Freshly explanted colon tissue had
somewhat higher levels of both GSTP1 (173 ? 26 relative
expression signal) and GSTT2 (152 ? 25) than LT97 (124 ? 14
and 55 ? 5) or HT29 cells (114 ? 7 and 101 ? 9), and these levels
were at least 1.5-fold higher than the next most abundant GST
which was MGST3 (99 ? 43, 17 ? 9 and 76 ? 18 relative expression
signals in primary colon tissue, LT97 cells and HT29 cells,
respectively). In primary cells, however, it must be borne in mind
that the expression levels vary considerably between cells from
different donors (Fig. 2) .
On the protein level expression patterns of the major GSTs
were comparable. Fig. 3 compares baseline GSTT1 and GSTP1
GSTP1 was the major GST subunit in all colon cells (194–805 ng/
106cells, Fig. 3), and GSTT1 protein was the next most abundant
GST protein for which antibodies were available. It was detected
in all cells, unless the GSTT1*0 polymorphism was identified, as
was the case for some of the primary colon cells obtained from a
total of 15 different donors. GSTA2 was expressed least
(1.2 ? 0.3 ng/106HT29 cells and 17 ? 15 ng/106primary colon cells
according to ). Moreover, GSTA2 was detectable in only nine of
the 10 analysed samples of primary colon cells (Fig. 2). Also, in HT29
cells GSTM1 and GSTM2 proteins were not detectable, although
HT29 cells do not bear the null-genotype for GSTM1. In contrast,
primary colon cells express considerable amounts of GSTM1
(62 ? 31 ng/106cells) and GSTM2 (48 ? 28 ng/106cells) as was
reportedby Ebert et al..Proteinlevelsof GSTT2, thesecondmost
abundant GST in colon cells according to mRNA expression levels
(see above), have not yet been determined since appropriate
antibodies were not available.
In primary cells, the total protein contents varied from 250 to
930 ng GST protein/106cells, demonstrating a large range of inter-
individually different GST protein expression levels in human
colonocytes . This is in good agreement with the mRNA
expression levels described above . The expression of GSTP1
protein in LT97 cells was approximately only one-half of the level
detected for HT29, but twice the average level detected in non-
transformed primary colon cells. Thus, LT97 cells took an
intermediate place in this order, which corresponds to their
the tumour marker property of increased GSTP1 expression in
colon cancer . Opposed to the findings with GST protein
expression, GST activity seems to be equally high in LT97 and HT29
cells and together these activities are higher than in non-
transformed primary cells (Fig. 3). There was thus no direct
correlation between GSTP1 protein levels and GST activity in LT97
and HT29 cells .
At present it can only be speculated why the enzyme activity is
comparable in HT29 cells and in LT97 cells, whereas the GSTP1
protein level are much higher in the former. One possible
explanation may be the high levels of expression of additional
GSTs which are also potent conjugators of CDNB. GSTM2 could be
one of these GSTs that conjugate CDNB . However, as is shown
in Table 2, the protein is not detectably expressed per se in HT29
cells, but it is highly inducible by butyrate . In LT97 cells
(Table 2), GSTM2 was expressed at low levels (thus possibly
explaining higher baseline GST activity) but was not induced by
butyrate . Additionally it has also to be borne in mind that GST
genotype and phenotype do not always necessarily correlate in
tumourcells.For example,Barkeret al.  observedan absenceof
GST mu protein in a number of colorectal tumour tissues with a
genotype positive for GSTM1
Fig. 2. Expression levels of selected GST mRNA in human primary colon cells
detected by cDNA macroarray.
Fig. 3. Baseline values for GST activities (white bars), GSTP1 (grey bars) and GSTT1
(black bars) protein expression levels in human primary colon cells, LT97 cells and
HT29 cells (data are from [72,77,78]).
GST protein-expression (ng/mg protein) and activity (nmol/min/mg protein) in colon cells representing different stages of cell transformation (data are from [83,156] and
were compiled by  (‘‘n.d.’, not detectable). Baseline protein contents per 106cells are 126 ? 27 (HT29 cells, n = 17), 154 ? 35 (LT97 cells, n = 13) and 133 ? 74 (primary
human colon cells, n = 15), as reported previously [82,83].
9 ? 3
10 ? 6
92 ? 78
682 ? 396
13 ? 7
427 ? 280
5768 ? 1344
2775 ? 1313
1885 ? 1208
912 ? 307
1121 ? 294
541 ? 245
319 ? 68
332 ? 185
159 ? 76
D. Scharlau et al./Mutation Research 682 (2009) 39–53
3. Activation of GSTs by butyrate
3.1. GST-gene activation by butyrate may be mediated by different
A number of different promoter elements and transcription
factors are involved in regulation of GST gene activation. The best
studied mechanism of GST gene activation involves the antiox-
idant/electrophile-responsive element (ARE/ERE), a promoter
element which can be found in the 50upstream region of many
rodent GST genes, e.g. mouse GSTA1, rat GSTA2 and rat GSTP .
Different members of the helix-loop-helix basic leucine zipper
(bZIP) family of transcription factors, including Nrf, Jun, Fos, Maf,
can bind to the ARE sequence and induce transcription of ARE-
mediated gene expression of drug metabolism enzymes including
GSTs . The mechanisms of transcriptional activation of GSTs by
the transcription factor Nrf2 (nuclear factor E2-related factor 2)
have been analysed in numerous studies . Under homeostatic
conditions Nrf2 is bound to the Kelch-like ECH associated protein 1
(Keap1) and Nrf2-mediated transcription is repressed (Fig. 4).
When challenged with chemoprotective phytochemicals, e.g.
sulforaphane or phenylethylisothiocyanate, binding of Nrf2 to
Keap1 is disrupted and Nrf2 translocates to the nucleus thereby
activating expression of genes containing an ARE in their promoter
sequence . The dissociation of Nrf2 from Keap1 is induced by
covalent modification of thiol groups of Keap1 or by post-
transcriptional modifications of Nrf2. Post-transcriptional mod-
ifications include phosphorylation of Nrf2 by phosphatidylinositol
3-kinase, protein kinase C, and extracellular signal-regulated
protein kinase (ERK). These mechanisms have been mainly studied
using rodent in vivo and in vitro models or cell free in vitro models.
Up to now, evidence that comparable mechanisms are involved in
transcriptional regulation of GSTs or other enzymes of drug
metabolism, in humans is scarce. However, recently a few studies
have been published which demonstrate activation of e.g. haem
oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and GSTP1, by
Nrf2-mediated mechanisms in human cell culture models [91–93].
These studies represent first hints, but more research has to be
done to understand the regulation of drug metabolism enzyme
expression, especially GSTs.
Butyrate, in general, can modulate gene expression by acting as
an inhibitor of histone deacetylases (HDACs). HDACs are enzymes
that deacetylatehistones, butalso otheracetylatedsubstrates .
Deacetylation of histones leads to a tighter binding of DNA and
histones in the nucleosomes. Therefore, inhibition of HDACs by
butyrate and other histone deacetylase inhibitors (HDACi) results
in hyperacetylation and a more relaxed DNA. Regulatory elements
of the DNA are thus more accessible for transcription factors,
resulting in an increase of transcriptional activity. Consequently
HDACi like butyrate can induce changes in gene expression.
In this context we have recently shown that mixtures of SCFA,
composed according to physiologically available concentrations in
the gut lumen increased acetylation of histone H4 in HT29 colon
adenocarcinoma cells and that this effect was induced by butyrate,
but also by propionate. In addition, complex in vitro fermentation
samples of inulin reduced histone deacetylase activity thereby
increasing acetylation of histone H4 in colon cancer cells . An
increased acetylation of histone H4  and inhibition of histone
deacetylases in general  by SCFA has also been found by others.
The fermentation samples were more effective than synthetic
mixtures, containing equal amounts of SCFA (acetate, propionate
and butyrate), or butyrate alone, suggesting the presence of
additional factors in fermentation samples that can influence
histone acetylation. Since comparable fermentation samples of
inulin increased mRNA expression of GSTM2 and GSTM5 in
primary colon cells , the induction of histone hyperacetylation
by fermentation samples may be involved in up-regulation of GST
expression. Further studies are needed to unravel the possible
Fig. 4. Schematic diagram for drug metabolism enzyme regulation by different substances. A major mechanism for induction of drug metabolism gene expression is
transcriptional activation through promoters containing ARE motifs. ARE motifs bind Nrf2, a transcription factor of the basic leucine zipper (bZip) transcription factor family.
By interaction with the redox sensor Keap1 in its reduced state, Nrf2 is maintained in the cytoplasm and targeted for degradation in the proteasome. Modification of Keap1
cysteine(s) by xenobiotics leads to disruption of the complex, stabilization, and nuclear translocation of Nrf2. Nrf2 has activity only as heterodimer with other transcriptional
regulators, as indicated. Its activity can further be modified by different signal transduction pathways, which, for example, mediate Nrf2 phosphorylation. The illustrated
pathways are explained in more detail in the text. ARE/ERE, antioxidant/electrophile response element; C/EBPb, CCAAT/enhancer-binding protein beta; ERK, extracellular
signal-regulated kinase; Keap1, Kelch-like ech-associated protein1; NRF2, NF-E2-related factor-2; PI3K, phosphatidylinositol 3-kinase; NQO-1, NAD(P)H:quinone
oxidoreductase 1; QR, quinone reductase; HO-1, haeme oxygenase-1; GATA, gata-binding protein; Sp1, specific protein-1; GSTA2 and GSTA4, glutathione S-transferase
alpha2 and 4; GSTP1, glutathione S-transferase Pi 1
D. Scharlau et al./Mutation Research 682 (2009) 39–53
mechanisms of modulation of GST expression by the induction of
A number of transcription factors are possibly activated during
modulation of GST expression, because the promoter regions of
sites for several transcription factors, e.g. activator protein-1 (AP-
1), STAT, GATA1, NF-kB and C/EBPb [39,41,97]. Work from our
group demonstrated that butyrate increased phosphorylation of
ERK . Activation of ERK by phospho?rylation occurs during
mitogen-activated protein kinase (MAPK) signalling, a signal
transduction pathway important for the regulation of key cellular
functions including apoptosis, differentiation, proliferation and
many more. SinceERK activates AP-1transcription factors  and
since GST genes contain AP-1 binding sites in their promoter
sequences, MAPK signalling may be involved in activation of GST
gene expression by butyrate. Support for this hypothesis comes
by MAPK signalling [99,100] or activation of ERK by butyrate
[41,101,102]. However, direct evidence that this signal transduc-
tion pathway is involved in butyrate-induced activation of GST
expression is still missing.
Taken together, mechanisms of GST gene activation are well
understood in rodent models and subject of research in human cell
culture models. The latter will certainly increase knowledge on
these mechanisms in humans in the future. This knowledge will
help to design experiments which will further elucidate the
molecular details of GST gene activation by butyrate and other
products formed during gut flora-mediated fermentation of
3.2. Butyrate treatment enhanced mRNA gene expression, protein
expression and enzyme activity of GSTs and other stress response
Tomeasurebutyrate-modulated expressionof GSTscDNA array
analyses were used. It is apparent that certain isoenzymes of the
GSTs were induced with different patterns of responses in each of
the cell types. The main genes of interest were the up-regulated
and primary cells. The analysis also revealed a relative GSTP1
induction in primary cells that was not considered to be significant
on the basis of the ‘‘fold’’ data used to characterise the genes
Fig. 5 compares the baseline expression levels and induction
levels of the two most abundant GSTs (GSTP1, GSTT2) in the three
cell types. Butyrate markedly increased GSTP1 in HT29 cells,
whereas GSTT2 was increased in primary human colon cells and in
LT97 adenoma derived cells. Some of these data were verified on
mRNA level by real-time RT-PCR or Northern blot as shown in
Fig. 6. We next investigated the effects of butyrate on expression of
selected proteins and on GST activity in the three cell types treated
in analogous manner. One of the first studies on effects with
butyrate had dealt with expression of GSTP1 protein, a major GST
in HT29 . It was found that GSTP1 protein, GST activity, total
protein and glutathione levels were increased (1.2–2.5-fold) after
24–72 h of incubation with 4 mM butyrate. In a follow up study
, it was shown that butyrate significantly enhanced protein
expression of GSTA1/2 (3.5-fold), GSTM2 (manifold, since it was
not detectable in medium controls), GSTP1 (1.5-fold) and activity
Fig. 5. Comparison of baseline expression levels (medium control, white bars) and induced expression levels (butyrate treatment groups, black bars; primary cells: 10 mM,
LT97 cells: 2 mM, HT29 cells: 4 mM) of two genes (GSTP1 and GSTT2) in human colon cells in different stages of cell transformation according to superarray, setting GAPDH
equal to 100% (data from ).
Fig. 6. Comparison of induced expression levels shown as fold change of two genes (GSTP1/GSTT2) in human colon cells of different stages of transformation according to real-
time RT-PCR, setting the medium control equal to 1.
D. Scharlau et al./Mutation Research 682 (2009) 39–53
of GST (1.4-fold), but not of GSTM1 or GSTT1 in HT29 cells.
Therefore, butyrate, consistently enhanced GST activity in HT29
cells in the same order of magnitude . Opposed to this, in
LT97 cells treated with butyrate, there was a trend for a reduced
GST activity resulting from 2 mM butyrate exposure and this was
reflected by a reduced expression of GSTA1, GSTP1 and GSTM2
proteins . The latter two monomeric GST proteins are those
which form the dimeric GST enzymes (hGSTP1-1, hGSTM2-2)
with highest specific activity for CDNB, the substrate used to
measure enzyme activity in this study [104,105]. Protein
expression could not yet be determined conclusively in primary
cells following butyrate exposure due to technical limitations,
but the determination of GST activity has revealed that treatment
of colon cells with butyrate slightly reduced the enzyme activity
resulting in only 88 ? 34% of the activity in the medium control
which was set to equal 100% . Since GST activity only reflects
GSH conjugation of CDNB and since this reaction is only catalysed
(with different quality) by some of the GST isoenzymes, the
parameter GST activity is not, on its own, sufficiently reliable to
indicate a regulated GST system. Glutathione (GSH) also increases
upon butyrate incubation with time, as was shown in detail
previously . Thus, the concentration of enzymes, of GSH and the
specificity of the substrate (in this case CDNB) are mutually
influencing activity levels found in cells under given conditions. On
the basis of available data generated in our laboratory so far, Table 3
summarises the major data on GST expression at mRNA, protein and
activity level in the three cell types representing different stages of
colon cell transformation. Some of the data was reviewed in the
preceding paragraphs; other information is directly taken from the
references, as indicated.
The data obtained using different techniques and approaches
clearly show a consistent pattern of GST regulation by butyrate
in the three different cell models. GSTP1 mRNA, which is the
most abundant GST in colon cells, has been shown to be
inducible in HT29, LT97 and in primary colon cells. The level of
induction was higher in HT29 cells than in the other two cell
types. There also seems to be a marked difference in post-
transcriptional modifications, since protein expression (and
with this enzyme activity) was increased in HT29 but decreased
in LT97. Furthermore, GSTA4 could be an interesting target for
chemoprotection, since it has high potential for deactivating
HNE, a physiologically relevant genotoxic compound and
product associated with oxidative stress. GSTA4 is transcrip-
tionally induced in HT29 cells as well as in primary cells. Since
levels were relatively low, at least for HT29 cells, more studies
are needed to assess modulation and importance for non-
transformed primary cells or for LT97 cells.
Until now effects of butyrate on GST expression in colon cancer
cells was exclusively investigated by our group, but butyrate has
been shown to induce several isoforms of GST, including GSTP1, in
vascular smooth muscle cells, too . To verify the physiological
significance of these in vitro results, animal or human studies are
important. However, up to date in vivo studies on effects of
butyrate or SCFA on GST expression are scarce. Helsby et al. have
demonstrated that diets containing wheat bran increased the
expression of GSTA1 and A2 in the colon of rats . Additionally,
rats fed the probiotic bacterium Bifidobacterium longum had higher
colonic mucosal GST levels than control rats . Since this
bacterium is known to increase levels of butyrate and SCFA in the
colon, the observed up-regulation of GST may be caused by these
In primary human colon cells short term treatment with
butyrate has been shown to protect against damage caused by
H2O2[109,110], which could be related to activation of enzymes
not necessarily related to GSTs. To study this possibility in more
detail gene expression was determined with a pathway specific
cDNA array for genes related to stress response after treating
colon epithelium stripes with non-toxic doses of butyrate
(10 mM, 12 h). Changes of COX-2, SOD2 and CAT expression were
confirmed with real-time RT-PCR and by measuring catalase-
enzyme activity. Expression of CAT was enhanced, whereas COX-2
and SOD2 were lowered according to both array and real-time RT-
PCR analysis. Butyrate also decreased COX-2 expression in oral
squamous cell carcinoma in vitro and in vivo . An enhanced
catalase-enzyme activity was detected after 2 h of butyrate
COX-2 could reduce inflammatory processes, whereas more
catalase improves detoxification of the cellular oxidant H2O2
. Related to this are earlier findings in primary rat colon cells
showing that pre-treatment of these cells with butyrate, followed
by subsequent challenge with H2O2 significantly reduced the
genotoxic response . It may well be possible that sustained
changes of this type could reduce damaging effects by oxidants
and protect cells from initiation.
n.d., not determined; (") up-regulated; (#) down-regulated; ($) not regulated.
ParameterPrimary colon cellsLT97 cellsHT29 cellsReference
mRNA (Northern blot)
mRNA (real-time RT-PCR)GSTP1"
GST proteins (Western blot)[37,77]
Enzyme activity (against CDNB)
D. Scharlau et al./Mutation Research 682 (2009) 39–53
4. The modulation of gene and protein expression by butyrate
may protect cells from genotoxic carcinogens
4.1. DNA damage caused by H2O2and HNE was reduced by butyrate
The prevention or inhibition of genotoxic effects by DNA
damaging agents is collectively called ‘‘antigenotoxicity’’. To
explore this experimentally in cell culture, different mechanisms
and experimental protocols are feasible. We used a new treatment
protocol to depict different types of effects on genetic damage and
DNA repair . For this, the putative chemoprotective is added
to 3 phases of an experimental protocol originally developed for
molecular epidemiological studies . Phase a: cells are
incubated in culture flasks for different time periods, Phase b:
cells are treated with a genotoxic model compound to induce DNA
damage, Phase c: repair is assessed in the cells by measuring the
persistence of damage after the incubation. Test compounds may
be added to the cells to achieve relevant concentrations during the
individual periods a, b or c or they can be present throughout
periods a–c. This protocol has enabled us to discriminate between
different effects of the compounds: (a) incubation with the cells
before genotoxic treatment is used to detect induction of
protective factors, (b) incubation together with the genotoxic
agent detects direct interactions (scavenging reactions), and (c)
incubation with the cells following removal of the agent enables
detection of an influence on DNA repair.
One study has dealt with the comparison of genotoxic activities
of two genotoxic agents, namely H2O2and HNE, in three different
cell types. The comparisons were made using treatment protocol a
and determining DNA damage with the single cell microgelelec-
trophoresis assay (Comet assay) in primary human colon cells,
LT97 cells, and a differentiated clone of HT29 cells (HT29 clone
The genotoxic activities of the compounds were compared to
the key parameters of GST expression in the three different cell
types, GST activities, GSH levels and GSTP1 protein amounts, as
described in more detail above. The Comet assay experiments
showed that both HNE and H2O2were clearly genotoxic in the
different human colon cells. HNE was more genotoxic in LT97 than
in HT29clone19A and primary human colon cells. Since GST
expression was significantly lower in LT97 than in HT29clone19A
cells, this was considered to be an explanation for the higher
genotoxicity of HNE in the colon adenoma cells .
Connected to this was the interest to determine whether a
butyrate-mediated modulation of GST expression and of GST
enzyme activity would result in a reduced genotoxicity of model
compounds. The first theory was that the metabolic GSH
conjugation of HNE would decrease the genotoxicity of the alkenal
by forming a more stable conjugate. Accordingly, butyrate
treatment would result in antigenotoxic effects. This was first
shown by Ebert et al. , who were not only able to demonstrate
that butyrate (4 mM) induces ERK1/2 phosphorylation after 5–
30 min, but also that after 24–72 h incubation, GST mRNA, GSTP1
protein, GSTP1 activity and total protein were increased (1.2–2.5-
fold) and GSH levels were maintained as well. Moreover, a marked
reduction of HNE-induced genotoxicity was caused by pre-
incubation with butyrate. Most effects were more pronounced
in HT29 parent cells than in HT29 clone cells. In a follow up study
by Knoll et al. , it was shown that incubation of HT29 cells
with butyrate (2–4 mM) significantly elicited a 1.8–3-fold up-
regulation of steady state GSTA4 mRNA over 8–24 h after
treatment. Moreover, 4 mM butyrate tended to increase GSTA4-
4 protein concentrations. Opposed to this, incubation with
100 mM L-buthionine-(S,R)-sulfoximine (BSO) decreased cellular
GSH levels by 77% without significant changes in cell viability.
Associated with this reduction of cellular GSH amounts was a 2-
fold higher level of HNE-induced DNA damage as measured by the
Comet assay. Collectively,the results of this study and the previous
work described above indicate that the genotoxicity of HNE is
GSTs that conjugate HNE, including GSTA4 and GSTP1. This is
supported by a study from Yadav et al. who found that depletion of
GSH in human erythroleukemia cells significantly increased HNE-
induced DNA damage and overexpression of GSTA4-4 significantly
prevented HNE-induced DNA damage . Additionally, a
decrease in GSH content also increased HNE-induced DNA lesions
in human THP1 monocytes . Since HNE contributes to colon
carcinogenesis, the favourable modulation of the GSH/GST system
by butyrate may lead to chemoprevention and reduction of the
4.2. Is it a double edged sword to enhance detoxification in both
normal cells and in tumour cells?
Consequences of the typical expression patterns induced by
butyrate considering normal and tumour cells remain to be
elucidated. For one, there are substrates which are activated by
GSTs rather than deactivated. Thus, GSTT1-enzyme is a metabolic
activator for halogenated compounds, producing a variety of
intermediates potentially dangerous for DNA and cells. For
example, mice exposed to dichloromethane showed a dose-
dependent incidence of cancer via the GSTT1-1 pathway .
To investigate the role of GSTs experimentally, CDNB was chosen
as a model compound, since it is a substrate for GST in biochemical
determinations and has genotoxic potential [83,118]. In butyrate
pre-treated HT29 colon cells, there was an approximately 1.7- or
1.3-fold increase of genotoxicity by 37, and 50 mM CDNB,
respectively . The second point to consider is that in the case
of colon tumour cells, an increased activity of GSTs may not be
beneficial for the organism and can be one of the factors conveying
resistance towards cytotoxic drugs during cancer chemotherapy.
prevent chemoresistance caused by GSTP induction [119,120]. For
the case of cancer chemoprevention, however, this enhanced
chemoresistance in colon cells, which are either non-initiated or in
early stages of transformation could be a beneficial mechanism to
reduce the impact of exposure towards carcinogenic compounds
and to avoid initiation or progression of cancer. Therefore, an
induction of GSTs in primary cells seems straightforward and
favourable since this should result in an enhanced detoxificationof
risk factors. Connected to this is a reduced probability of cancer
initiationintheunderlyingstemcells(see for a reviewonthe
importance of colon stem cells in cancer initiation). In this context
it is important to mention differential effects of GST up-regulation,
because detoxification is only one of the functions of GSTs. For
example, GSTs can directly conjugate isothiocyanates without
prior activation resulting in the depletion of both GSH and
isothiocyanate levels . Since isothiocyanates can act chemo-
preventive by various mechanisms, this can be considered
unfavourable in terms of chemoprevention. Moreover GSTs can
lead to activation of compounds instead of detoxification. For
example, conjugation of GSH with the solvent dichloromethane
results in formation of the highly unstable S-chloromethylglu-
tathione, which can result in modification of DNA . An up-
regulation of GSTs is therefore not always beneficial, and the
effects on the cells have to be critically analysed in further
In tumour and in adenoma cells, GST induction could
theoretically enhance the survival of transformed cells .
Therefore our findings may mirror an adverse property, which
needs to be discussed in light of the in vivo situation. In one animal
D. Scharlau et al./Mutation Research 682 (2009) 39–53
study, e.g. protective effects were not found from treatment with
slow-release pellets of sodium butyrate (100 mg/day) in a rat
model of azoxymethane (AOM)-induced intestinal carcinogenesis,
but adverse effects were not detected either .
All together, based on a number of considerations, protection of
tumour cells may not be probable in vivo. For one, the luminal
concentrations of butyrate (in the millimolar range) are probably
much too high to result in survival of neoplastic cells that may
profit from the GST induction. As example, the feeding of inulin-
type dietary fibre to rats resulted in total SCFA yields of 70–75 mM
. When incubating the same type of inulin with gut flora in
vitro, SCFA yields were comparable in batch culture fermentation
systems (?90 mM) of which the relative butyrate concentrations
amounted to at least 10 mM . The concentrations are well
tolerated by primary, non-transformed colon cells  but are 5-
and 2.5-fold higher than maximal EC50values for HT29 or LT97
cells after 24 and 72 h exposure, respectively. Under these
exposure situations, which are expected not only to occur for a
moment but should be maintained over long durations (?72 h) in
people consuming sufficient dietary fibres, early stages of tumour
cells possibly undergo apoptosis and are inhibited from dividing.
Thus, the physiological gut luminal butyrate concentrations would
impair survival of neoplastic cells and thus decrease availability of
such cells for GST induction. Another reflection is that, not only
butyrate, but also propionate is produced during gut fermentation,
and this SCFA adds on to the growth inhibitory properties of gut
luminal products . Beyond that, in vivo, colon tissue is probably
more protected from the gut luminal components than cells in
culture by a number of barrier functions of the mucosa .
Finally, animal studies seem to support the contention that dietary
a higher efficacy of protecting from colon tumours in animals than
fibres resulting in lower amounts of butyrate. In particular, an in
vivo study by Perrin et al. demonstrated that those fibres, which
promoteda stable butyrate-producing
decreased the rate of AOM-initiated ACF in rats, thus adding on
to the line of evidence that a stable butyrate-producing colonic
ecosystem related to dietary plant foods reduced risks of
developing colon cancer .
5. Effects of complete gut fermentation samples better reflect
in vivo exposure conditions than butyrate alone
Dietary fibres reach the colon unaltered, where they are
fermented by the gut flora to yield products such as SCFA. Of these,
butyrate is physiologically relevant to the colonic epithelium in
which it serves as a principle energy source . Interest in its
role as a possible protective agent has arisen from its properties to
inhibit survival of cells in vitro , including colon tumour cell
lines [68,132]. Other findings show that it also protects from H2O2-
induced genetic damage in primary rat and human colon cells and
in human colon tumour cell lines [109,110].
The theory of butyrate’s efficacy in reducing cancer risks,
however, has several flaws, the major one being that there is
vivo .Finally,anothershortcominginunderstanding butyrate’s
efficacy in chemoprevention is that little is known on the impact of
how butyrate affects human colon cells in conjunction with the
other fermentation products, which are formed simultaneously.
The additional components may theoretically enhance, inhibit or
even synergistically increase the activities that butyrate has been
shown to extend as an individual compound. Recent studies are
therefore addressing the possibility of these combination effects.
Some of these studies have, for instance, shown that butyrate’s
impact on inhibiting cell survival, is additive with propionate (but
not with acetate). Thus it was shown that propionate also inhibits
tumour cell survival at low concentrations and mixtures of
propionate and butyrate were more effective than each individual
compound on its own . The combination of SCFA (as present in
the complex fermentation mixture) was, however, less effective
than the complete fermentation mixture itself. Therefore,probably
can inhibit survival of tumour cells. These could include additional
minor SCFA, which are, however, probably formed (depending on
dietary fibre) at such low concentrations that they may not have
significant impact on the measured parameters. Examples for
these minor SCFA are valerate, hexanoate and the branched SCFA
(isobutyrate and isovalerate). Qualities (relative molar composi-
tion) and quantities (absolute molar concentrations) of SCFA in
general are highly different for different fibres and thus must be
considered when explaining why different fibres have different
impacts in the gut . Moreover, formation of toxic and faecal
associated products, such as bile acids, may be suppressed in the
presence of dietary fibre and thus their absence could have
toxicological consequences as well.
Next to SCFA, there are still other fermentation products that
arise from a wide variety of plants and animal nutrients. An
example of such a fibre source is wheat bran that also contains a
number of secondary plant ingredients or phytoprotectants, which
have antioxidative potential  and which are concentrated in
the aleuron layer . These possible chemoprotective com-
pounds include phytic acid [135,136], alkylresorcinols ,
apigenin , lignans , lipids  and hydroxycinnamic
acids , in particular ferulic acid. Their diverse biological
activities include scavenging of radicals , acting anti-
inflammatory , and anticarcinogenic in the rat colon 
and mouse skin .
Altogether very little is known on the quality and quantity of
the many diverse compounds from most plant sources of dietary
fibre and which fate they have in the gut. But experimental studies
with individual components of e.g. defined components of dietary
fibre or of plant extracts do point to the possibility that they could
substantially contribute to biological activities in cells of the gut
mucosa and potentially result in chemoprotective, anticancer
types of effects.
5.1. Experimental approaches to obtain samples simulating gut lumen
Different methods have been established to simulate digestion
in vitro, resulting in samples resembling the contents of the gut
lumen. These methods can either simulate the passage of foods
through the whole digestive tract (including mouth, stomach,
small and large intestine) or simulate only a part of the whole
passage. The type of food ingredient that is supposed to be
analysed and its special physiological properties during digestion
have to be considered to choose the appropriate digestion model.
Dietary fibre sources, which contain ingredients that do not
fully reach the colon because they may be digested and absorbed,
can be pre-digested using conditions, which simulate the upper
regions of the gastrointestinal tract. This can be done according to
an in vitro-batch-technique which simulates the enzymatic
degradation of starch and proteins in the upper gastrointestinal
tract (stomach and duodenum), using pepsin anda-amylase .
Dietary fibres, which completely reach the colon in an undigested
manner, can be directly subjected to the simulation of the colon.
Fermentation products can be generated in vitro using
anaerobic ‘‘batch culture’’ protocols that simulate the conditions
of the human colon lumen. This provides an experimental
approach to compare biological activities of different dietary fibre
sources or rather more of their fermentation products . For
this, samples are mixed with human gut flora and incubated in
D. Scharlau et al./Mutation Research 682 (2009) 39–53
is used as a bacterial source and mixed with buffer and the food
samples to give a final fibre content of 10 g/l and faecal suspension
performed for 24 h and subsequently insoluble parts are removed
from the samples by centrifugation. The obtained fermentation
supernatants represent the composition of the gut lumen and can
be used for further studies.
Alternatively, different types of gut models can be used, which
are more refined in their anaerobic culture conditions and in their
microbiological turnover and thus better mimic the in vivo
situation. The three stage type of gut model, for instance can be
used to simulate in vivo fermentation in the various colon
segments (proximal, sigmoid, distal) . The most advanced
in vitro model of the gastrointestinal tract allows considering
concentrations and activities of intestinal contents (e.g. digestive
enzymes, microbes) and the passage of food products through the
gastrointestinal tract as a dynamic process. The TNO, computer
controlled, gastrointestinal models (nicknamed TIM) simulate to a
high degree these successive dynamic processes in the stomach
and the small intestine (TIM-1) and the large intestine (TIM-2).
These complex models are highly elaborate tools to study the
stability, release, absorption and metabolism of food stuffs or food
5.2. Some key results on complex fermentation samples and their
effects on GSTs
The addition of 10% of inulin fermentation supernatants
obtained by a batch model simulating fermentation in the colon
to the medium (resulting in an effective concentration of 2 mM
butyrate) resulted in a marked induction of GST enzyme activity
(57.0 ? 5.1 nmol/min ml/106cells) in comparison to the medium
control (43.1 ? 4.6 nmol/min ml/106cells) . Some further key
results on comparative activities of fermentation samples derived
from inulin-type fructans and how they effectively modulate a
diverse range of biological parameters (including induction of GSTs)
in comparison to the corresponding mixtures of SCFA and of butyrate
are summarised in Table 4.
inulin, soy and wheat) were fermented with human faecal slurries
in vitro, analysed for SCFA, and corresponding synthetic SCFA
mixtures were prepared . HT29 colon tumour cells were
treated for 72 h with individual SCFA or complex samples. Growth
of cells, GST activity, and chemoresistance towards HNE were
determined. Fermentation products inhibited cell growth more
than corresponding SCFA mixtures, and the SCFA mixtures were
more active than butyrate, probably due to phytoprotectants and
to propionate, respectively, which also inhibit cell growth. Only
butyrate induced GST activity, whereas chemoresistance was
caused by SCFA mixtures from all four fibre sources, but
significantly only by the corresponding fermentation samples
produced from soy. This may mean that a GST isoenzyme was
induced that does not efficiently conjugate CDNB, but which may
deactivate HNE. The chemoprotective activities, consisting of GST
induction and antigenotoxicity towards HNE, of the fermentation
samples were, however,lowerthan thoseof the SCFA mixtures and
these were also less pronounced than the activities of butyrate. In
contrast effects on inhibition of cell growth followed the opposite
pattern, where the complex fermentation samples were more
effective than the SCFA mixtures and these were more effective
than butyrate alone. It must be borne in mind that the
fermentation of whole vegetables releases a broader range of
compounds than expected for inulin alone, and especially
polyphenols may also contribute to chemoprevention. Recently,
a study has shown that e.g. intervention with a compound related
to green tea, polyphenon E, modulated GST activity and GSTP1
will therefore self evidently be of interest, to study the bio-
activation capacity of polyphenols in the gut more in depth, and
how they may interact with butyrate in this context.
In an extension of the studies with fermentation of grains, we
next investigated effects of wheat bran-derived arabinoxylans and
fermentation products on similar parameters of chemoprevention.
Newly isolated water extractable (WeAx) and alkali extractable
arabinoxylans (AeAx) were fermented under anaerobic conditions
with human faeces. Resulting fermentation supernatants were
analysed for SCFA and used to treat HT29 colon cancer cells. Cell
growth, cytotoxicity, antigenotoxicity against H2O2or HNE, and
GST activity were determined. WeAx decreased H2O2-induced
DNA damage by 64%, thus demonstrating chemoprotective
properties by this wheat bran fibre. The fermentation of WeAx
and AeAx resulted in 3-fold increases of SCFA, but all fermentation
supernatants (including the control without arabinoxylans)
inhibited the growth of the HT29 cells, reduced the genotoxicity
of HNE, and enhanced the activity of GSTs (FS WeAx, 2-fold; FS
AeAx, 1.7-fold; and control FS, 1.4-fold), which detoxify HNE. Thus,
increases in SCFA were not reflected by enhanced functional
effects. The conclusion was that fermentation mixtures contain
modulatory compounds that arise from the faeces and might add
to the effectiveness of SCFA . In a study on fermentation
samples derived from resistant starch, it was shown that batch
Relative efficacies of fermentation supernatants, SCFA mixtures and butyrate for modulating various parameters (SFS = Synergy1fermentation supernatant, SCFA
mixture = acetate, butyrate, propionate in the concentrations present in SFS).
Cell type ParameterFermented
Relative activities based on medium controlsReference
HT29 cellsInhibition of cell survival
Increased histone H4 acetylation
Stimulation of GST activity
Inhibition of cell survival
Stimulation of GST activity
Protection from HNE genotoxicity
SFS > SCFA mixture > butyrate > faeces control
SCFA-Mix > butyrate
SFS = SCFA mixture > butyrate > faeces control
SFS > SCFA mixture > butyrate > faeces control
Only butyrate stimulates GST activity
Butyrate > SCFA mixture >SFS
LT97 cells Inhibition of cell survival
Increased histone H4 acetylation
Stimulation of GST activity
SFS > SCFA mixture > butyrate > faeces control
Butyrate > SFS (10%) or SCFA-Mix (100%) > butyrate > SFS
Was only measured in HT29
Primary colon cells Supporting cell survival (trophic effects)
Stimulation of GST activity
Induced GSTA4 mRNA
Induced catalase mRNA
Induced catalase activity
SFS > faeces control > SCFA mixture = butyrate
No effect by any treatment (12 h)
Butyrate = faeces control > SFS = SCFA mixture
Only butyrate was investigated
Only butyrate was investigated
D. Scharlau et al./Mutation Research 682 (2009) 39–53
fermentation resulted in antigenotoxicity towards H2O2. Moreover
there was also an improvement of integrity across the intestinal
barrier and the authors concluded that fermentation products of
selected resistant starches were capable of inhibiting the initiation
and promotionstages of carcinogenesis in vitro . These results
from in vitro studies are supported by some in vivo studies. Dietary
resistant starch, especially in the butyrylated form, effectively
reduced DNA damage induced by high-protein diets in rats
[153,154]. Moreover DNA single-strand and double-strand breaks
caused by white or red meat were also reduced by including
resistant starch in the diet of rats . These results and the in
vitro results described above therefore suggest that dietary fibres
or their metabolites could efficiently reduce DNA damage and may
reduce colon cancer risk. However results from human studies to
prove this are still deficient.
6. Summary and conclusions
It is well known that nutrition leads to a considerable burden of
toxic and genotoxic factors in the gut lumen. Faecal samples for
instance have been shown to contain bile acids, amines, sulfates,
bacterial toxins as well as additional products of bacterial
biotransformation, non-digested food residues, excretable meta-
bolites, and toxic compounds.
Beneficial effects of fibres are seen in their ability to shorten the
toxins. Additionally, fibre adsorbs toxic metabolites of endogenous
and/or bacterial origin (e.g. secondary bile acids, biogenic amines,
bacterial toxins), thereby also reducing exposure of the enter-
ocytes to these faecal compounds. Moreover, dietary fibre
selectively may enhance the growth of non-pathogenic gut
bacteria (e.g. lactic acid producing bacteria), such as Bifidobacter-
ium. These bacteria and others consume the dietary fibre, produce
lactic acid or SCFA, such as butyrate, acetate and propionate. They
metabolites and protect the enterocytes by different molecular
Yetanotherpostulated mechanismofdietaryfibreisthat itmay
act chemoprotective through its microbial production of butyrate.
This compound has been investigated in many model systems and
has been found to act beneficial by inhibiting growth of tumour
cells, inducing tumour cell differentiation or causing the elimina-
tion of these cells by apoptosis. In non-transformed colon cells,
butyrate is a growth factor and a nutrient. An important
mechanism by which butyrate causes biological effects in colon
carcinoma cells has been proposed to be the hyperacetylation of
histones by inhibiting HDAC, thereby compensating an imbalance
of histone acetylation which can lead to transcriptional dysregula-
tion and silencing of genes that are involved in the control of cell-
cycle progression, differentiation, apoptosis and cancer develop-
A new mechanism, addressed in this review, however, is the
been shown for the example of GSTs that they were induced on
mRNA, protein and activity levels in colon cells representing
different stages of the neoplastic cell transformation process.
Importantly, this modulation of gene and protein expression may
protect cells from genotoxic carcinogens, such as H2O2and HNE. In
relation to the in vivo situation, however, it must be remembered
that butyrate is not available as a sole compound but probably acts
in unison with other metabolites of dietary fibre and of the faecal
gut flora. In this context the new approaches to investigate
complete gut fermentation samples are of importance, since they
self evidently better reflect in vivo exposure conditions than
butyrate alone. Some first key results on biological activities of
complex fermentation samples have for instance shown that
fermentation samples derived from inulin-type fructans may
increase level of butyrate and induce GST activities; whereas
fermentation supernatants derived from other fibres do not
necessarily have similar effects. It must be borne in mind that
most of the results come from in vitro studies and few come from
animal studies. Further animal and human studies are thus needed
to define the exact role of dietary fibres and butyrate in inducing
GST activity and thereby reducing the risk of colon cancer. In any
case, especially in light of controversial epidemiological findings
on cancer prevention, further research is needed on the contribu-
tion of butyrate and other metabolites to chemoprevention. This
research should include the identification of chemoprotection
markers that are sensitive to butyrate and these could include the
up-regulation of detoxifying enzymes.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Work on primary cells was supported by the German Research
Council, Deutsche Forschungsgemeinschaft, Germany (DFG PO
284/8-1). The work on LT97 cells was supported by the German
Research Council, BMBF, Germany (FKZ 01EA0103) and the work
on HT29 cells by ORAFTI, Tienen, Belgium (PRECANTOO). The
intervention study with synbiotics was supported by EU (QCRT-
1999-00346). Research on prebiotics was supported by the
0313829C/D’O), Deutsche Forschungsgemeinschaft,
(PO 284/8-2), EU project SEAFOODPLUS (FQS-506359) and the
Food Standards Agency, Biomics (N 12013). We thank all donors of
biopsies and tissue samples for giving their informed consent and
supporting the work. In addition we thank Dr. K. Richter
(Department of General, Visceral and Vascular Surgery, Friedrich
Schiller University of Jena) for supplying samples from tumour
resections. We thank Dr. K. Tuohy (Department of Food Microbial
Sciences, School of Food Biosciences, University of Reading, United
Kingdom) who kindly provided the fermentation supernatants.
Additionally, we thank Prof. G. Jahreis (Department of Nutritional
Physiology, Institute for Nutrition, Friedrich Schiller University
Jena, Germany) for identifying the short-chain fatty acids in the
fermentation samples and Prof. B. Marian (Institute for Cancer
Research, Medical University of Vienna, Austria) for the kindly gift
of LT97 cells.
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